CA2464239C - Psma antibodies and protein multimers - Google Patents

Psma antibodies and protein multimers Download PDF

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Publication number
CA2464239C
CA2464239C CA2464239A CA2464239A CA2464239C CA 2464239 C CA2464239 C CA 2464239C CA 2464239 A CA2464239 A CA 2464239A CA 2464239 A CA2464239 A CA 2464239A CA 2464239 C CA2464239 C CA 2464239C
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psma
antigen
antibody
binding fragment
seq
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CA2464239A1 (en
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Paul J. Maddon
Gerald P. Donovan
William C. Olson
Norbert Schuelke
Jason Gardner
Dangshe Ma
Jaspal S. Kang
Larry Green
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Amgen Fremont Inc
PSMA Development Co LLC
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Amgen Fremont Inc
PSMA Development Co LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A61K51/1096—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1072—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Abstract

The invention includes antibodies or antigen-binding fragments thereof which bind specifically to conformational epitopes on the extracellular domain of PSMA, compositions containing one or a combination of such antibodies or antibodies or antigen-binding fragments thereof, hybridoma cell lines that produce the antibodies, and methods of using the antibodies or antigen-binding fragments thereof for cancer diagnosis and treatment. The invention also includes oligomeric forms of PSMA proteins, compositions comprising the multimers, and antibodies that selectively bind to the multimers.

Description

PSIVIA ANTIBODIES AND PROTEIN MULTIMERS
Field of the Invention This invention relates generally to the field of cancer associated polypeptides and antibodies that recognize native epitopes on the polypeptides. In particular, the invention relates in part to antibodies or antigen-binding fragments thereof which bind specifically to conformational epitopes on the extracellular domain of PSMA, rnultimeric forms of PSMA
proteins, antibodies that selectively bind to the m.ultimers, and compositions containing such antibodies or multimers.
Background of the Invention Prostate cancer is the most prevalent type of cancer and the second leading cause of death from cancer in American men, with an estimated 179,000 cases and 37,000 deaths in 1999, (Landis, S.H. et al. CA Cancer J. Clin. 48:6-29 (1998)). The number of men diagnosed with prostate cancer is steadily increasing as a result of the increasing population of older men as well as a greater awareness of the disease leading to its earlier diagnosis (Parker et al., 1997, CA Cancer .1. Clin. 47:5-280). The life time risk for men developing prostate cancer is about 1 in 5 for Caucasians, 1 in 6 for African Americans. High risk groups are represented by those with a positive family history of prostate cancer or African Americans.
Over a lifetime, more than 2/3 of the men diagnosed with prostate cancer die of the disease (Wingo et al., 1996, CA Cancer J. Clin. 46:113-25). Moreover, many patients who do not succumb to prostate cancer require continuous treatment to ameliorate symptoms such as pain, bleeding and urinary obstruction. Thus, prostate cancer also represents a major cause of suffering and increased health care expenditures.
Where prostate cancer is localized and the patient's life expectancy is 10 years or more, radical prostatectomy offers the best chance for eradication of the disease.
- 2 -Historically, the drawback of this procedure is that most cancers had spread beyond the bounds of the operation by the time they were detected. Patients with bulky, high-grade tumors are less likely to be successfully treated by radical prostatectomy.
Radiation therapy has also been widely used as an alternative to radical prostatectomy. Patients generally treated by radiation therapy are those who are older and less healthy and those with higher-grade, more clinically advanced tumors.
Particularly preferred procedures are external-beam therapy which involves three dimensional, confocal radiation therapy where the field of radiation is designed to conform to the volume of tissue treated;
interstitial-radiation therapy where seeds of radioactive compounds are implanted using ultrasound guidance; and a combination of external-beam therapy and interstitial-radiation therapy.
For treatment of patients with locally advanced disease, hormonal therapy before or following radical prostatectomy or radiation therapy has been utilized.
Hormonal therapy is the main form of treating men with disseminated prostate cancer. Orchiectomy reduces serum testosterone concentrations, while estrogen treatment is similarly beneficial.
Diethylstilbestrol from estrogen is another useful hormonal therapy which has a disadvantage of causing cardiovascular toxicity. When gonadotropin-releasing hormone agonists are administered testosterone concentrations are ultimately reduced. Flutamide and other nonsteroidal, anti-androgen agents block binding of testosterone to its intracellular receptors.
As a result, it blocks the effect of testosterone, increasing serum testosterone concentrations and allows patients to remain potent--a significant problem after radical prostatectomy and radiation treatments.
Cytotoxic chemotherapy is largely ineffective in treating prostate cancer. Its toxicity makes such therapy unsuitable for elderly patients. In addition, prostate cancer is relatively resistant to cytotoxic agents.
Relapsed or more advanced disease is also treated with anti-androgen therapy.
Unfortunately, almost all tumors become hormone-resistant and progress rapidly in the absence of any effective therapy.
Accordingly, there is a need for effective therapeutics for prostate cancer which are not overwhelmingly toxic to normal tissues of a patient, and which are effective in selectively eliminating prostate cancer cells.
3 Summary of the Invention The present invention relates to antibodies or antigen-binding fragments thereof which specifically bind the extracellular domain of prostate specific membrane antigen (PSMA), compositions containing one or a combination of such antibodies or antigen-binding fragments thereof, hybridoma cell lines that produce the antibodies, and methods of using the antibodies or antigen-binding fragments thereof for cancer diagnosis and treatment.
According to one aspect of the invention, isolated antibodies or an antigen-binding fragments thereof are provided. The antibodies or fragments thereof specifically bind to an extracellular domain of prostate specific membrane antigen (PSMA), and competitively inhibit the specific binding of a second antibody to its target epitope on PSMA. In a second aspect of the invention, isolated antibodies or antigen-binding fragments thereof are provided which specifically bind to an epitope on prostate specific membrane antigen (PSMA) defined by a second antibody. In each of the forgoing aspects of the invention, the second antibody is selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA
5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix
4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, Abgenix 4.152.1, and antibodies comprising (a) a heavy chain encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as SEQ lD
NOs: 8-13.
In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11 PSMA 5.4, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, and Abgenix 4.152.1.

In other embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of antibodies comprising (a) a heavy chain encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as SEQ ID
NOs: 8-13, and antigen-binding fragments thereof.
In further embodiments, the antibody or antigen-binding fragments thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least about 90%
identical to the nucleotide sequence encoding the foregoing antibodies, preferably at least about 95% identical, more preferably at least about 97% identical, still more preferably at least about 98% identical, and most preferably is at least about 99%
identical.
In some embodiments of the foregoing aspects, antigen-binding fragments of the isolated antibodies are provided. The antigen-binding fragments include (a) a heavy chain variable region encoded by a nucleic acid molecule comprising the coding regions or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as: SEQ ID NOs: 14, 18, 22, 26 and 30, and (b) a light chain variable region encoded by a nucleic acid molecule comprising the coding region or region of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as: SEQ
ID NOs: 16, 20, 24, 28 and 32. In other embodiments, the antigen-binding fragment includes (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of amino acid sequences set forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of nucleotide sequences set forth as: SEQ ID NOs: 17, 21, 25, 29 and 33.
In a further embodiments of the invention, isolated antigen-binding fragments of antibodies, which include a CDR of the foregoing antigen-binding fragments are provided.
Preferably the CDR is CDR3.
According another aspect of the invention, expression vectors including an isolated nucleic acid molecule encoding the foregoing isolated antibodies or antigen-binding fragments is provided. Host cells transformed or transfected by these expression vectors also are provided.
- 5 -In certain embodiments, the antibody or antigen-binding fragment thereof is selected for its ability to bind live cells, such as a tumor cell or a prostate cell, preferably LNCaP cells.
In other embodiments, the antibody or antigen-binding fragment thereof mediates cytolysis of cells expressing PSMA. Preferably cytolysis of cells expressing PSMA is mediated by effector cells or is complement mediated in the presence of effector cells.
In other embodiments, the antibody or antigen-binding fragment thereof inhibits the growth of cells expressing PSMA. Preferably the antibody or antigen-binding fragment thereof does not require cell lysis to bind to the extracellular domain of PSMA.
In further embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE or has immunoglobulin constant and/or variable domain of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, TgA2, IgAsec, IgD or IgE. In other embodiments, the antibody is a bispecific or multispecific antibody.
In still other embodiments, the antibody is a recombinant antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody or a chimeric antibody, or a mixture of these. In particularly preferred embodiments, the antibody is a human antibody, e.g., a monoclonal antibody, polyclonal antibody or a mixture of monoclonal and polyclonal antibodies. In still other embodiments, the antibody is a bispecific or multispecific antibody.
Preferred antigen-binding fragments include a Fab fragment, a F(ab)2 fragment, and a Fv fragment CDR3.
In further embodiments, the isolated antibody or antigen-binding fragment is a monoclonal antibody produced by a hybridoma cell line selected from the group consisting of PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA

5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA 10.3 (PTA-3247) , PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389), Abgenix
-6-4.2923 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix 4.152.1(PTA-4653).
In certain other embodiments, the antibody or antigen-binding fragment thereof binds to a conformational epitope and/or is internalized into a cell along with the prostate specific membrane antigen. In other embodiments, the isolated antibody or antigen-binding fragment thereof is bound to a label, preferably one selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.
In still other embodiments, the isolated antibody or antigen-binding fragment thereof is bound to at least one therapeutic moiety, such as a drug, preferably a cytotoxic drug, a replication-selective virus, a toxin or a fragment thereof, or an enzyme or a fragment thereof.
Preferred cytotoxic drug include: calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin, paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin PHE. In other embodiments, the therapeutic moiety is an immunostimulatory or immunomodulating agent, preferably one selected from the group consisting of: a cytokine, chemokine and adjuvant.
In some embodiments, the antibodies or antigen-binding fragments of the invention specifically bind cell-surface PSMA and/or rsPSMA with a binding affinity of about 1 X
10-9M or less. Preferably, the binding affinity is about 1 X 10-1 M or less, more preferably the binding affinity is about 1 X 1041M or less. In other embodiments the binding affinity is less than about 5 X 1040M.
In additional embodiments, the antibodies or antigen-binding fragments of the invention mediate specific cell killing of PSMA-expressing cells with an IC50s of less than about 1 X 10-1 M. Preferably the IC50s is less than about 1 X 1041M. More preferably the IC50s is less than about 1 X 10-12M. In other embodiments the IC5os is less than about 1.5 X
10-"M.
In yet other embodiments, the isolated antibody or antigen-binding fragment thereof is bound to a radioisotope. The radioisotope can emit a radiations, 13 radiations, or y radiations.
Preferably the radioisotope is selected from the group consisting of 225Ac,2mmAt,212Bi, 213Bi, 186Rh, 188Rh, 177th, 90y, 131L 67c,u, 125L 123L 77Br, 153sm, 166H0,64Cu, 212pb, 224Ra and 223Ra.
- 7 -According to another aspect of the invention, hybridoma cell lines are provided that produce an antibody selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1 and Abgenix 4.152.1. In some embodiments, the hybridoma cell line is selected from the group consisting of PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA
7.3 (PTA-3293), PSMA 10.3 (PTA-3247) , PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix 4.152.1(PTA-4653).
According to a further aspect of the invention, compositions are provided that include the foregoing antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier, excipient, or stabilizer. Other compositions include a combination of two or more of the foregoing antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier, excipient, or stabilizer. In some embodiments, the compositions also include an antitumor agent, an immunostimulatory agent, an immunomodulator, or a combination thereof. Preferred antitumor agents include a cytotoxic agent, an agent that acts on tumor neovasculature, or a combination thereof.
Preferred immunomodulators include a-interferon, 7-interferon, tumor necrosis factor-a or a combination thereof. Preferred immunostimulatory agents include interleukin-2, immunostimulatory oligonucleotides, or a combination thereof.
According to another aspect of the invention, kits for detecting prostate cancer for diagnosis, prognosis or monitoring are provided. The kits include the foregoing isolated
- 8 -labeled antibody or antigen-binding fragment thereof, and one or more compounds for detecting the label. Preferably the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.
The invention in another aspect provides one or more of the foregoing isolated antibodies or antigen-binding fragments thereof packaged in lyophilized form, or packaged in an aqueous medium.
In another aspect of the invention, methods for detecting the presence of PSMA, or a cell expressing PSMA, in a sample are provided. The methods include contacting the sample with any of the foregoing antibodies or antigen-binding fragments thereof which specifically bind to an extracellular domain of PSMA, for a time sufficient to allow the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the PSMA-antibody complex or PSMA-antigen-binding fragment complex. The presence of a complex in the sample is indicative of the presence in the sample of PSMA or a cell expressing PSMA.
In another aspect, the invention provides other methods for diagnosing a PSMA-mediated disease in a subject. The methods include administering to a subject suspected of having or previously diagnosed with PSMA-mediated disease an amount of any of the foregoing antibodies or antigen-binding fragments thereof which specifically bind to an extracellular domain of prostate specific membrane antigen. The method also includes allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the PSMA-antibody complex or PSMA-antigen-binding fragment antibody complex to the target epitope. The presence of a complex in the subject suspected of having or previously diagnosed with prostate cancer is indicative of the presence of a PSMA-mediated disease.
In certain embodiments of the methods, the PSMA-mediated disease is prostate cancer.
- 9 -In preferred embodiments of the foregoing methods, the antibody or antigen-binding fragment thereof is labeled. In other embodiments of the foregoing methods, a second antibody is administered to detect the first antibody or antigen-binding fragment thereof.
In a further aspect of the invention, methods for assessing the prognosis of a subject with a PSMA-mediated disease are provided. The methods include administering to a subject suspected of having or previously diagnosed with PSMA-mediated disease an effective amount of an antibody or antigen-binding fragment thereof according to claim Al or Bl, allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the complex to the target epitope. The amount of the complex in the subject suspected of having or previously diagnosed with PSMA-mediated disease is indicative of the prognosis.
In another aspect of the invention, methods for assessing the effectiveness of a treatment of a subject with a PSMA-mediated disease are provided. The methods include administering to a subject suspected treated for a PSMA-mediated disease an effective amount of the foregoing antibodies or antigen-binding fragments thereof, allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the complex to the target epitope. The amount of the complex in the subject suspected of having or previously diagnosed with PSMA-mediated disease is indicative of the effectiveness of-the treatment.
In certain embodiments of these two aspects of the invention, the PSMA-mediated disease is prostate cancer. "
- 10 -fragment thereof is labeled. In further embodiments, a second antibody is administered to detect the first antibody or antigen-binding fragment thereof.
According to yet another aspect of the invention, methods for inhibiting the growth of a cell expressing PSMA are provided. The methods include contacting a cell expressing PSMA with an amount of at least one of the foregoing antibodies or antigen-binding fragments thereof which specifically binds to an extracellular domain of PSMA
effective to inhibit the growth of the cell expressing PSMA.
According to another aspect of the invention, methods for inducing cytolysis of a cell expressing PSMA are provided. The methods include contacting a cell expressing PSMA
with an amount of at least one of the foregoing antibodies or antigen-binding fragments thereof which specifically binds to an extracellular domain of PSMA effective to induce cytolysis of the cell expressing PSMA. In certain embodiments, the cytolysis occurs in the presence of an effector cell. In other embodiments, the cytolysis is complement mediated.
According to still another aspect of the invention, methods for treating or preventing a PSMA-mediated disease are provided. The methods include administering to a subject having a PSMA-mediated disease an effective amount of at least one of the forgoing antibodies or antigen-binding fragments thereof to treat or prevent the PSMA-mediated .
disease. In some embodiments, the PSMA-mediated disease is a cancer, such as prostate cancer.
In yet a further aspect of the invention, methods for treating or preventing a PSMA-mediated disease are provided. The methods include administering to a subject having a PSMA-mediated disease or at risk of having a PSMA-mediated disease an amount of at least one of the foregoing antibodies or antigen-binding fragments thereof effective to treat or prevent the PSMA-mediated disease.
In some embodiments, the PSMA-mediated disease is a cancer, such as prostate cancer.
In other embodiments, the method also includes administering another therapeutic agent to treat or prevent the PSMA-mediated disease at any time before, during or after the administration of the antibody or antigen-binding fragment thereof. In some of these
- 11 -embodiments, the therapeutic agent is a vaccine, and preferably the vaccine immunizes the subject against PSMA.
In still other embodiments, the antibody or antigen-binding fragment thereof is bound to at least one therapeutic moiety, preferably a cytotoxic drug, a drug which acts on the tumor neovasculature and combinations thereof. Preferred cytotoxic drugs are selected from the group consisting of: calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin, paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin PHE.
In other embodiments, the antibody or antigen-binding fragment thereof is bound to a radioisotope and the radiations emitted by the radioisotope is selected from the group consisting of a, i3 and y radiations. Preferably, the radioisotope is selected from the group consisting of 225Ac, 211m, 212Bi, 213Bi, 186Rh, 188Rh, 177u, 90y, 1311, 67cu, 1251, 123-, 1 77Br, 153sm, 166H0, 64ca, 212pb, 224Ra and 223R a.
The present invention provides methods for modulating at least one enzymatic activity of PSMA. As used in preferred embodiments of the methods, "modulating" an enzymatic activity of PSMA means enhancing or inhibiting the enzymatic activity. Thus in certain aspects of the invention, methods for inhibiting an enzymatic activity of PSMA are provided, and in other aspects of the invention, methods for enhancing an enzymatic activity of PSMA are provided. The terms "enhancing' and "inhibiting" in this context indicate that the enzymatic activity of PSMA is enhanced or inhibited in the presence of an antibody that specifically binds PSMA, or antigen-binding fragment thereof, relative to the level of activity in the absence of such an antibody or antigen-binding fragment thereof.
Enzymatic activities of PSMA include folate hydrolase activity, N-acetylated a-linked acidic dipeptidase (NAALADase) activity, dipeptidyl dipeptidase IV activity and y-glutamyl hydrolase activity.
Thus the invention in another aspect provides methods for modulating folate hydrolase activity. In certain embodiments of these methods, the activity is inhibited and in other embodiments, the activity is enhanced. The methods include contacting a folate hydrolase polypeptide with an amount of the foregoing isolated antibody or antigen-binding fragment thereof, under conditions wherein the isolated antibody or antigen-binding fragment thereof modulates the folate hydrolase activity. The folate hydrolase polypeptide can be isolated, contained in a sample such as a cell, a cell homogenate, a tissue, or a tissue
- 12 -homogenate, or contained in an organism. The organism preferably is an animal, particularly preferably a mammal.
In another aspect of the invention, methods for modulating N-acetylated a-linked acidic dipeptidase (NAALADase) activity are provided. In certain embodiments of these methods, the activity is inhibited and in other embodiments, the activity is enhanced. The methods include contacting a NAALADase polypeptide with an amount of the foregoing isolated antibody or antigen-binding fragment thereof under conditions wherein the isolated antibody or antigen-binding fragment thereof modulates NAALADase activity. The NAALADase polypeptide can be isolated, contained in a sample such as a cell, a cell homogenate, a tissue, or a tissue homogenate, or contained in an organism. The organism preferably is an animal, particularly preferably a mammal.
In yet another aspect of the invention, methods for modulating dipeptidyl dipeptidase IV activity are provided. In certain embodiments of these methods, the activity is inhibited and in other embodiments, the activity is enhanced. The methods include contacting a dipeptidyl dipeptidase IV polypeptide with an amount of the foregoing isolated antibody or antigen-binding fragment thereof under conditions wherein the isolated antibody or antigen-binding fragment thereof modulates dipeptidyl dipeptidase IV activity. The dipeptidyl dipeptidase IV polypeptide can be isolated, contained in a sample such as a cell, a cell homogenate, a tissue, or a tissue homogenate, or contained in an organism. The organism preferably is an animal, particularly preferably a mammal.
In yet another aspect of the invention, methods for modulating 7-glutamy1 hydrolase activity are provided. In certain embodiments of these methods, the activity is inhibited and in other embodiments, the activity is enhanced. The methods include contacting a 7-glutamyl hydrolase polypeptide with an amount of the foregoing isolated antibody or antigen-binding fragment thereof under conditions wherein the isolated antibody or antigen-binding fragment thereof modulates 7-glutamyl hydrolase activity. The 7-glutamyl hydrolase polypeptide can be isolated, contained in a sample such as a cell, a cell homogenate, a tissue, or a tissue homogenate, or contained in an organism. The organism preferably is an animal, particularly preferably a mammal.
Methods of specific delivery of at least one therapeutic agent to PSMA-expressing cells are provided according to another aspect of the invention. The methods include administering an effective amount of at least one of the foregoing antibodies or antigen-
- 13 -binding fragments thereof conjugated to the at least one therapeutic agent. In some embodiments, the therapeutic agent is a nucleic acid molecule, an antitumor drug, a toxin or a fragment thereof, an enzyme or a fragment thereof, a replication-selective virus, or an immunostimulatory or immunomodulating agent. Preferred antitumor drugs include cytotoxic drugs, drugs which act on the tumor neovasculature and combinations thereof.
Preferred cytotoxic drugs include calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin, paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin PHE. Preferred immunostimulatory or immunomodulating agent included cytokines, chemokines and adjuvants.
In still another aspect of the invention, isolated antibodies that selectively bind a PSMA protein multimer are provided. In preferred embodiments, the PSMA protein multimer is a dimer, and preferably at least one of the PSMA proteins forming the multimer is a recombinant, soluble PSMA (rsPSMA) polypeptide. Preferably the rsPSMA
polypeptide consists essentially of amino acids 44-750 of SEQ ID NO: 1.
In a further aspect of the invention, isolated antibodies are provided that selectively , bind a PSMA protein multimer and modulate one or more enzymatic activities of the PSMA
protein multimer. As used in preferred embodiments of this aspect of the invention, "modulating" an enzymatic activity of a PSMA multimer means enhancing or inhibiting the enzymatic activity. Thus in certain aspects of the invention, antibodies that inhibit an enzymatic activity of PSMA multimers are provided, and in other aspects of the invention, antibodies that inhibit an enzymatic activity of PSMA multimers are provided.
The terms "enhancing' and "inhibiting" in this context indicate that the enzymatic activity of a PSMA
multimer is enhanced or inhibited in the presence of an antibody that specifically binds the PSMA multimers, or antigen-binding fragment thereof; relative to the level of activity in the absence of such an antibody or antigen-binding fragment thereof. In some embodiments, the enzymatic activity is selected from the group consisting of folate hydrolase activity, NAALADase activity, dipeptidyl dipeptidase IV activity and y-glutamyl hydrolase activity.
In other embodiments, the enzymatic activity is in the extracellular domain of the PSMA
molecule. In still other embodiments, the antibody or antigen-binding fragment thereof specifically binds to an extracellular domain of PSMA.
- 14 -In a further aspect, an isolated antibody or antigen-binding fragment thereof is provided that selectively binds a PSMA protein multimer. In this aspect, the isolated antibody is raised by immunizing an animal with a preparation comprising a PSMA protein multimer. Preferred preparations used in raising the antibody include those having at least about 10%, 20%, 30%, 40%, 50%, 75%, 90%, or 95% PSMA protein multimer.
Preferably the PSMA protein multimer is a dimer.
In yet another aspect of the invention, compositions are provided that include one or more of the foregoing isolated antibodies, and an immunostimulatory molecule, such as an adjuvant and/or and a cytokine. Preferably the immunostimulatory molecule is IL-2 or an immunostimulatory oligonucleotide. In certain embodiments, the foregoing compositions also include a pharmaceutically-acceptable carrier.
The invention also includes methods for inducing an immune response, including administering to a subject in need of such treatment an effective amount of the foregoing isolated antibodies or compositions.
The invention provides, in another aspect, isolated antibodies or antigen-binding fragments thereof that selectively bind a PSMA protein multimer and modulate at least one enzymatic activity of PSMA. As used in preferred embodiments of this aspect of the invention, "modulating" an enzymatic activity of a PSMA means enhancing or inhibiting the enzymatic activity. Thus in certain aspects of the invention, antibodies that inhibit an enzymatic activity of PSMA are provided, and in other aspects of the invention, antibodies that inhibit an enzymatic activity of PSMA are provided. The terms "enhancing' and "inhibiting" in this context indicate that the enzymatic activity of PSMA is enhanced or inhibited in the presence of an antibody that specifically binds PSMA, or antigen-binding fragment thereof, relative to the level of activity in the absence of such an antibody or antigen-binding fragment thereof. The enzyme, in certain embodiments, is selected from the group consisting of hydrolases and peptidases. Preferred hydrolases include folate hydrolase and y-glutamyl hydrolase. In a particularly preferred embodiment of PSMA
inhibition, the hydrolase is folate hydrolase and the antibody is mAb 5.4 or mAb 3.9.
Preferred peptidases include NAALADase and dipeptidyl dipeptidase IV. In some embodiments, the enzyme is active in cancer cells and has lesser activity in normal cells than in cancer cells or, preferably, no activity in normal cells. In preferred embodiments, the cancer cells in which the enzyme is active are prostate cancer cells. Compositions including the foregoing isolated antibodies
- 15 -or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier, also are provided by the invention.
In another aspect of the invention, compositions are provided that include an isolated PSMA protein multimer. Preferably the PSMA protein multimer is a dimer. In certain embodiments, the compositions include at least about 10%, 20%, 30%, 40%, 50%, 75%, 90%, or 95% PSMA protein multimer. In other embodiments, the PSMA protein multimer comprises noncovalently associated PSMA proteins. The PSMA proteins preferably are noncovalently associated under nondenaturing conditions.
In certain embodiments of the foregoing compositions, at least one of the PSMA
proteins forming the multimer is a recombinant, soluble PSMA (rsPSMA) polypeptide. In other embodiments, the PSMA protein multimer is reactive with a conformation-specific antibody that specifically recognizes PSMA. Preferably, the PSMA protein multimer comprises PSMA proteins in a native conformation and/or the PSMA multimer is enzymatically active. In preferred embodiments, the enzymatic activity is folate hydrolase activity, NAALADase activity, dipeptidyl dipeptidase IV activity and/or y-glutamyl hydrolase activity.
In still other embodiments, the foregoing compositions also include an adjuvant and/or a cytokine or other immunostimulatory molecule. Preferred cytokines include IL-2, 1L-12, IL-18 and GM-CSF. In further embodiments, the foregoing compositions also include a pharmaceutically acceptable carrier.
According to yet another aspect of the invention, methods for inducing an immune response are provided. The methods include administering to a subject in need of such treatment an effective amount of one or more of the foregoing compositions.
In a further aspect, the invention includes isolated recombinant soluble PSMA
(rsPSMA) protein multimers, and isolated rsPSMA protein dimers. In some embodiments, the dimer includes noncovalently associated rsPSMA proteins, and preferably the rsPSMA
proteins are noncovalently associated under nondenaturing conditions. In other embodiments, the isolated rsPSMA dimer is reactive with a conformation-specific antibody that specifically recognizes PSMA.
In a certain preferred embodiment, the isolated rsPSMA dimer is enzymatically active, with the enzymatic activity selected from the group consisting of folate hydrolase
- 16 -activity, NAALADase activity, dipeptidyl dipeptidase IV activity and y-glutamyl hydrolase activity.
In still another aspect of the invention, methods of screening for a candidate agent that modulates at least one enzymatic activity of a PSMA enzyme are provided. As used in preferred embodiments of the methods, "modulating" an enzymatic activity of PSMA means enhancing or inhibiting the enzymatic activity. Thus in certain aspects of the invention, methods for screening for a candidate agent that inhibits an enzymatic activity of PSMA are provided, and in other aspects of the invention, methods for screening for a candidate agent that enhances an enzymatic activity of PSMA are provided. The terms "enhancing" and "inhibiting" in this context indicate that the enzymatic activity of PSMA is enhanced or inhibited in the presence of a candidate agent relative to the level of activity in the absence of such an agent. The methods include mixing the candidate agent with an isolated PSMA
protein multimer to form a reaction mixture, followed by adding a substrate for the PSMA
enzyme to the reaction mixture, and determining the amount of a product formed from the substrate by the PSMA enzyme. A change in the amount of product formed in comparison to a control is indicative of an agent capable of modulating at least one enzymatic activity of the PSMA enzyme. A decrease in the amount of product formed in comparison to a control is indicative of an agent capable of inhibiting at least one enzymatic activity of the PSMA
enzyme. An increase in the amount of product formed in comparison to a control is indicative of an agent capable of enhancing at least one enzymatic activity of the PSMA
enzyme. In some embodiments the PSMA enzyme is selected from the group consisting of NAALADase, folate hydrolase, dipeptidyl dipeptidase IV and y-glutamyl hydrolase. In other embodiments the PSMA multimer comprises recombinant, soluble PSMA. In yet other embodiments the candidate agent is selected from the group consisting of an antibody, a small organic compound, or a peptide.
In another aspect of the invention, candidate agents that modulate at least one enzymatic activity of PSMA are provided. The candidate agents are identified according to the foregoing methods. Thus in certain aspects of the invention, candidate agents that inhibit an enzymatic activity of PSMA are provided, and in other aspects of the invention, candidate agents that enhance an enzymatic activity of PSMA are provided. In certain embodiments, the agent is selected from a combinatorial antibody library, a combinatorial protein library, or a small organic molecule library.

=
- 17 -The invention also provides methods for identifying compounds that promote dissociation of PSMA dimers. The methods include contacting a PSMA dimer with a compound under conditions that do not promote dissociation of the PSMA dimer in the absence of the compound, measuring the amount of PSMA monomer and/or dimer;
and comparing the amount of PSMA monomer and/or dimer measured in the presence of the compound with that observed in the absence of the compound. An increase in the amount of PSMA monomer measured in the presence of the compound indicates that the compound is capable of promoting dissociation of the PSMA dimer. A decrease in the amount of PSMA
dimer measured in the presence of the compound indicates that the compound is capable of promoting dissociation of the PSMA dimer. When the amounts of PSMA monomer and PSMA dimer are measured, the methods can include calculating a ratio of PSMA
monomer to PSMA dimer and comparing the ratio obtained in the presence of the compound with that obtained in the absence of the compound. In such methods, an increase in the ratio measured in the presence of the compound indicates that the compound is capable of promoting dissociation of the PSMA dimer.
The use of the foregoing compositions, molecules and agents in the preparation of medicaments also is provided. In preferred embodiments, the medicaments are useful in the treatment of conditions related to hyperproliferative diseases including cancer, and diseases of inappropriate NAALADase activity, folate hydrolase activity, dipeptidyl dipeptidase IV activity and/or y-glutamyl hydrolase activity.
Specific aspects of the invention include:
- an isolated antibody or antigen-binding fragment thereof that selectively binds a prostate specific membrane antigen (PSMA) protein dimer, wherein each of the PSMA proteins forming the dimer comprises amino acids 44-750 of SEQ ID NO:1;
- a composition comprising the isolated antibody or antigen-binding fragment thereof as described herein and an immunostimulatory oligonucleotide;

- 17a -- a composition comprising the isolated antibody or antigen-binding fragment thereof as described herein and a cytokine;
- a composition comprising the isolated antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer;
- an expression vector comprising an isolated nucleic acid molecule encoding the isolated antibody or antigen-binding fragment as described herein;
- a host cell transformed or transfected by the expression vector as described herein;
- a composition comprising: the antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer;
- a composition comprising: a combination of two or more antibodies or antigen-binding fragments thereof as described herein, and a pharmaceutically acceptable carrier, excipient or stabilizer;
- a kit for detecting PSMA-expressing cancer for diagnosis, prognosis or monitoring comprising: the isolated labeled antibody or antigen-binding fragment thereof of as described herein, and one or more compounds for detecting the label;
- a method for detecting the presence of PSMA protein dimers, or a cell expressing PSMA protein dimer, in a sample comprising: contacting the sample with the antibody or antigen-binding fragment thereof as described herein for a time sufficient to allow the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the PSMA-antibody complex or PSMA-antigen-binding fragment complex, wherein the presence of a complex in the sample is indicative of the presence in the sample of PSMA protein dimers or a cell expressing PSMA protein dimer;
- a method for diagnosing a PSMA-expressing cancer in a subject, said method comprising: administering to a subject suspected of having, or previously diagnosed with, =

- 17b -PSMA-expressing cancer an amount of the isolated antibody or antigen-binding fragment thereof as described herein; allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the PSMA-antibody complex or PSMA-antigen-binding fragment complex, wherein the presence of a complex in the subject suspected of having, or previously diagnosed with, PSMA-expressing cancer is indicative of the presence of a PSMA-expressing cancer;
- a method for assessing the prognosis of a subject with a PSMA-expressing cancer, said method comprising: administering the antibody or antigen-binding fragment thereof as described herein to a subject suspected of having or previously diagnosed with PSMA-expressing cancer, allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the complex, wherein the amount of the complex in the subject suspected of having or previously diagnosed with PSMA-expressing cancer is indicative of the prognosis;
- a method for assessing the effectiveness of a treatment of a subject with a PSMA-expressing cancer, said method comprising: administering the antibody or antigen-binding fragment thereof as described herein to a subject treated for a PSMA-expressing cancer, allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the complex, wherein the amount of the complex in the subject suspected of having or previously diagnosed with PSMA-expressing cancer is indicative of the effectiveness of the treatment;
- use of an effective amount of the antibody or antigen-binding fragment thereof as described herein for inhibiting the growth of a cell expressing PSMA protein dimer;
- use an effective amount of the antibody or antigen-binding fragment thereof as described herein for inducing cytolysis of a cell expressing PSMA protein dimer;
- use of the antibody or antigen-binding fragment thereof as described herein for treating for preventing a PSMA-expressing cancer in a subject having a PSMA-expressing cancer or at risk of having a PSMA-expressing cancer;

=

- 17c -- use of the isolated antibody or antigen-binding fragment thereof as described herein for inhibiting folate hydrolase, N-acetylated a-linked acidic dipeptidase (NAALADase), dipeptidyl dipeptidase IV or y-glutamyl hydrolase activity, under conditions wherein the isolated antibody or antigen-binding fragment thereof inhibits the folate hydrolase, NAALADase, dipeptidyl dipeptidase IV or y-glutamyl hydrolase activity;
- use of the antibody or antigen-binding fragment thereof as described herein conjugated to at least one therapeutic agent for specific delivery of said at least one therapeutic agent to PSMA-expressing cells;
- a plasmid which encodes the antibody or antigen-binding fragment thereof as described herein;
- a plasmid selected from the group consisting of AB-PG1-XG1-006 Heavy Chain (SEQ ID NO: 2) (ATCC patent deposit designation PTA-4403), AB-PG1-XG1-Light Chain (SEQ ID NO: 8) (ATCC patent deposit designation PTA-4404), AB-PG1-XG1-026 Heavy Chain (SEQ ID NO: 3) (ATCC patent deposit designation PTA-4405), AB-PG1-XG1-026 Light Chain (SEQ ID NO: 9) (ATCC patent deposit designation PTA-4406), AB-PG1-XG1-051 Heavy Chain (SEQ ID NO: 4) (ATCC patent deposit designation PTA-4407), AB-PG1-XG1-051 Light Chain (SEQ ID NO: 10) (ATCC patent deposit designation PTA-4408), AB-PG1- XG1-077 Heavy Chain (SEQ
ID
NO: 6) (ATCC patent deposit designation PTA-4411), and AB-PG1-XG1-077 Light Chain (SEQ ID NO: 12) (ATCC patent deposit designation PTA-4412);
- a host cell transformed or transfected by the plasmid as described herein;
and - a hybridoma cell line that produces an antibody selected from the group consisting of PSMA 3.9 (ATCC patent deposit designation PTA-3258) and PSMA 5.4 (ATCC patent deposit designation PTA-3268).
These and other aspects of the invention will be described in further detail in connection with the detailed description of the invention.

' 64371-608 - 17d -Brief Description of the Drawings Figure 1 depicts PSMA reactivity of mAbs as determined by flow cytometry.
Anti-PSMA mAbs (3.7, 3.9, 3.11, 3.12, 5.4, and 10.3) incubated with either parental 3T3 cells (denoted by black lines) or 3T3 cells engineered to express cell-surface PSMA
(3T3-PSMA;
gray lines).
Figure 2 shows a digitized image of immunoprecipitation of PSMA by mAbs.
Lysates from 3T3-PSMA cells or parental 3T3 cells were incubated with each mAb and then precipitated using Protein A/G agarose beads. After washing, proteins were resolved on a
- 18 -polyacrylamide gel, blotted onto nitrocellulose membranes and visualized using the MAl3544 anti-PSMA mAb.
Figure 3 shows the recognition of non-denatured PSMA by several PSMA
antibodies that recognize PSMA conformation.
Figure 4 is a digitized image of a Western blot that shows the recognition of denatured PSMA by two PSMA antibodies and shows that antibodies that recognize PSMA
conformation do not recognize denatured PSMA.
Figure 5 is a digitized image of a polyacrylamide gel that shows an analysis of purified recombinant, soluble PSMA (rsPSMA) and of full-length PSMA from 3T3 cells (3T3 PSMA) or LNCaP cells (LNCaP PSMA) by reduced and non-reduced SDS-PAGE.
Figure 6 is a digitized image of a polyacrylamide gel that depicts a Blue Native PAGE
analysis of purified recombinant, soluble PSMA (Purified rsPSMA) and of full-length PSMA
extracted from 3T3 cells (3T3 PSMA) or LNCaP cells (LNCaP PSMA).
Figure 7 shows the effect of four antibodies (mAb 3.9, mAb 5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate hydrolase through measuring the rate of cleavage of glutamate from methotrexate di-gamma glutamate by folate hydrolase present in 0.0002 jig rsPSMA #7.
Figure 8 shows the effect of four antibodies (mAb 3.9, mAb 5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate hydrolase through measuring the rate of cleavage of glutamate from methotrexate di-gamma glutamate by folate hydrolase present in 0.0002 Kg rsPSMA #8.
Figure 9 shows the effect of four antibodies (mAb 3.9, mAb 5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate hydrolase through measuring the rate of cleavage of glutamate from methotrexate di-gamma glutamate by folate hydrolase present in lysates of C4-2 cells.
Figure 10 depicts the cloning protocol for IgG1 antibody cloning into pcDNA.
Figure 11 provides the plasmid map of a nucleic acid molecule encoding the heavy chain of antibody AB-PG1-XG1-006.
Figure 12 provides the plasmid map of a nucleic acid molecule encoding the heavy chain of antibody AB-PG1-XG1-026.
Figure 13 provides the plasmid map of a nucleic acid molecule encoding the heavy chain of antibody AB-PG1-XG1-051.
- 19 -Figure 14 provides the plasmid map of a nucleic acid molecule encoding the heavy chain of antibody AB-PG1-XG1-069.
Figure 15 provides the plasmid map of a nucleic acid molecule encoding the heavy chain of antibody AB-PG1-XG1-077.
Figure 16 provides the plasmid map of a nucleic acid molecule encoding the heavy chain of antibody PSMA 10.3.
Figure 17 provides the plasmid map of a nucleic acid molecule encoding the light chain of antibody AB-PG1-XG1-006.
Figure 18 provides the plasmid map of a nucleic acid molecule encoding the light chain of antibody AB-PG1-XG1-026.
Figure 19 provides the plasmid map of a nucleic acid molecule encoding the light chain of antibody AB-PG1-XG1-051.
Figure 20 provides the plasmid map of a nucleic acid molecule encoding the light chain of antibody AB-PG1-XG1-069.
Figure 21 provides the plasmid map of a nucleic acid molecule encoding the light chain of antibody AB-PG1-XG1-077.
Figure 22 provides the plasmid map of a nucleic acid molecule encoding the light chain of antibody PSMA 10.3.
Figure 23 depicts the cytotoxicity of225Ac-3.9 on LNCaP target cells.
Figure 24 illustrates the reactivity of anti-PSMA monoclonal antibodies XG-006, XG-051, 4.40.1, 4.49.1,4.292.1 and 4.304.1 incubated with either parent 3T3 cells (black histogram) or 3T3 cells enigineered to express cell-surface human PSMA (grey histogram) and analyzed by flow cytometry.
Figure 25 illustrates the binding of the anti-PSMA Abs. Figure 25A shows that anti-PSMA mAbs bind to 3T3-PSMA cells and not 3T3 cells. One representative experiment from at least ten determinations is shown. Figure 25B illustrates that binding to cell-surface PSMA using serial dilutions of anti-PSMA mAb-containing culture supernatants occurred.
One representative experiment from five is shown. Figure 25C shows binding to cell-surface PSMA using serial dilutions of purified anti-PSMA mAbs, XG-006 and 10.3 One representative experiment is shown.
Figure 26 illustrates the immunotoxin cytotoxicity of murine anti-PSMA
antibodies on C4-2 prostate cancer cells. SJ25C-1 as a control antibody is a murine anti-CD19 IgG.
- 20 -The LD 50s (M) for 5.4, 3.9, and mJ591 antibodies were 2.27 x 10-11, 2.29 x 10-11 and 8.82 x --11, respectively.
Figure 27 illustrates the immunotoxin cytotoxicity of murine anti-PSMA
antibodies on PSMA-3T3 cells. SJ25C-1 as a control antibody is a murine anti-CD19 IgG.
The LD 50s (M) for 5.4, 3.9, and mJ591 antibodies were 1.64 x 10-11, 1.96 x 10-11 and 8.90 x 10-11, respectively.
Figure 28 provides the cytotoxicity of direct conjugated human 4.304 anti-PSMA

antibodies with saporin on PSMA-3T3. The LD50 was 1.48 x 10-11M for direct conjugated 4.304 anti-PSMA antibodies with saporin.
Figure 29 illustrates the results of the competition assay of unmodified 4.304, 4.40, m1591 anti-PSMA antibodies used to compete with In-111 radiolabeled 4.40 and 4.304 anti-PSMA antibodies.
Figure 30 illustrates the results of the competition assay of unmodified 4.304, mJ591 anti-PSMA antibodies used to compete with In-111 radiolabeled mJ591 anti-PSMA
antibodies.
Figure 31 shows an analysis of antibody PRGX1-XG-006 in association phase and dissociation phase at different concentrations of rsPSMA from 100 n1V1 to 6.25 nM.
Figure 32 shows the results of the comparison of the fully human anti-PSMA
antibodies 4.40.1,4.49.1, 051 and 006 and the murine anti-PSMA antibody 3.9 performed TM
using Biacore analysis.
Figure 33 provides results from the Scatchard analysis using In-111 labeled anti-PSMA antibody 3.9 of the PSMA-3T3, LNCaP and C4-2 cell lines.
Figure 34 shows in vitro cytotoxicity of Ac-225 labeled human anti-PSMA
antibody 4.40 on prostate cancer cells.
Figure 35 shows the results of in vivo radioimmunotherapy with Lu-177 labeled human anti-PSMA antibodies.
Figure 36 is a series of graphs that show flow cytometry data for the binding of anti-PSMA antisera to PSMA-3T3 cells. Antisera from mice immunized with a rsPSMA
dimer preparation (ABIM151, ABIM152, ABM/1153, ABIM154 and ABB/1155) exhibited strong binding to PSMA-expressing cells. Antisera from mice immunized with a rsPSMA
monomer preparation (ABM/1156, ABIM157, AMM158, ABM4159 and ABIM160) exhibited little or no binding to PSMA-expressing cells.
- 21 -Detailed Description of the Invention The present invention provides antibodies or antigen-binding fragments thereof which bind specifically to conformational epitopes on the extracellular domain of PSMA, compositions containing one or a combination of such antibodies or antigen-binding fragments thereof, hybridoma cell lines that produce the antibodies, and methods of using the antibodies or antigen-binding fragments thereof for cancer diagnosis and treatment.
Prostate specific membrane antigen (PSMA) is a 100 kD Type II membrane glycoprotein expressed in prostate tissues and was originally identified by reactivity with a monoclonal antibody designated 7E11-05 (Horoszewicz et al., 1987, Anticancer Res. 7:927-935; U.S. Pat. No. 5,162,504). PSMA was obtained in purified form (Wright et al., 1990, Antibody Immunoconjugates and Radio Pharmaceuticals 3:Abstract 193) and characterized as a type II transmembrane protein having sequence identity with the transferrin receptor (Israeli et al., 1994, Cancer Res. 54:1807-1811) and with NAALADase activity (Carter et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:749-753). More importantly, PSMA is expressed in increased amounts in prostate cancer, and elevated levels of PSMA are also detectable in the sera of these patients (Horoszewicz et al., 1987; Rochon et al., 1994, Prostate 25:219-223;
Murphy et al., 1995, Prostate 26:164-168; and Murphy et al., 1995, Anticancer Res. 15:1473-1479). PSMA expression increases with disease progression, becoming highest in metastatic, hormone-refractory disease for which there is no present therapy. Provocative recent data indicates that PSMA is also abundantly expressed on the neovasculature of a variety of other important tumors, including bladder, pancreas, sarcoma, melanoma, lung, and kidney tumor cells, but not on normal vasculature.
One aspect of the invention provides an isolated antibody or an antigen-binding fragment thereof which specifically binds to an extracellular domain of PSMA
wherein the antibody or the antigen-binding fragment thereof competitively inhibits the specific binding of a second antibody to its target epitope on PSMA, and wherein the second antibody is selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA
5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, 4.248.2, . 4.360.3, 4.7.1, 4.4.1, 4.177.3, 4.16.1, 4.22.3, 4.28.3, 4.40.2, 4.48.3, 4.49.1, 4.209.3, 4.219.3, 4.288.1, 4.333.1, 4.54.1, 4.153.1, 4.232.3, 4.292.3, 4.304.1, 4.78.1, and 4.152.1.
-22 -Another aspect of the invention provides an isolated antibody or an antigen-binding fragment thereof that specifically binds to an epitope on PSMA defined by an antibody selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA
5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, 4.248.2, 4.360.3, 4.7.1, 4.4.1, 4.177.3, 4.16.1, 4.22.3, 4.28.3, 4.40.2, 4.48.3, 4.49.1, 4.209.3, 4.219.3, 4.288.1, 4.333.1, 4.54.1, 4.153.1, 4.232.3, 4.292.3, 4.304.1, 4.78.1, and 4.152.1.
In particular embodiments, these antibodies are produced by hybridomas referred to herein as PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA
7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, and Abgenix 4.152.1, respectively.
These hybridomas were deposited with ATCC as an International Depository Authority and given the following Patent Deposit Designations (Table 1):
Table 1.
Antibody Hybridoma/Plasmid Patent Deposit Date of Deposit Designation PSMA 3.7 PSMA 3.7 PTA-3257 April 5, PSMA 3.9 PSMA 3.9 PTA-3258 April 5, PSMA 3.11 PSMA 3.11 PTA-3269 April 10, PSMA 5.4 PSMA 5.4 PTA-3268 April 10, PSMA 7.1 PSMA 7.1 PTA-3292 April 18, PSMA 7.3 PSMA 7.3 PTA-3293 April 18, PSMA 10.3 PSMA 10.3 PTA-3347 May 1, 2001 PSMA 10.3 HC in PTA-4413 May 29, 2002 pcDNA (SEQ ID NO: 7) PSMA 10.3 Kappa in PTA-4414 May 29, 2002 pcDNA (SEQ ID NO: 13) PSMA 1.8.3 PSMA 1.8.3 PTA-3906 Dec. 5, 2001 PSMA A3.1.3 PSMA A3.1.3 PTA-3904 Dec. 5, 2001
-23 -PSMA A3.3.1 PSMA A3.3.1 PTA-3905 Dec. 5, 2001 Abgenix 4.248.2 Abgenix 4.248.2 PTA-4427 June 4, 2002 Abgenix 4.360.3 Abgenix 4.360.3 PTA-4428 June 4, 2002 Abgenix 4.7.1 Abgenix 4.7.1 PTA-4429 June 4, 2002 Abgenix 4.4.1 Abgenix 4.4.1 PTA-4556 July 18, 2002 Abgenix 4.177.3 Abgenix 4.177.3 PTA-4557 July 18, 2002 Abgenix 4.16.1 Abgenix 4.16.1 PTA-4357 May 16, 2002 _ Abgenix 4.22.3 Abgenix 4.22.3 PTA-4358 May 16, 2002 Abgenix 4.28.3 Abgenix 4.28.3 PTA-4359 May 16, 2002 Abgenix 4.40.2 Abgenix 4.40.2 PTA-4360 May 16, 2002 Abgenix 4.48.3 Abgenix 4.48.3 PTA-4361 May 16, 2002 Abgenix 4.49.1 Abgenix 4.49.1 PTA-4362 May 16, 2002 Abgenix 4.209.3 Abgenix 4.209.3 PTA-4365 May 16, 2002 Abgenix 4.219.3 Abgenix 4.219.3 PTA-4366 May 16, 2002 ' Abgenix 4.288.1 Abgenix 4.288.1 PTA-4367 May 16, 2002 Abgenix 4.333.1 Abgenix 4.333.1 PTA-4368 May 16, 2002 Abgenix 4.54.1 Abgenix 4.54.1 PTA-4363 May 16, 2002 Abgenix 4.153.1 Abgenix 4.153.1 PTA-4388 May 23, 2002 Abgenix 4.232.3 Abgenix 4.232.3 PTA-4389 May 23, 2002 Abgenix 4.292.3 Abgenix 4.292.3 PTA-4390 May 23, 2002 Abgenix 4.304.1 Abgenix 4.304.1 PTA-4391 May 23, 2002 AB-PGI -XGI -006 AB-PG1-XG1-006 Heavy PTA-4403 May 29, 2002 Chain (SEQ ID NO: 2) AB-PG1-XG1-006 Light PTA-4404 Chain (SEQ ID NO: 8) AB-PG1-XG1-026 AB-PG1-XG1-026 Heavy PTA-4405 May 29, 2002 Chain (SEQ ID NO: 3) AB-PG1-XG1-026 Light PTA-4406 Chain (SEQ ID NO: 9) _ AB-PG1-XG1-051 AB-PG1-XG1-051 Heavy PTA-4407 May 29, 2002 Chain (SEQ ID NO: 4) AB-PGI-XGI-051 Light PTA-4408 _ ,
- 24 -Chain (SEQ ID NO: 10) AB-PG1-XG1-069 AB-PG1-XG1-069 Heavy PTA-4409 May 29, 2002 Chain (SEQ ID NO: 5) AB-PG1-XG1-069 Light PTA-4410 Chain (SEQ ID NO: 11) AB-PG1-XG1-077 AB-PG1-XG1-077 Heavy PTA-4411 -May 29, 2002 Chain (SEQ ID NO: 6) AB-PG1-XG1-077 Light PTA-4412 Chain (SEQ ID NO: 12) In another aspect of the invention, antibodies having particular sequences are provided. Specifically, the antibodies are selected from the group consisting of antibodies comprising: a heavy chain encoded by a nucleic acid molecule comprising the heavy chain .
coding region or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as SEQ ID NOs: 2-7, and a light chain encoded by a nucleic acid molecule comprising the light chain coding region or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as SEQ ID
NOs: 8-13.
Also provided are antigen-binding fragments of the foregoing antibodies.
The plasmids encoding the heavy and light chains of antibodies PSMA 10.3, A1B-PG1-XG1-006, AB-PG1-XG1-026, AB-PG1-XG1-051, AB-PG1-XG1-069, AB-PG1-XG1-077 were also deposited with ATCC and are shown in Table 1 above. As used herein, the names of the deposited hybridomas or plasmids may be used interchangeably with the names of the antibodies. It would be clear to one of skill in the art when the name is intended to refer to the antibody or when it refers to the plasmids or hybridomas that encode or produce the antibodies, respectively. Additionally, the antibody names may be an abbreviated form of the name shown in Table 1. For instance antibody AB-PG1-XG1-006 may be referred to as AB-PG1-XG1-006, PG1-XG1-006, XG1-006, 006, etc. In another example, the antibody name PSMA 4.232.3 may be referred to as PSMA 4.232.1, 4.232.3, 4.232.1, 4.232, etc. It is intended that all of the variations in the name of the antibody refer to the same antibody and not a different one.
Antibodies are also provided that are encoded by particular sets of heavy and light chain sequences. In one embodiment an antibody (AB-PG1-X01-006) encoded by a nucleic
- 25 -acid molecule which comprises the coding region or regions of the nucleic acid sequences set forth as :SEQ ID NOs: 2 and 8 is provided. In another embodiment the antibody (AB-PG1-XG1-026) is encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ ID NOs: 3 and 9. In still another embodiment the antibody (AB-PG1-XG1-051) is encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ ID NOs: 4 and 10. In yet another embodiment the antibody (AB-PG1-XG1-069) is encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ
ID NOs: 5 and 11. In another embodiment the antibody (AB-PG1-XG1-077) is encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ ID NOs: 6 and 12. In yet another embodiment the antibody (PSMA 10.3) is encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ ID NOs: 7 and 13.
In particularly preferred embodiments, the antibodies include a heavy chain variable region encoded by a nucleic acid molecule comprising the coding regions or regions of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as:
SEQ ID NOs: 14, 18, 22,26 and 30, and a light chain variable region encoded by a nucleic acid molecule comprising the coding region or region of a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as: SEQ ID NOs: 16, 20, 24, 28 and 32.
As used herein, a "coding region" refers to a region of a nucleotide sequence that encodes a polypeptide sequence; the coding region can include a region coding for a portion of a protein that is later cleaved off, such as a signal peptide.
Those of skill in the art will appreciate that the invention includes nucleic acids and polypeptides that include nucleotide and amino acid sequences presented herein. In some instances, the nucleotide and amino acid sequences may include sequences that encode or that are signal peptides. The invention embraces each of these sequences with, or without, the portion of the sequence that encodes or is a signal peptide.
Antibodies also are provided that include particular sets of heavy and light chain variable sequences. In one embodiment an antibody (AB-PG1-XG1-006) includes an inununoglobulin variable sequence encoded by nucleic acid molecules which included the coding region or regions of the nucleic acid sequences set forth as :SEQ ID
NOs: 14 and 16 is provided Likewise the antibody may include an immunoglobulin variable sequence which
- 26 -comprises the amino acid sequences set forth as SEQ ID NOs: 15 and 17. In another embodiment the antibody (AB-PG1-XG1-026) includes an immunoglobulin variable sequence encoded by nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ ID NOs: 18 and 20 or includes an imrnunoglobulin variable sequence which comprises the amino acid sequences set forth as SEQ ID
NOs: 19 and 21. In still another embodiment the antibody (AB-PG1-XG1-051) includes an immunoglobulin variable sequence encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ ID NOs: 22 and 24 or includes an immunoglobulin variable sequence which comprises the amino acid sequences set forth as SEQ ID NOs: 23 and 25. In yet another embodiment the antibody (AB-XG1-069) includes an immunoglobulin variable sequence encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ
ID NOs: 26 and 28 or includes an immunoglobulin variable sequence which comprises the amino acid sequences set forth as SEQ ID NOs: 27 and 29. In another embodiment the antibody (AB-PG1-XG1-077) includes an immunoglobulin variable sequence encoded by the nucleic acid molecules comprising the coding region or regions of nucleotide sequences set forth as: SEQ ID NOs: 30 and 32 or includes an immunoglobulin variable sequence which comprises the amino acid sequences set forth as SEQ JD NOs: 31 and 33.
In certain embodiments, the antibody is encoded by a nucleic acid molecule that is highly homologous to the foregoing nucleic acid molecules. Preferably the homologous nucleic acid molecule comprises a nucleotide sequence that is at least about 90% identical to the nucleotide sequence provided herein. More preferably, the nucleotide sequence is at least about 95% identical, at least about 97% identical, at least about 98%
identical, or at least about 99% identical to the nucleotide sequence provided herein. The homology can be calculated using various, publicly available software tools well known to one of ordinary skill in the art. Exemplary tools include the BLAST system available from the website of the National Center for Biotechnology Information (NCBI) at the National Institutes of Health.
One method of identifying highly homologous nucleotide sequences is via nucleic acid hybridization. Thus the invention also includes antibodies having the PSMA-binding properties and other functional properties described herein, which are encoded by nucleic acid molecules that hybridize under high stringency conditions to the foregoing nucleic acid molecules. Identification of related sequences can also be achieved using polymerase chain
- 27 -reaction (PCR) and other amplification techniques suitable for cloning related nucleic acid sequences. Preferably, PCR primers are selected to amplify portions of a nucleic acid sequence of interest, such as a CDR.
The term "high stringency conditions" as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley &
Sons, Inc., New York. One example of high-stringency conditions is hybridization at 65 C in TM
hybridization buffer (3.5X SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH2PO4(pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, p117; SDS is sodium dodecyl sulphate; and EDTA
is ethylenediaminetetracetic acid. After hybridi7ation, a membrane upon which the nucleic acid is transferred is washed, for example, in 2X SSC at room temperature and then at 0.1 - 0.5X
SSC/0.1X SDS at temperatures up to 68 C.
In other preferred embodiments, the antibodies include a heavy chain variable region comprising an amino acid sequence selected from the group consisting of amino acid sequences set forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and a light chain variable region comprising an amino acid sequence selected from the group consisting of nucleotide sequences set forth as: SEQ ID NOs: 17,21, 25, 29 and 33. Antigen-binding fragments of the foregoing also are provided, as described elsewhere herein.
As used herein, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The
- 28 -variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complement system.
The term "antigen-binding fragment" of an antibody as used herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., PSMA). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL
and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V
and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv);
see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA
85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional procedures, such as proteolytic fragmentation procedures, as described in J.
Goding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (N.Y.
Academic Press 1983). The fragments are screened for utility in the same manner as are intact antibodies.
An "isolated antibody", as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to PSMA is substantially free of antibodies that specifically bind antigens other than PSMA). An isolated antibody that specifically binds to an epitope, isoform or variant of PSMA may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., PSMA species homologs). Moreover, an isolated antibody may
- 29 -be substantially free of other cellular material and/or chemicals. As used herein, "specific binding" refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
The isolated antibodies of the invention encompass various antibody isotypes, such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, IgE. As used herein, "isotype"
refers to the antibody class (e.g. IgM or IgG1) that is encoded by heavy chain constant region genes. The antibodies can be full length or can include only an antigen-binding fragment such as the antibody constant and/or variable domain of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD or IgE or could consist of a Fab fragment, a F(ab1)2 fragment, and a Fv fragment.
The antibodies of the present invention can be polyclonal, monoclonal, or a mixture of polyclonal and monoclonal antibodies. The antibodies can be produced by a variety of techniques well known in the art. Procedures for raising polyclonal antibodies are well known. For example anti-PSMA polyclonal antibodies are raised by administering PSMA.
protein subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The PSMA can be injected at a total volume of 100 tl per site at six different sites, typically with one or more adjustments. The rabbits are thembled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is collected 10 days after each boost. Polyclonal antibodies are recovered from the serum, preferably by affinity chromatography using PSMA to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in E.
Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988).
Monoclonal antibody production may be effected by techniques which are also well known in the art. The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. The process of monoclonal antibody production involves obtaining immune somatic cells with the potential for producing antibody, in particular B lymphocytes, which have been previously immunized with the antigen of interest either in vivo or in vitro and that are suitable for fusion with a B-cell myeloma line.
- 30 -Mammalian lymphocytes typically are immunized by in vivo immunization of the animal (e.g., a mouse) with the desired protein or polypeptide, e.g., with PSMA in the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Once immunized, animals can be used as a source of antibody-producing lymphocytes. Following the last antigen boost, the animals are sacrificed and spleen cells removed. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myeloma lines described herein. Of these, the BALB/c mouse is preferred.
However, other mouse strains, rabbit, hamster, sheep and frog may also be used as hosts for preparing antibody-producing cells. See; Goding (in Monoclonal Antibodies:
Principles and Practice, 2d ed., pp. 60-61, Orlando, Fla., Academic Press, 1986). In particular, mouse strains that have human imm-unoglobulin genes inserted in the genome (and which cannot produce mouse immunoglobulins) are preferred. Examples include the HuMAb mouse strains produced by Medarex/GenPharm International, and the XenoMouse strains produced by Abgenix. Such mice produce fully human iinmunoglobulin molecules in response to immunization.
Those antibody-producing cells that are in the dividing plasmablast stage fuse preferentially. Somatic cells may be obtained from the lymph nodes, spleens and peripheral blood of antigen-primed animals, and the lymphatic cells of choice depend to a large extent on their empirical usefulness in the particular fusion system. The antibody-secreting lymphocytes are then fused with (mouse) B cell myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies.
Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature 256:495 (1975).
Alternatively, human somatic cells capable of producing antibody, specifically B
lymphocytes, are suitable for fusion with myeloma cell lines. While B
lymphocytes from biopsied spleens, tonsils or lymph nodes of an individual may be used, the more easily accessible peripheral blood B lymphocytes are preferred. The lymphocytes may be derived from patients with diagnosed prostate carcinomas or another PSMA-expressing cancer. In
-31 -addition, human B cells may be directly immortalized by the Epstein-Barr virus (Cole et al., 1995, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed such as viral or oncogenic transformation of B lymphocytes.
Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of the desired hybridomas. Examples of such myeloma cell lines that may be used for the production of fused cell lines include P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4.1, Sp2/0-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7, S194/5XXO Bul, all derived from mice;
R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 derived from rats and U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6, all derived from humans (Goding, in Monoclonal Antibodies:
Principles and Practice, 2d ed., pp. 65-66, Orlando, Fla., Academic Press, 1986; Campbell, in Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology Vol. 13, Burden and Von Knippenberg, eds. pp. 75-83, Amsterdam, Elseview, 1984).
Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol ("PEG") or other fusing agents (See Milstein and Kohler, Eur.
Immunol. 6:511 (1976).
In other embodiments, the antibodies can be recombinant antibodies. The term "recombinant antibody", as used herein, is intended to include antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for another species' immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.
In yet other embodiments, the antibodies can be chimeric or humanized antibodies.
As used herein, the term "chimeric antibody" refers to an antibody, that combines the murine variable or hypervariable regions with the human constant region or constant and variable
- 32 -framework regions. As used herein, the term "humanized antibody" refers to an antibody that retains only the antigen-binding CDRs from the parent antibody in association with human framework regions (see, Waldmann, 1991, Science 252:1657). Such chimeric or humanized antibodies retaining binding specificity of the murine antibody are expected to have reduced immunogenicity when administered in vivo for diagnostic, prophylactic or therapeutic applications according to the invention.
According to an alternative embodiment, the monoclonal antibodies of the present invention can be modified to be in the form of a bispecific antibody, or a multispecific antibody. The term "bispecific antibody" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities which bind to, or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific antibody" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities which bind to, or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component.
Accordingly, the invention includes, but is not limited to, bispecific, trispecifie, tetraspecific, and other multispecific antibodies which are directed to cell surface antigens, such as PSMA, and to Fc receptors on effector cells. The term "bispecific antibodies" further includes diabodies.
Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polyp eptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poijak, R.J., et al. (1994) Structure 2:1121-1123).
A bispecific antibody can be formed of an antigen-binding region specific for the extracellular domain of PSMA and an antigen-binding region specific for an effector cell which has tumoricidal or tumor inhibitory activity. The two antigen-binding regions of the bispecific antibody are either chemically linked or can be expressed by a cell genetically engineered to produce the bispecific antibody. (See generally, Fanger et al., 1995 Drug News & Perspec. 8(3):133-137). Suitable effector cells having tumoricidal activity include but are not limited to cytotoxic T-cells (primarily CD8+ cells), natural killer cells, etc. An effective amount of a bispecific antibody according to the invention is administered to a prostrate
- 33 -cancer patient and the bispecific antibody kills and/or inhibits proliferation of the malignant cells after localization at sites of primary or metastatic tumors bearing PSMA.
In certain embodiments, the antibodies are human antibodies. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse have been grafted onto human framework sequences (referred to herein as "humanized antibodies"). Human antibodies directed against PSMA are generated using transgenic mice carrying parts of the human immune system rather than the mouse system.
Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. patents 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals results in the production of fully human antibodies to the antigen of interest.
Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies are prepared according to standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.
Preferably, the mice are 6-16 weeks of age upon the first immunization. For example, a purified or enriched preparation of PSMA antigen (e.g., recombinant PSMA or PSMA-expressing cells) is used to immunize the mice intraperitoneally (IP), although other routes of immunization known to one of ordinary skill in the art are also possible. PSMA
antigen is injected in combination with an adjuvant, such as complete Freund's adjuvant, and preferably the initial injection is followed by booster immunizations with antigen in an adjuvant, such as
- 34 -incomplete Freund's adjuvant. The immune response is monitored over the course of the immunization protocol with plasma samples obtained by, for example, retroorbital bleeds.
The plasma is screened by ELISA (as described below), and mice with sufficient titers of anti-PSMA human immunoglobulin are used for fusions. Mice are boosted intravenously with antigen 3 days before sacrifice and removal of the spleen.
In particular embodiments, the antibodies are produced by hybridomas referred to herein as PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA
10.3 (PTA-3247) , PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix 4288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix 4.152.1 (PTA-4653). These hybridomas were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection ("ATCC") as an International Depository Authority and given the Patent Deposit Designations shown above and in Table 1.
The present invention further provides nucleic acid molecules encoding anti-PSMA
antibodies and vectors comprising the nucleic acid molecules as described herein. The vectors provided can be used to transform or transfect host cells for producing anti-PSMA
antibodies with the specificity of antibodies described herein. In a preferred embodiment the antibodies produced will have the specificity of the antibodies AB-PG1-XG1-006, AB-PG1-XG1-026, AB-PG1-XG1-051, AB-PG1, XG1-069, AB-PG1-XG1-077 and PSMA 10.3. In one embodiment the vectors can comprise an isolated nucleic acid molecule encoding the heavy chain of the antibodies listed above encoded by a nucleic acid molecules comprising the coding region or regions of the nucleic acid sequences set forth as SEQ ID
NO: 2-7. In another embodiment, the vectors can comprise the nucleic acid sequences encoding the light chain of the antibodies set forth as SEQ ID NOs: 8-13. In a further embodiment the vectors
- 35 -of the invention may comprise a heavy chain and a light chain sequence. In a further embodiment, plasmids are given which produce the antibodies or antigen binding fragments described herein. Plasmids of the invention include plasmids selected from the group consisting of: AB-PG1-XG1-006 Heavy Chain (SEQ ID NO: 2), AB-PG1-XG1-006 Light Chain (SEQ ID NO: 8), AB-PG1-XG1-026 Heavy Chain (SEQ ID NO: 3), AB-PG1-XG1-026 Light Chain (SEQ ID NO: 9), AB-PG1-XG1-051 Heavy Chain (SEQ JD NO: 4), AB-PG1-XG1-051 Light Chain (SEQ ID NO: 10), AB-PG1-XG1-069 Heavy Chain (SEQ ID
NO: 5), AB-PG1-XG1-069 Light Chain (SEQ ID NO: 11), AB-PG1-XG1-077 Heavy Chain (SEQ JD NO: 6), AB-PG1-XG1-077 Light Chain (SEQ ID NO: 12), PSMA 10.3 Heavy Chain (SEQ ID NO: 7), and PSMA 10.3 Kappa (SEQ ID NO: 13).
The isolated antibody or antigen-binding fragment thereof preferably is selected for its ability to bind live cells expressing PSMA. In order to *demonstrate binding of monoclonal antibodies to live cells expressing the PSMA, flow cytometry can be used. For example, cell lines expressing PSMA (grown under standard growth conditions) or prostate cancer cells that express PSMA are mixed with various concentrations of monoclonal antibodies in PBS
TM
containing 0.1% Tween 80 and 20% mouse serum, and incubated at 37 C for 1 hour. After washing, the cells are reacted with fluorescein-labeled anti-human IgG
secondary antibody (if human anti-PSMA antibodies were used) under the same conditions as the primary antibody staining._ The samples can be analyzed by a fluorescence activated cell sorter (FACS) instrument using light and side scatter properties to gate on single cells. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished sensitivity depending on the density of the antigen.
Binding of the antibody or antigen-binding fragment thereof to live cells expressing PSMA can inhibit the growth of the cells or mediate cytolysis of the cells.
Cytolysis can be complement mediated or can be mediated by effector cells. In a preferred embodiment, the cytolysis is carried out in a living organism, preferably a mammal, and the live cell is a tumor cell. In a preferred embodiment the tumor cell is a prostate cancer cell.
- 36 -The testing of antibody cytolytic activity in vitro by chromium release assay can provide an initial screening prior to testing in vivo models. This testing can be carried out using standard chromium release assays. Briefly, polymorphonuclear cells (PMN), or other effector cells, from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed PMNs can be suspended in RPMI
supplemented with 10% heat-inactivated fetal calf serum and mixed with 51Cr labeled cells expressing PSMA, at various ratios of effector cells to tumor cells (effector cells:tumor cells).
Purified anti-PSMA IgGs can then be added at various concentrations.
Irrelevant IgG can be used as negative control. Assays can be carried out for 0-120 minutes at 37 C.
Samples can be assayed for cytolysis by measuring 51Cr release into the culture supernatant. Anti-PSMA
monoclonal antibodies can also be tested in combinations with each other to determine whether cytolysis is enhanced with multiple monoclonal antibodies.
Antibodies which bind to PSMA also can be tested in an in vivo model (e.g., in mice) to determine their efficacy in mediating cytolysis and killing of cells expressing PSMA, e.g., tumor cells. These antibodies can be selected, for example, based on the following criteria, which are not intended to be exclusive:
1) binding to live cells expressing PSMA;
2) high affinity of binding to PSMA;
3) binding to a unique epitope on PSMA (to eliminate the possibility that antibodies with complimentary activities when used in combination would compete for binding to the same epitope);
4) opsonization of cells expressing PSMA;
5) mediation of growth inhibition, phagocytosis and/or killing of cells expressing PSMA in the presence of effector cells;
6) modulation (inhibition or enhancement) of NAALADase, folate hydrolase, dipeptidyl peptidase IV and/or 7-glutamyl hydrolase activities;
7) growth inhibition, cell cycle arrest and/or cytotoxicity in the absence of effector cells;
8) internalization of PSMA;
9) binding to a conformational epitope on PSMA;
- 37 -10) minimal cross-reactivity with cells or tissues that do not express PSMA; and 11) preferential binding to dimeric forms of PSMA rather than monomeric forms of PSMA.
Preferred antibodies of the invention meet one or more, and preferably all, of these criteria. In a particular embodiment, the antibodies are used in combination, e.g., as a pharmaceutical composition comprising two or more different anti-PSMA
antibodies or binding fragments thereof. For example, anti-PSMA antibodies having different, but complementary activities can be combined in a single therapy to achieve a desired therapeutic or diagnostic effect. An illustration of this would be a composition containing an anti-PSMA
antibody that mediates highly effective killing of target cells in the presence of effector cells, combined with another anti-PSMA antibody that inhibits the growth of cells expressing PSMA.
In a preferred aspect of the invention, the antibody or antigen-binding fragment thereof binds to a conformational epitope within the extracellular domain of the PSMA
molecule. To determine if the selected human anti-PSMA antibodies bind to conformational epitopes, each antibody can be tested in assays using native protein (e.g., non-denaturing immunoprecipitation, flow cytometric analysis of cell surface binding) and denatured protein (e.g., Western blot, immunoprecipitation of denatured proteins). A comparison of the results will indicate whether the antibodies bind conformational epitopes. Antibodies that bind to native protein but not denatured protein are those antibodies that bind conformational epitopes, and are preferred antibodies.
To determine if the selected human anti-PSMA antibodies bind preferentially (i.e., selectively and/or specifically) to a PSMA dimer, each antibody can be tested in assays (e.g., immunoprecipitation followed by Western blotting) using native dimeric PSMA
protein and dissociated monomeric PSMA protein. A comparison of the results will indicate whether the antibodies bind preferentially to the dimer or to the monomer. Antibodies that bind to the PSMA dimer but not to the monomeric PSMA protein are preferred antibodies.
Preferred antibodies include antibodies that competitively inhibit the specific binding of a second antibody to its target epitope on PSMA. To determine competitive inhibition, a variety of assays known to one of ordinary skill in the art can be employed.
For example, the cross-competition assays set forth in Examples 4 and 21 can be used to determine if an
- 38 -antibody competitively inhibits binding to PSMA by another antibody. These examples provide cell-based methods employing flow cytometry or solid phase binding analysis. Other assays that evaluate the ability of antibodies to cross-compete for PSMA
molecules that are not expressed on the surface of cells, in solid phase or in solution phase, also can be used.
These assays preferably use the PSMA multimers described herein.
Certain preferred antibodies competitively inhibit the specific binding of a second antibody to its target epitope on PSMA by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Inhibition can be assessed at various molar ratios or mass ratios; for example competitive binding experiments can be conducted with a 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold or more molar excess of the first antibody over the second antibody.
Other preferred antibodies include antibodies that specifically (i.e., selectively) bind to an epitope on PSMA defined by a second antibody. To determine the epitope, one can use standard epitope mapping methods known in the art. For example, fragments (peptides) of PSMA antigen (preferably synthetic peptides) that bind the second antibody can be used to determine whether a candidate antibody binds the same epitope. For linear epitopes, overlapping peptides of a defined length (e.g., 8 or more amino acids) are synthesized. The peptides preferably are offset by 1 amino acid, such that a series of peptides covering every 8 amino acid fragment of the PSMA protein sequence are prepared. Fewer peptides can be prepared by using larger offsets, e.g., 2 or 3 amino acids. In addition, longer peptides (e.g., 9-, 10- or 11-mers) can be synthesized. Binding of peptides to antibodies can be determined using standard methodologies including surface plasmon resonance (BIACORE; see Example 22) and ELISA assays. For examination of conformational epitopes, larger PSMA
fragments can be used. Other methods that use mass spectrometry to define conformational epitopes have been described and can be used (see, e.g., Baerga-Ortiz et al., Protein Science 11:1300-1308, 2002 and references cited therein). Still other methods for epitope determination are provided in standard laboratory reference works, such as Unit 6.8 ("Phage Display Selection and Analysis of B-cell Epitopes") and Unit 9.8 ("Identification of Antigenic Determinants Using Synthetic Peptide Combinatorial Libraries") of Current Protocols in Immunology, Coligan et al., eds., John Wiley & Sons. Epitopes can be confirmed by introducing point mutations or deletions into a known epitope, and then testing binding with one or more antibodies to determine which mutations reduce binding of the antibodies.
- 39 -In one embodiment of the invention the antibody or antigen-binding fragment thereof binds to and is internalized with PSMA expressed on cells. The mechanism by which the antibody or antigen-binding fragment thereof is internalized with the prostate specific membrane antigen is not critical to the practice of the present invention. For example, the antibody or antigen-binding fragment thereof can induce internalization of PSMA.
Alternatively, internalization of the antibody or antigen-binding fragment thereof can be the result of routine internalization of PSMA. The antibody or antigen-binding fragment thereof can be used in an unmodified form, alone or in combination with other compositions.
Alternatively, the antibody or antigen-binding fragment thereof can be bound to a substance effective to kill the cells upon binding of the antibody or antigen-binding fragment thereof to prostate specific membrane antigen and upon internalization of the biological agent with the prostate specific membrane antigen.
The human PSMA antibodies of the present invention specifically bind cell-surface PSMA and/or rsPSMA with sub-nanomolar affinity. The human PSMA antibodies of the present invention have binding affinities of about 1 X 10-9M or less, preferably about 1 X
10-19M or less, more preferably 1 X 10-11M or less. In a particular embodiment the binding affinity is less than about 5 X 10-10M.
An antibody can be linked to a detectable marker, an antitumor agent or an immunomodulator. Antitumor agents can include cytotoxic agents and agents that act on tumor neovasculature. Detectable markers include, for example, radioactive or fluorescent markers. Cytotoxic agents include cytotoxic radionuclides, chemical toxins and protein toxins.
The cytotoxic radionuclide or radiotherapeutic isotope preferably is an alpha-emitting isotope such as 225Ac, 211At 212Bi, 213Bi, 212pb, 224Ra or 223Ra.
Alternatively, the cytotoxic radionuclide may a beta-emitting isotope such as 186Rh, issRh, 177La, 90y, 1311, 67cu., 64cu, 153Sm or 166Ho. Further, the cytotoxic radionuclide may emit Auger and low energy electrons and include the isotopes 1251, 123-1 or 77Br.
Suitable chemical toxins or chemotherapeutic agents include members of the enediyne family of molecules, such as calicheamicin and esperamicin. Chemical toxins can also be taken from the group consisting of methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.
Other antineoplastic agents that may be conjugated to the anti-PSMA antibodies of the
-40 -present invention include dolastatins (U.S. Patent Nos. 6,034,065 and 6,239,104) and derivatives thereof. Of particular interest is dolastatin 10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine) and the derivatives auristatin PHE (dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester) (Pettit, G.R. et al., Anticancer Drug Des. 13(4):243-277, 1998; Woyke, T. et al., Antimicrob. Agents Chemother. 45(12):3580-3584, 2001), and aurastatin E and the like. Toxins that are less preferred in the compositions and methods of the invention include poisonous lectins, plant toxins such as ricin, abrin, modeccin, botulina and diphtheria toxins. Of course, combinations of the various toxins could also be coupled to one antibody molecule thereby accommodating variable cytotoxicity. Other chemotherapeutic agents are known to those skilled in the art.
Toxin-conjugated forms of the PSMA antibodies of the present invention mediate specific cell killing of PSMA-expressing cells at picomolar concentrations.
The toxin-conjugated PSMA antibodies of the present invention exhibit IC50s at concentrations of less than about 1 X 10-1 M, preferably less than about I X 10-11M, more preferably less than about 1 X 10-12M. In a particular embodiment an 1050 is achieved at a concetration of less than about 1.5 X 10-11M.
Agents that act on the tumor vasculature can include tubulin-binding agents such as combrestatin A4 (Griggs et al., Lancet OncoL 2:82, 2001), angiostatin and endostatin (reviewed in Rosen, Oncologist 5:20, 2000) and interferon inducible protein 10 (U.S. Patent No. 5,994,292). A number of antiangiogenic agents currently in clinical trials are also contemplated. Agents currently in clinical trials include:
2ME2, Angiostatin, Angiozyme, Anti-VEGF RhuMAb, Apra (CT-2584), Avicine, Benefin, BMS275291, Carboxyamidotriazole, CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC 412), CM101, Combretastatin A-4 Prodrug, EMD 121974, Endostatin, Flavopiridol, Genistein (GCP), Green Tea Extract, 1M-862, ImrnTher, Interferon alpha, Interleukin-12, Iressa (ZD1839), Marirnastat, Metastat (Col-3), Neovastat , Octreotide, Paclitaxel, Penicillamine, Photofrin, Photopoint, P1-88, Prinomastat (AG-3340), PTK787 (ZK22584), R0317453, Solimastat, Squalamine, SU 101, SU 5416, SU-6668, Suradista (FCE
26644), Suramin (Metaret), Tetrathiomolybdate, Thalidomide, TNP-470 and Vitaxin.
additional antiangiogenic agents are described by Kerbel, J. Clin. Oncol. 19(18s):45s-51s, 2001.
Immunomodulators suitable for conjugation to anti-PSMA antibodies include a-interferon, y-interferon, and tumor necrosis factor alpha (TNFa).
- 41 -The coupling of one or more toxin molecules to the anti-PSMA antibody is envisioned to include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding, and complexation. The toxic compounds used to prepare the anti-PSMA immunotoxins are attached to the antibodies or PSMA-binding fragments thereof by standard protocols known in the art.
The covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent agents are useful in coupling protein molecules to other proteins, peptides or amine functions, etc. For example, the literature is replete with coupling agents such as carbodiimides, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylene diamines. This list is not intended to be exhaustive of the various coupling agents known in the art but, rather, is exemplary of the more common coupling agents.
In preferred embodiments, it is contemplated that one may wish to first derivatize the antibody, and then attach the toxin component to the derivatized product.
Suitable cross-linking agents for use in this manner include, for example, SPDP (N-succinimidy1-3-(2-pyridyldithio)propionate), and SMPT, 4-succinimidyl-oxycarbonyl-methyl-(2-pyridyldithio)toluene.
In addition, protein toxins can be fused to the anti-PSMA antibody or PSMA
binding fragment by genetic methods to form a hybrid irnmunotoxin fusion protein. To make a fusion immunotoxin protein in accordance with the invention, a nucleic acid molecule is generated that encodes an anti-PSMA antibody, a fragment of an anti-PSMA antibody, a single chain anti-PSMA antibody, or a subunit of an anti-PSMA antibody linked to a protein toxin. Such fusion proteins contain at least a targeting agent (e.g., anti-PSMA antibody subunit) and a toxin of the invention, operatively attached. The fusion proteins may also include additional peptide sequences, such as peptide spacers which operatively attach the targeting agent and toxin compound, as long as such additional sequences do not appreciably affect the targeting or toxin activities of the fusion protein. The two proteins can be attached by a peptide linker or spacer, such as a glycine-serine spacer peptide, or a peptide hinge, as is well known in the art. Thus, for example, the C-terminus of an anti-PSMA antibody or fragment thereof can be fused to the N-terminus of the protein toxin molecule to form an imrnunotoxin that retains the binding properties of the anti-PSMA antibody. Other fusion arrangements will be known to one of ordinary skill in the art.
- 42 -To express the fusion immunotoxin, the nucleic acid encoding the fusion protein is inserted into an expression vector in accordance with standard methods, for stable expression of the fusion protein, preferably in mammalian cells, such as CHO cells. The fusion protein can be isolated and purified from the cells or culture supernatant using standard methodology, such as a PSMA affinity column.
Radionuclides typically are coupled to an antibody by chelation. For example, in the case of metallic radionuclides, a bifunctional chelator is commonly used to link the isotope to the antibody or other protein of interest. Typically, the chelator is first attached to the antibody, and the chelator-antibody conjugate is contacted with the metallic radioisotope. A
number of bifunctional chelators have been developed for this purpose, including the diethylenetriamine pentaacetic acid (DTPA) series of amino acids described in U.S. patents 5,124,471, 5,286,850 and 5,434,287. As another example, hydroxamic acid-based bifunctional chelating agents are described in U.S. patent 5,756,825. Another example is the chelating agent termedp-SCN-Bz-HEHA (1,4,7,10,13,16-hexaazacyclo-octadecane-N,N',N",N"',N",N"'"-hexaacetic acid) (Deal et al., J. Med. Chen2. 42:2988, 1999), which is an effective chelator of radiometals such as 225AC. Yet another example is DOTA
(1,4,7,10-tetraazacyclododecane N,N',N",N"'-tetraacetic acid), which is a bifunctional chelating agent (see McDevitt et al., Science 294:1537-1540, 2001) that can be used in a two-step method for labeling followed by conjugation.
In another aspect, the invention provides compositions comprising an isolated antibody, an antibody derivatized or linked to other functional moieties, or an antigen-binding fragment thereof or a combination of one or more of the aforementioned antibodies or antigen-binding fragments thereof. The compositions include a physiologically or pharmaceutically acceptable carrier, excipient, or stabilizer mixed with the isolated antibody or antigen-binding fragment thereof. In a preferred embodiment, the compositions include a combination of multiple (e.g., two or more) isolated antibodies or antigen-binding portions thereof of the invention. Preferably, each of the antibodies or antigen-binding portions thereof of the composition binds to a distinct conformational epitope of PSMA.
In one embodiment, anti-PSMA antibodies having complementary activities are used in combination, e.g., as a pharmaceutical composition, comprising two or more anti-PSMA
antibodies. For example, an antibody that mediates highly effective cytolysis of target cells
-43 -in the presence of effector cells can be combined with another antibody that inhibits the growth of cells expressing PSMA. As used herein, "target cell" shall mean any undesirable cell in a subject (e.g., a human or animal) that can be targeted by a composition of the invention. In preferred embodiments, the target cell is a cell expressing or overexpressing PSMA.
Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one anti-tumor agent, immunomodulator, immunostimulatory agent, or other conventional therapy. The agent may be bound or conjugated to or formed as a recombinant fusion molecule with the PSMA =
antibodies of the present invention for directed targeting of the agent to PSMA-expressing cells.
The PSMA antibodies of the present invention may be used as a targeting moiety for delivery of replication-selective virus to PSMA-expressing cells for tumor therapy.
Replication-competent virus such as the p53 pathway targeting adenovirus mutant d11520, =
ONYX-015, kill tumor cells selectively (Biederer, C. et al., J. Mol. Med.
80(3):163-175, 2002).
As used herein, "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" includes any and all salts, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
Depending on the route of administration, the active compound, i.e., antibody may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents, such as supplementary immune potentiating agents including
- 44 -adjuvants, chemokines and cytokines. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
A salt retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al.
(1977)1 Pharm. Sci.
66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chioroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
An anti-PSMA antibody composition may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
The pharmaceutical compositions may contain suitable buffering agents, including:
acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy.
All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly
-45 -and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of anti-PSMA antibodies, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, intratumor, or transdermal.
When antibodies are used therapeutically, preferred routes of administration include intravenous and by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp.
1694-1712;
-46 -). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resorting to undue experimentation.
The compositions of the invention are administered in effective amounts. An "effective amount" is that amount of an anti-PSMA antibody composition that alone, or together with further doses, produces the desired response, e.g. treats a malignancy in a subject. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.
Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. R is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of anti-PSMA antibodies for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by determining the physiological effects of the anti-PSMA
antibody composition, such as regression of a tumor or decrease of disease symptoms.
Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
The doses of anti-PSMA antibodies administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied,
-47 -higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
In general, doses can range from about 10 1.1g/kg to about 100,000 Kg/kg.
Based upon the composition, the dose can be delivered continuously, such as by continuous pump, or at periodic intervals. Desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art. Other protocols for the administration of anti-PSMA antibody compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from the foregoing.
In general, doses of radionuclide delivered by the anti-PSMA antibodies of the invention can range from about 0.01 mCi/Kg to about 10 mCi/kg. Preferably the dose of radionuclide ranges from about 0.1 mCi/Kg to about 1.0 mCi/kg. The optimal dose of a given isotope can be determined empirically by simple routine titration experiments well known to one of ordinary skill in the art.
Administration of anti-PSMA antibody compositions to mammals other than humans, e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above.
The compositions (antibodies to PSMA and derivatives/conjugates thereof) of the present invention have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders. As used herein, the term "subject" is intended to include humans and non-human animals. Preferred subjects include a human patient having a disorder characterized by expression, typically aberrant expression (e.g., overexpression) of PSMA.
One aspect of the present invention relates to a method of detecting cancerous cells or portions thereof in a biological sample (e.g., histological or cytological specimens, biopsies and the like), and, in particular, to distinguish malignant tumors from normal tissues and non-malignant tumors. This method involves providing an antibody or an antigen-binding binding fragment thereof, probe, or ligand, which binds to an extracellular domain of PSMA
of such cells, e.g., an anti-PSMA antibody. The anti-PSMA antibody is bound to a label that permits the detection of the cells or portions thereof (e.g., PSMA or fragments thereof liberated from such cancerous cells) upon binding of the anti-PSMA antibody to the cells or
-48 -portions thereof. The biological sample is contacted with the labeled anti-PSMA antibody under conditions effective to permit binding of the anti-PSMA antibody to the extracellular domain of PSMA of any of the cells or portions thereof in the biological sample. The presence of any cells or portions thereof in the biological sample is detected by detection of the label. In one preferred form, the contact between the anti-PSMA antibody and the biological sample is carried out in a living mammal and involves administering the anti-PSMA antibody to the mammal under conditions that permit binding of the anti-PSMA
antibody to PSMA of any of the cells or portions thereof in the biological sample. Again, such administration can be carried out by any suitable method known to one of ordinary skill in the art.
In addition, the anti-PSMA antibodies of the present invention can be used in immunofluorescence techniques to examine human tissue, cell and bodily fluid specimens. In a typical protocol, slides containing cryostat sections of frozen, unfixed tissue biopsy samples or cytological smears are air dried, formalin or acetone fixed, and incubated with the monoclonal antibody preparation in a humidified chamber at room temperature.
The slides are then washed and further incubated with a preparation of a secondary antibody directed against the monoclonal antibody, usually some type of anti-mouse immunoglobulin if the monoclonal antibodies used are derived from the fusion of a mouse spleen lymphocyte and a mouse myeloma cell line. This secondary antibody is tagged with a compound, for instance rhodamine or fluorescein isothiocyanate, that fluoresces at a particular wavelength. The staining pattern and intensities within the sample are then determined by fluorescent light microscopy and optionally photographically recorded.
As yet another alternative, computer enhanced fluorescence image analysis or flow cytometry can be used to examine tissue specimens or exfoliated cells, i.e., single cell preparations from aspiration biopsies of tumors using the anti-PSMA antibodies of this invention. The anti-PSMA antibodies of the invention are particularly useful in quantitation of live tumor cells, i.e., single cell preparations from aspiration biopsies of prostate tumors by computer enhanced fluorescence image analyzer or with a flow cytometer. The antibodies of the invention are particularly useful in such assays to differentiate benign from malignant prostate tumors since the PSMA protein to which the anti-PSMA antibodies bind is expressed in increased amounts by malignant tumors as compared to benign prostate tumors. The percent PSMA positive cell population, alone or in conjunction with determination of other
- 49 -attributes of the cells (e.g., DNA ploidy of these cells), may, additionally, provide very useful prognostic information by providing an early indicator of disease progression.
In yet another alternative embodiment, the antibodies of the present invention can be used in combination with other known antibodies to provide additional information regarding the malignant phenotype of a cancer.
The method of the present invention can be Used to screen patients for diseases associated with the presence of cancerous cells or portions thereof.
Alternatively, it can be used to identify the recurrence of such diseases, particularly when the disease is localized in a particular biological material of the patient. For example, recurrence of prostatic disease in the prostatic fossa may be encountered following radical prostatectomy. Using the method of the present invention, this recurrence can be detected by administering a short range radiolabeled antibody to the mammal and then detecting the label rectally, such as with a transrectal detector probe.
Alternatively, the contacting step can be carried out in a sample of serum or urine or other body fluids, including but not limited to seminal fluid, prostatic fluid, ejaculate, and the like, such as to detect the presence of PSMA in the body fluid. When the contacting is carried out in a serum or urine sample, it is preferred that the biological agent recognize substantially no antigens circulating in the blood other than PSMA. Since intact cells do not excrete or secrete PSMA into the extracellular environment, detecting PSMA in serum, urine, or other body fluids generally indicates that cells are being lysed or shed.
Thus, the biological agents and methods of the present invention can be used to determine the effectiveness of a cancer treatment protocol by monitoring the level of PSMA
in serum, urine or other body fluids.
In a particularly preferred embodiment of the method of detecting cancerous cells in accordance with the present invention, the anti-PSMA antibodies or an antigen-binding fragment thereof, binds to and is internalized with the prostate specific membrane antigen of such cells. Again, the biological agent is bound to a label effective to permit detection of the cells or portions thereof upon binding of the biological agent to and internalization of the biological agent with the prostate specific membrane antigen.
Biological agents suitable for detecting cancerous cells include anti-PSMA
antibodies, such as monoclonal or polyclonal antibodies. In addition, antibody fragments, half-antibodies, hybrid derivatives, probes, and other molecular constructs may be utilized. These
- 50 -biological agents, such as antibodies, antigen-binding fragments thereof, probes, or ligands, bind to extracellular domains of prostate specific membrane antigens or portions thereof in cancerous cells. As a result, the biological agents bind not only to cells which are fixed or cells whose intracellular antigenic domains are otherwise exposed to the extracellular environment. Consequently, binding of the biological agents is concentrated in areas where there are prostate cells, irrespective of whether these cells are fixed or unfixed, viable or necrotic. Additionally or alternatively, these biological agents bind to and are internalized with prostate specific membrane antigens or portions thereof in normal, benign hyperplastic, and to a greater degree in cancerous cells.
The antibodies or antigen-binding fragments thereof can also be utilized in in vivo therapy of cancer. The antibodies can be used alone or covalently attached, either directly or via linker, to a compound which kills and/or inhibits proliferation of the malignant cells or tissues following administration and localization of the conjugates. When the antibody is used by itself, it may mediate tumor destruction by complement fixation or antibody-dependent cellular cytotoxicity. Alternatively, the antibody may be administered in combination with a chemotherapeutic drug to result in synergistic therapeutic effects (Baslya and Mendelsohn, 1994 Breast Cancer Res. and Treatment 29:127-138). A variety of , different types of substances can be directly conjugated to the antibody for therapeutic uses, including radioactive metal and non-metal isotopes, chemotherapeutic drugs, toxins, etc. as described above and known in the art (see, e.g., Vitetta and Uhr, 1985, Annu.
Rev. Immunol.
3:197).
The antibodies or antigen-binding fragments thereof of the invention can also be administered together with complement. Accordingly, within the scope of the invention are compositions comprising antibodies or antigen-binding fragments thereof and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies or antigen-binding fragments thereof.
Alternatively, the antibodies or antigen-binding fragments thereof of the invention and the complement or serum can be administered separately.
The antibodies can be administered with one or more immunostimulatory agents to induce or enhance an immune response, such as IL-2 and immunostimulatory oligonucleotides (e.g., those containing CpG motifs). Preferred immunostimulatory agents
- 51 -stimulate specific arms of the immune system, such as natural killer (NK) cells that mediate antibody-dependent cell cytotoxicity (ADCC).
Antigens, such as the PSMA dimers described herein, can be administered with one or more adjuvants to induce or enhance an immune response. An adjuvant is a substance which potentiates the immune response. Adjuvants of many kinds are well known in the art.
Specific examples of adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham); saponins including QS21 (SmithKline Beecham); immunostimulatory oligonucleotides (e.g., CpG oligonucleotides described by Kreig et al., Nature 374:546-9, 1995);incomplete Freund's adjuvant; complete Freund's adjuvant; montanide;
vitamin E and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol, Quil A, Ribi Detox, CRL-1005, L-121, and combinations thereof.
Other agents which stimulate the immune response of the subject to PSMA
multimer antigens can also be administered to the subject. For example, cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties.
Many cytoldnes useful for such purposes will be known to one of ordinary skill in the art, including interleukin-2 (IL-2); IL-4; IL-5; IL-12, which has been shown to enhance the protective effects of vaccines (see, e.g., Science 268: 1432-1434, 1995); GM-CSF; IL-15;
IL-18;
combinations thereof, and the like. Thus cytoldnes can be administered in conjunction with antibodies, antigens, chemokines and/or adjuvants to increase an immune response.
Chemokines useful in increasing immune responses include but are not limited to SLC, ELC, MIP3a, MIP313, IP-10, MIG, and combinations thereof.
The antibodies or antigen-binding fragments thereof of the present invention can be used in conjunction with other therapeutic treatment modalities. Such other treatments include surgery, radiation, cryosurgery, thermotherapy, hormone treatment, chemotherapy, vaccines, and other immunotherapies.
Also encompassed by the present invention is a method which involves using the antibodies or antigen-binding fragments thereof for prophylaxis. For example, these materials can be used to prevent or delay development or progression of cancer.
Use of the cancer therapy of the present invention has a number of benefits.
Since the anti-PSMA antibodies or antigen-binding fragments thereof according to the present invention preferentially target prostate cancer cells, other tissue is spared.
As a result, treatment with such biological agents is safer, particularly for elderly patients. Treatment
- 52 -according to the present invention is expected to be particularly effective, because it directs high levels of anti-PSMA antibodies or antigen-binding fragments thereof to the bone marrow and lymph nodes where prostate cancer metastases predominate. Moreover, tumor sites for prostate cancer tend to be small in size and, therefore, easily destroyed by cytotoxic agents. Treatment in accordance with the present invention can be effectively monitored with clinical parameters such as serum prostate specific antigen and/or pathological features of a patient's cancer, including stage, Gleason score, extracapsular, seminal, vesicle or perineural invasion, positive margins, involved lymph nodes, etc. Alternatively, these parameters can be used to indicate when such treatment should be employed.
Because the antibodies or antigen-binding fragments thereof of the present invention bind to living cells, therapeutic methods using these biological agents are much more effective than those which target lysed cells. For the same reasons, diagnostic and imaging methods which determine the location of living normal, benign hyperplastic, or cancerous cells are much improved by employing the antibodies or antigen-binding fragments thereof of the present invention. In addition, the ability to differentiate between living and dead cells can be advantageous, especially to monitor the effectiveness of a particular treatment regimen.
Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. The kits can further contain at least one additional reagent, such as complement, or one or more additional antibodies of the invention (e.g., an antibody having a complementary activity which binds to an epitope in PSMA
antigen distinct from the first antibody). Other kits can include the PSMA multimers described hereinbelow.
Kits containing the antibodies or antigen-binding fragments thereof of the invention can be prepared for in vitro diagnosis, prognosis and/or monitoring cancer by the immunohistological, immunocytological and immunoserological methods described above.
The components of the kits can be packaged either in aqueous medium or in lyophilized form. When the antibodies or antigen-binding fragments thereof are used in the kits in the form of conjugates in which a label moiety is attached, such as an enzyme or a radioactive metal ion, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user or the kit.
- 53 -A kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like. A first of said container means or series of container means may contain one or more anti-PSMA antibodies or antigen-binding fragments thereof or PSMA. A second container means or series of container means may contain a label or linker-label intermediate capable of binding to the primary anti-PSMA
antibodies (or fragment thereof).
Kits for use in in vivo tumor localization and therapy method containing the anti-PSMA antibodies or antigen-binding fragments thereof conjugated to other compounds or substances can be prepared. The components of the kits can be packaged either in aqueous medium or in lyophilized form. When the antibodies or antigen-binding fragments thereof are used in the kits in the form of conjugates in which a label or a therapeutic moiety is attached, such as a radioactive metal ion or a therapeutic drug moiety, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user of the kit.
In one aspect of the invention, a method for modulating at least one enzymatic activity of PSMA, the activity selected from the group consisting of N-acetylated a-linked acidic dipeptidase (NAALADase), folate hydrolase, dipeptidyl dipeptidase IV
and y-glutamyl hydrolase activity or combination thereof in vitro or in vivo. The modulation may be enhancement or inhibition of at least one enzymatic activity of PSMA.
In a preferred embodiment, the invention provides methods for inhibiting at least one enzymatic activity of PSMA, the activity selected from the group consisting of N-acetylated a-linked acidic dipeptidase (NAALADase), folate hydrolase, dipeptidyl dipeptidase IV and y-glutamyl hydrolase activity or combination thereof in vitro or in vivo. The method comprises contacting a NAALADase, a folate hydrolase, a dipeptidyl dipeptidase IV and/or a y-glutamyl hydrolase with an amount of an isolated antibody or antigen-binding fragment thereof of the invention under conditions wherein the isolated monoclonal antibody or antigen-binding fragment thereof inhibits NAALADase, folate hydrolase, dipeptidyl dipeptidase IV or y-glutamyl hydrolase activity.
Tissue levels of NAALADase can be determined by detergent solubilizing homogenizing tissues, pelleting the insoluble material by centrifugation and measuring the NAALADase activity in the remaining supernatant. Likewise, the NAALADase activity in
- 54 -bodily fluids can also be measured by first pelleting the cellular material by centrifugation and performing a typical enzyme assay for NAALADase activity on the supernatant.
NAALADase enzyme assays have been described by Frieden, 1959, J. Biol, Chem., 234:2891. In this assay, the reaction product of the NAALADase enzyme is glutamic acid.
This is derived from the enzyme catalyzed cleavage of N-acetylaspartylglutamate to yield N-acetylaspartic acid and glutamic acid. Glutamic acid, in a NAD(P)+ requiring step, yields 2-oxoglutarate plus NAD(P)H in a reaction catalyzed by glutamate dehydrogenase.
Progress of the reaction can easily and conveniently be measured by the change in absorbance at 340 nm due to the conversion of NAD(P)+ to NAD(P)H.
Folate hydrolase activity of PSMA can be measured by performing enzyme assays as described by Heston and others (e.g., Clin. Cancer Res. 2(9):1445-51, 1996;
Urology 49(3A
Suppl):104-12,1997). Folate hydrolases such as PSMA remove the gamma-linked glutamates from polyglutamated folates. Folate hydrolase activity can be measured using substrates such as rnethotrexate tri-gamma glutamate (MTXG1u3), methotrexate di-gamma glutamate (MTXG1u2) and pteroylpentaglutamate (PteGlu5), for example using capillary electrophoresis (see Clin. Cancer Res. 2(9):1445-51, 1996). Timed incubations of PSMA
with polyglutamated substrates is followed by separation and detection of hydrolysis products.
The invention also includes isolated antibodies and binding fragments thereof that selectively bind PSMA multimers. As used herein, particularly with respect to the binding of PSMA multimers by the anti-PSMA antibodies and binding fragments, "selectively binds"
means that an antibody preferentially binds to a PSMA protein multimer (e.g., with greater avidity, greater binding affinity) rather than to a PSMA protein monomer. In preferred embodiments, the antibodies of the invention bind to a PSMA protein multimer with an avidity and/or binding affinity that is 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 70-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold or more than that exhibited by the antibody for a PSMA protein monomer. Preferably, the antibody selectively binds a PSMA protein multimer, and not a PSMA protein monomer, i.e., substantially exclusively binds to a PSMA protein multimer. Most preferably, the antibody selectively binds a PSMA protein dimer.
- 55 -The isolated antibody or binding fragment that selectively binds a PSMA
protein multimer can, in some embodiments, modulate enzymatic activity of the PSMA
protein multimer. In one such embodiment, the antibody inhibits at least one enzymatic activity such as NAALADase activity, folate hydrolase activity, dipeptidyl dip eptidase IV
activity, y-glutamyl hydrolase activity, or combinations thereof. In another embodiment, the antibody enhances at least one enzymatic activity such as NAALADase activity, folate hydrolase activity, dipeptidyl dipeptidase IV activity, y-glutamyl hydrolase activity, or combinations thereof.
A PSMA protein multimer, as used herein, is a protein complex of at least two PSMA
proteins or fragments thereof. The PSMA protein multimers can be composed of various combinations of full-length PSMA proteins (e.g., SEQ ID NO:1), recombinant soluble PSMA
(rsPSMA, e.g., amino acids 44-750 of SEQ ID NO:1) and fragments of the foregoing that form multimers (i.e., that retain the protein domain required for forming dimers and/or higher order multimers of PSMA). In preferred embodiments, at least one of the PSMA
proteins forming the multimer is a recombinant, soluble PSMA (rsPSMA) polypeptide.
Preferred PSMA protein multimers are dimers, particularly those formed from recombinant soluble PSMA protein. A particularly preferred embodiment is a rsPSMA homodimer.
The PSMA protein multimers referred to herein are believed to assume a native conformation and preferably have such a conformation. The PSMA proteins in certain embodiments are noncovalently bound together to form the PSMA protein multimer. For example, it has been discovered that PSMA protein noncovalently associates to form dimers under non-denaturing conditions, as described in the Examples below.
The PSMA protein multimers can, and preferably do, retain the activities of PSMA.
The PSMA activity may be an enzymatic activity, such as folate hydrolase activity, NAALADase activity, dipeptidyl peptidase IV activity and y-glutamyl hydrolase activity.
Methods for testing the PSMA activity of multimers are well known in the art (reviewed by O'Keefe et al. in: Prostate Cancer: Biology, Genetics, and the New Therapeutics, L.W.K.
Chung, W.B. Isaacs and J.W. Simons (eds.) Humana Press, Totowa, NJ, 2000, pp.
307-326), some of which are described in the Examples herein below.
In certain aspects, the invention also includes compositions including one or more of the isolated PSMA protein multimers described herein, such as the PSMA protein dimer. In preferred embodiments, a PSMA protein multimer composition contains at least about 10%
-56-.
PSMA protein multimer. In other embodiments, the PSMA protein multimer composition contains at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%
or 99.5% PSMA protein multimer. In a preferred embodiment, the PSMA protein multimer composition contains substantially pure PSMA protein multimer, with substantially no PSMA protein monomer. It is understood that the list of specific percentages includes by inference all of the unnamed percentages between the recited percentages.
As used herein with respect to polypeptides, proteins or fragments thereof, "isolated"
means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may be, but need not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use.
Substantially pure polypeptides may be produced by techniques well known in the art. Because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight of the preparation.
The protein is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, i.e. isolated from other proteins.
Fragments of a PSMA protein preferably are those fragments which retain a distinct functional capability of the PSMA protein. Functional capabilities which can be retained in a fragment include binding of other PSMA molecules to form dimers and higher order multimers, interaction with antibodies, interaction with other polypeptides or fragments thereof, and enzymatic activity. Other PSMA protein fragments, e.g., other recombinant soluble fragments of SEQ ID NO:1, can be selected according to their functional properties.
For example, one of ordinary skill in the art can prepare PSMA fragments recombinantly and test those fragments according to the methods exemplified below.
Modifications to a PSMA polypeptide are typically made to the nucleic acid which encodes the PSMA polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties.
Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a
- 57 -fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the PSMA amino acid sequence.
In general, modified PSMA polypeptides include polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a PSMA
polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
Modifications conveniently are prepared by altering a nucleic acid molecule that encodes the PSMA polypeptide. Mutations of a nucleic acid which encode a PSMA
polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the modified polypeptide.
Modifications can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the PSMA
polypeptide.
Modified PSMA polypeptides then can be expressed and tested for one or more activities (e.g., antibody binding, enzymatic activity, multimeric stability) to determine which mutation provides a modified polypeptide with the desired properties. Further mutations can be made to modified PSMA polypeptides (or to non-modified PSMA polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coil, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a PSMA coding sequence or cDNA clone to enhance expression of the polypeptide. The activity of modified PSMA polypeptides can be tested by cloning the gene encoding the modified PSMA polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the modified PSMA polypeptide, and testing for a functional capability of the PSMA

polypeptides as disclosed herein. The foregoing procedures are well known to one of ordinary skill in the art.
The skilled artisan will also realize that conservative amino acid substitutions may be made in PSMA polypeptides to provide functionally equivalent PSMA
polypeptides, i.e.,
- 58 -modified PSMA polypeptides that retain the functional capabilities of PSMA
polypeptides.
As used herein, a "conservative amino acid substitution" refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Modified PSMA polypeptides can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M.
Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplary functionally equivalent PSMA polypeptides include conservative amino acid substitutions of SEQ lD
NO:1, or fragments thereof, such as the recombinant soluble PSMA polypeptide (amino acids 44-750 of SEQ ID NO:1). Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (e) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
Conservative amino-acid substitutions in PSMA polypeptides typically are made by alteration of a nucleic acid encoding a PSMA polypeptide. Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis, or by chemical synthesis of a gene encoding a PSMA polypeptide. Where amino acid substitutions are made to a small fragment of a PSMA polypeptide, the substitutions can be made by directly synthesizing the peptide. The activity of functionally equivalent fragments of PSMA
polypeptides can be tested by cloning the gene encoding the altered PSMA
polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered PSMA polypeptide, and testing for a functional capability of the PSMA polypeptides as disclosed herein.
The PSMA protein multimers as described herein have a number of uses, some of which are described elsewhere herein. The multimers are useful for testing of compounds that modulate PSMA enzymatic activity or PSMA multimerization. The multimers can be used to isolate antibodies that selectively bind PSMA, including those selective for conformational epitopes, those selective for binding PSMA multimers and not PSMA
monomers, and those that selectively modulate an enzymatic activity of PSMA.
The
- 59 -multimers, particularly dimeric PSMA, also can be used to induce or increase immune responses to PSMA, as vaccine compositions.
Agents that selectively modulate an enzymatic activity of PSMA include agents that inhibit or enhance at least one enzymatic activity of PSMA, such as NAALADase activity, folate hydrolase activity, dipeptidyl dipeptidase IV activity, y-glutamyl hydrolase activity, or combinations thereof.
Thus methods of screening for candidate agents that modulate at least one enzymatic activity of a PSMA enzyme are provided in accordance with the invention. The methods can include mixing the candidate agent with an isolated PSMA protein multimer to form a reaction mixture, thereby contacting the PSMA enzyme with the candidate agent.
The methods also include adding a substrate for the PSMA enzyme to the reaction mixture, and determining the amount of a product formed from the substrate by the PSMA
enzyme. Such methods are adaptable to automated, high-throughput screening of compounds. A
decrease in the amount of product formed in comparison to a control is indicative of an agent capable of inhibiting at least one enzymatic activity of the PSMA enzyme. An increase in the amount of product formed in comparison to a control is indicative of an agent capable of enhancing at least one enzymatic activity of the PSMA enzyme. The PSMA enzyme can be NAALADase, folate hydrolase, dipeptidyl dipeptidase IV and/or y-glutamyl hydrolase. The PSMA enzyme preferably is a PSMA multimer that includes recombinant soluble PSMA, most preferably a noncovalently associated dimer of PSMA in a native conformation.
The reaction mixture comprises a candidate agent. The candidate agent is preferably an antibody, a small organic compound, or a peptide, and accordingly can be selected from combinatorial antibody libraries, combinatorial protein libraries, or small organic molecule libraries. Typically, a plurality of reaction mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations.
Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.
Candidate agents encompass numerous chemical classes, although typically they are organic compounds, proteins or antibodies (and fragments thereof that bind antigen). In some preferred embodiments, the candidate agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate agents comprise functional
- 60 -chemical groups necessary for structural interactions with polypeptides and/or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random or non-random peptides, combinatorial libraries of proteins or antibodies, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-agent binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
The mixture of the foregoing reaction materials is incubated under conditions whereby, the candidate agent interacts with the PSMA enzyme. The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4 C and 40 C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
- 61 -After incubation, the presence or absence of PSMA enzyme activity is detected by any convenient method available to the user. For example, the reaction mixture can contain a substrate for the PSMA enzyme. Preferably the substrate and/or the product formed by the action of the PSMA enzyme are detectable. The substrate usually comprises, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical, or electron density, etc) or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, etc.). The label may be bound to the substrate, or incorporated into the structure of the substrate.
A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. For example, the label may be detected while bound to the substrate or subsequent to separation from the substrate. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc.
or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for detecting a variety of labels are well known in the art.
Examples Materials and Methods DNA constructs. All secreted PSMA constructs were derived from the original human PSMA
clone p55A provided by Dr. W.D.W. Heston (Israeli et al., Cancer Res. 53: 227-230, 1993).
The constructs were subcloned into expression vector PPI4 (Trkola et al., Nature 384: 184-187, 1996) for high-level expression and secretion in mammalian cells.
Recombinant soluble PSMA (rsPSMA) corresponds to the entire extracellular domain of PSMA (amino acids 44-750 of SEQ ID NO: 1 (GenBank Protein Accession number AAA60209)).
pcDNA Plasmid Constructs: Nucleic acid molecules encoding the anti-PSMA
antibodies 10.3, 006, 026, 051, 069 and 077 were cloned into plasmid pcDNA. The cloning protocol is given in Figure 10. Primers (SEQ ID NOs: 33-36, sense and anti-sense) used for the variable region amplifications are also shown. The plasmids constructed for anti-PSMA
antibodies 006, 026, 051, 069, 077 and 10.3 contain nucleotide sequences encoding the heavy chain of the antibodies (SEQ ID NOs: 2-7; PTA-4403, PTA-4405, PTA-4407, PTA-4409, PTA-4411,
- 62 -PTA-4413, respectively) or contain nucleotide sequences encoding light chain of the antibodies (SEQ ED NOs: 8-13; PTA-4404, PTA-4406, PTA-4408, PTA-4410, PTA-4412 and PTA-4414, respectively). Plasmid maps are given in Figures 11-22.
TM
Western blots. Cells were lysed in PBS containing 1mM EDTA, 1% NP-40, 1%
Triton X-100, and 5mg/m1 aprotinin and cell debris was removed by centrifugation at 3000g for 30 min at 4 C. Lysates were separated on a 5-20% gradient gel before transfer to nitrocellulose membranes. The resulting blots were blocked in PBS containing 5% milk, 0.02%
SDS and 0.1% Triton X-100 before incubation with MAB544 primary antibody (Maine Biotechnologies) at a concentration of 2mg/ml. After three washes, blots were incubated with a goat anti-mouse HRP-conjugated secondary antibody at a concentration of 0.2mg/ml.
Blots are visualized using the Renaissance chemiluminescence system (Perkin-Elmer Life Sciences, Boston, MA).
ELISA. Cells were lysed in PBS containing lraM EDTA, 1% NP-40, 1% Triton X-100, and 5mg/m1 aprotinin. The resulting cell membranes were plated onto 96-well plates and dried in a sterile hood overnight. The plates were then blocked with PBS containing casein and Tween-20 before addition of mouse sera or hybridoma supernatants, using purified MAB544 (Maine Biotechnologies) or 7E11 (Cytogen) as a standard. After washing in PBS, an alkaline phosphatase conjugated secondary antibody (subclass specific) was incubated and subsequently washed in PBS. The pNPP substrate was then added for colorimetric detection at a wavelength of 405 urn.
Flow cytometty. Wild-type 3T3 or PSMA-expressing 3T3 cells (106 cells per condition) were washed in PBS containing 0.1% NaN3. Antibodies or sera were then added (1:100 dilution in PBS) and incubated on ice for 30 minutes. After washing in PBS+0.1% NaN3, the cells were incubated with anti-mouse IgG+IgM (Calbiotech) for 30 minutes on ice. Cells were washed again in PBS+0.1% NaN3 and analyzed by flow cytometry.
Example 1: Generation of a panel of monoclonal antibodies (mAbs) to conformational gitopes on PSMA
- 63 -A panel of anti-PSMA mAbs that represent promising candidates for therapy was created. Briefly, the mAbs were generated as follows: BALB/c mice were immunized subcutaneously with recombinant PSMA at approximately three-week intervals.
After a total of 4 injections, mice were sacrificed and their splenocytes fused with a m.yeloma cell line using standard techniques in order to create hybridomas. Individual hybridoma supernatants were screened by ELISA for reactivity with PSMA derived from either LNCaP
human prostate tumor cells or from 3T3 cells engineered to express full-length human PSMA (3T3-PSMA cells). Positive clones were secondarily screened by flow cytometry for specific reactivity with intact 3T3-PSMA and LNCaP cells so as to select antibodies that recognize native, cell-surface PSMA and thus have the greatest therapeutic potential.
Mice having the ability to produce human antibodies (XenoMouse, Abgenix;
Mendez et al., Nature Genetics 15:146, 1997) were immunized subcutaneously once or twice weekly with 5 X 106 LNCaP cells adjuvanted with alum or Titermax Gold (Sigma Chemical Co., St.
Louis, MO). Animals were boosted twice with 10 lug of recombinant PSMA protein immunoaffinity captured onto protein G magnetic microbeads (Miltenyi Biotec, Auburn, CA). PSMA mAb 3.11 was used for capture. Splenocytes were fused with NSO
myeloma cells and the hybridomas that resulted were screened as above by flow cytometry to detect clones producing antibodies reactive with the extracellular portion of PSMA.
One clone, 10.3 (PTA-3347), produced such antibodies.
These methods have yielded a high proportion of mAbs that react exclusively with conformation-specific epitopes on cell-surface PSMA. As shown in Fig. 1, several (mAbs 3.7, 3.9, 3.11, 5.4, and 10.3) but not all (mAb 3.12) mAbs specifically bind viable PSMA-expressing cells. Using recombinant soluble PSMA proteins expressed in Chinese hamster ovary (CHO) cell lines, it further was demonstrated that the mAbs bind epitopes in the extracellular region of PSMA. The mAbs were also tested for their ability to immunoprecipitate native PSMA from 3T3-PSMA cell lysates. The mAbs positive in flow cytometry (Fig. 1) were also effective in immunoprecipitation (Fig. 2), whereas mAb 3.12 was unreactive. Fig. 3 shows the recognition of non-denatured full-length PSMA
and recombinant soluble PSMA by several PSMA antibodies that recognize PSMA
conformation.
This further confirms that these methods yield a preponderance of mAbs that efficiently recognize native PSMA.
- 64 -The mAbs were tested for reactivity with denatured PSMA by Western blot analysis (Fig. 4). Lysates from the indicated cells and samples (controls: 3T3 cells, PSMA-negative human prostate cell lines PC-3 and DU145, mock supernatant; PSMA-positive samples:
PSMA-expressing 3T3 cells, PSMA-positive human prostate cell line LNCaP, rsPSMA-positive supernatant) were resolved by SDS-PAGE, electroblotted, and probed with anti-PSMA mAbs 3.1 and 3.12 (ATCC Patent Deposit Designations PTA-3639 and PTA-3640, respectively). Four mAbs tested in parallel (3.7, 3.8, 3.9, 3.11) showed no reactivity to either full-length or secreted rsPSMA proteins. 7E11 mAb immunoprecipitated full-length but not secreted rsPSMA.
The mAbs reactive in flow cytometry and immunoprecipitation (mAbs 3.7, 3.9, 3.11, 5.4, and 10.3) were all unreactive in Western blot analysis, indicating that the mAbs do not recognize linear epitopes. Taken together, the data strongly suggest that these 5 mAbs recognize conformation-specific epitopes located in the extracellular domain of PSMA.
Since mAbs to conformational epitopes typically possess the greatest affinity and specificity for antigen, they represent preferred candidates for therapy.
The reactivities of certain anti-PSMA antibodies are described in Table 2:
Table 2: anti-PSMA antibody properties Reactivity mAb ELISA Flow EP Western Epitope Cytometry 3.1 Linear, Extracellular, exposed on native PSMA
3.7 Conformational, extracellular 3.8 Conformational, extracellular 3.9 Conformational, extracellular 3.11 Conformational, extracellular 3.12 Linear, Extracellular, not exposed on native PSMA
5.4 Conformational, extracellular 7.1 Linear, Extracellular, not exposed on
- 65 -native PSMA
7.3 Conformational, extracellular 10.3 Conformational, extracellular 1.8.3 Extracellular A3.1.3 Extracellular A3.3.1 Extracellular The mAbs were determined by ELISA to be primarily of the mouse IgG2a, mouse IgG2b and human IgG1 isotypes, which mediate potent effector functions.
Although a number of anti-PSMA mAbs have been described over the years and evaluated for therapeutic potential (see, e.g., Liu, H. et al. Cancer Res. 57: 3629-3634, 1997; Chang, S.S. et al. Cancer Res. 59: 3192-3198, 1999; Murphy, G.P. et al. J Urology 160: 2396-2401, 1998), none inhibit the enzymatic activity of PSMA and few recognize conformational determinants on PSMA.
to Example 2: Production of anti-PSMA mAbs To accurately and quantitatively assess the therapeutic potential of these mAbs, the mAbs are produced in a quantity and quality suitable for extensive in vitro and in vivo characterization. Briefly, the mAb-secreting hybridomas are cultured in roller bottles in DMEM/F12 medium supplemented with 10% PBS that has been depleted of bovine IgG
(Life Technologies). During the production phase of the culture, cells are maintained at ¨5 x 106 cells/mL via twice-weekly exchanges of media. Collected media are clarified by filtration through a 0.22 micron filter and stored at ¨95 C prior to purification. Given an average antibody expression levels of ¨25 mg/L, approximately 3L of roller bottle supernatants are required for each antibody to allow for losses in purification.
Culture supernatants from a given hybridoma are pooled and loaded onto a Protein A
TM
Sepharose affinity column. Mouse IgG2a, mouse IgG2b and human IgG1 antibodies are loaded directly, but supernatants containing mouse IgG1 antibodies are adjusted to pH 8.5 and 1M NaCI prior to loading in order to promote binding. After washing the column, the mAb is eluted with low pH buffer into fractions using 1M Tris, pH 8Ø Elution peak fractions are pooled, dialyzed against PBS buffer, concentrated to 5 mg/mL and stored in sterile aliquots at ¨95 C. All purification procedures are carried out using endotoxin-free
- 66 -buffers and sanitized chromatography columns. Purified mAbs are tested for purity by reducing and nonreducing SDS-PAGE, for PSMA binding affinity by ELISA, and for endotoxin levels by the limulus amebocyte lysate assay. These procedures routinely yield "animal-grade" antibody at >95% purity and <0.5 endotoxin units per milligram of protein.
Example 3: Evaluation of the therapeutic potential of the unlabeled mAbs in vitro Purified mAbs are tested in a battery of assays for therapeutically relevant properties, including affinity, specificity, enzyme inhibitory activity and effector functions. The ideal product candidate binds and inhibits PSMA activity at subnanomolar concentrations and mediates potent cell-killing through Fe-related effector functions.
First, the mAbs' affinity for cell-surface and secreted forms of PSMA is measured by flow cytometry and ELISA, respectively. In the flow cytometry assay, varying amounts of mAbs are incubated with 5x105 3T3-PSMA cells in FACS buffer (PBS containing 1%
FBS
and 0.1% NaN3) for 2 hr to allow for saturation binding. Cells are washed and incubated with a phycoerythrin-coupled goat antibody to mouse IgG (ICN/Cappel) for detection of bound mAb by flow cytometry. Specific binding is calculated by subtracting the fluorescence intensity observed with parental 3T3 cells.
For ELISA, CHO cell-derived recombinant soluble PSMA protein (rsPSMA, Progenies, Tarrytown, NY) is diluted to 1 )_tg/m1 in 50 mM carbonate buffer, pH 9.4, and coated overnight at 4 C onto 96-well Immulon II microtiter plates at 100 pl/well. The plates are then blocked for 2 hr with PBS buffer containing 5% BSA. mAbs are added in a range of concentrations in ELISA buffer (PBS buffer containing 2% BSA, 1% FBS and 0.5%
Tween 20) for 2 hours at room temperature. The plates are washed, and horseradish peroxidase conjugated goat antibody to mouse IgG is added for 1 hr at room temperature.
The plates are washed again and 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) substrate (Pierce, Rockford, IL) is added for colorimeffic readout at 450 nm using an ELISA
plate reader (Molecular Devices, Sunnyvale, CA).
Example 4: mAb cross-competition binding assay To identify whether a given group of mAbs recognize distinct or overlapping epitopes on PSMA, cross-competition binding assays are performed (Liu, H. et al. Cancer Res 57:
3629-3634, 1997). In this flow cytometry assay, a biotinylated test mAb is incubated with
- 67 -3T3-PSMA cells in the presence or absence of varying concentrations of unlabeled competitor mAbs as described above. Following washing, phycoerythrin-conjugated streptavidin is added to determine the amount of bound biotinylated mAb. The percent inhibition is defined relative to that observed in the presence of an isotype-matched mAb of irrelevant specificity (0% inhibition) and to that observed using excess unlabeled test mAb (100% inhibition).
Example 5: Effects of mAbs on PSMA enzymatic activity PSMA has been shown to possess both folate hydrolase and N-acetylated a-linked acidic dipeptidase (NAALADase) enzymatic activities, which may influence the proliferation and malignancies of the tumor cell (Heston, W.D.W. Prostate: Basic and Clinical Aspects (R.K. Naz, ed.). CRC Press, New York: 219-243, 1997). Several of the mAbs described above (mAb 3.9, mAb 5.4 and mAb 7.3) and mAb J591 (ATCC #HB-12126) were tested for folate hydrolase modulating activity using previously described assays for measuring PSMA
enzymatic activity (Pinto, J.T. et al. Clinical Cancer Res 2: 1445-1451, 1996).
Briefly, folate hydrolase activity was measured as follows. Fifty tM
methotrexate di-gamma glutamate and 10 pg/m1rsPSMA (premixed with anti-PSMA or irrelevant mAb) was incubated in pH 4.5 acetate buffer in a volume of 1000 for 2 hr at 37 C.
Reactions were terminated by boiling for 5 minutes prior to separation of free, mono- and di-gamma glutamate forms of methotrexate by capillary electrophoresis on a Spectra Phoresis 1000 (Thermo Separation, San Jose, CA). The various methotrexate derivatives were quantified based on their retention times and absorbance at 300 nm.
The data show that mAb 5.4 potently blocks the enzymatic activity of purified rsPSMA protein and in lysates of C4-2 cells. C4-2 is an androgen independent derivative of the LNaCP cell line (human prostate cancer line) which expresses endogenous PSMA. More details regarding the C4-2 cell line may be found in O'Keefe D.S. et al.
Prostate 45: 149-157, 2000). Figures 7 and 8 provide the results for two production lots of rsPSMA
(rsPSMA #7 and rsPSMA #8). The results for the C4-2 cell lysates are shown in Figure 9.
The figures illustrate the effect of four antibodies (mAb 3.9, mAb 5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate hydrolase by way of the rate of cleavage of glutamate from methotrexate di-gamma glutamate (MTXG1u2) by folate hydrolase present in the two
- 68 -=
production lots of rsPSMA and in the C4-2 cell lysates. In addition to the inhibitory effects of mAb 5.4, mAb 3.9 was also found to inhibit folate hydrolase activity.
For NAALADase activity assays, rsPSMA protein is incubated with varying amounts of anti-PSMA or control mAbs in 50mM Tris pH 7.4, 1mM CoC12 for 10 minutes at before adding 50 1 of 0.6iuM N-acetylaspartyl-[3H]glutamate. After 15 minutes, the reaction is stopped by adding 1 ml of 100mM NaPO4. Cleaved glutamate is separated from the substrate by ion exchange chromatography and detected by scintillation counting. Each measurement is performed in triplicate.
Example 6: Reactivity with normal and malignant human tissues by immunohistochemistry Anti-PSMA mAbs are tested by immunohistochemistry for reactivity with both normal and malignant human tissues using an avidin-biotin peroxidase method (Silver, D.A.
et al. Clin Cancer Res 3: 81-85,1997). Frozen or paraffin-embedded tissues can be used.
Paraffin-embedded tissue sections are deparaffinized and endogenous peroxidase activity is blocked by incubation with 1% H202 for 15 minutes. Sections are blocked in a 1:10 dilution of horse serum in 2% PBS-BSA (Sigma Chemical, St Louis, MO) for 30 minutes before overnight incubation with 21.1g/m1 anti-PSMA mAb in 2% PBS-BSA. After washing, sections are incubated with biotinylated secondary antibody, washed, and incubated with avidin:biotin peroxidase complexes (Vector Laboratories, Burlingame, CA) diluted 1:25 in PBS for 30 minutes. After washing, sections are visualized by immersion in PBS
containing 0.05% diaminobenzidine tetrachloride, 0.01% H202, and 0.5% Triton X-100.
Negative control sections are incubated with isotype-matched mAbs of irrelevant specificity. As a positive control, 7E11 (Cytogen, Princeton, NJ), a well-characterized anti-PSMA mAb, is used.
Example 7: Antibody-dependent cellular cytotoxicity (ADCC) In the ADCC assay, mAbs are serially diluted and combined with 51Cr-labeled PSMA cells or human prostate PC-3 cells that have been engineered to express human PSMA
(PC-3-PSMA cells). NK effector cells are purified from lymph nodes or spleens using anti-NK microbeads (Miltenyi Biotec). Sera, NK effector cells, and 51Cr-loaded target cells are co-incubated at effector:target cell ratios of 10:1, 20:1, and 40:1, with each condition performed in triplicate. Cells are incubated 4-5 hours at 37 C before supernatants are
- 69 -collected for measurement of 51Cr release by gamma counting. The percent specific lysis is determined relative to that observed in the presence of isotype-matched non-specific mAb (0% lysis) to that obtained using 10% sodium dodecyl sulfate (100% lysis).
Example 8: Complement-mediated lysis (CML) For CML, 51Cr-loaded 3T3-PSMA or PC-3-PSMA cells serve as target cells. Serial dilutions of mAbs are co-incubated with rabbit complement and target cells for 4-5 hours at 37 C, with each condition being performed in triplicate. Supernatants are then collected and counted with a gamma counter. Specific lysis is computed as previously done with the ADCC assay data.
Example 9: Anti-proliferative effects To test anti-proliferative effects of these antibodies, anti-PSMA mAbs are serially diluted and incubated with LNCaP, PC-3-PSMA and parental PC-3 cells in log-phase growth.
At 4 hr, 24 hr, and 72 hr intervals, cells are removed and analyzed for density and viability by =
trypan blue staining and WST-1 assay (Roche Biochemicals).
Example 10: Optimization of chelation and radiolabeling procedures The most promising mAbs identified using the procedures described in the foregoing examples will be optimized for biochemical and biological stability and activity after labeling prior to evaluation in animals. Success in in vitro experiments is defined as identification of a radiolabeled mAb that specifically kills PSMA-expressing tumor cells at >10-fold lower concentrations than unlabeled or similarly labeled isotype control mAb.
Because the preferred a- and n-emitting isotopes are all radiometals, each of the mAbs is first conjugated with an appropriate metal chelating agent. Based on the favorable in vivo stability data and its proven use in human clinical trials, the bifunctional chelating agent C-functionalized trans cyclohexyldiethylenetriaminepentaacetic acid (p-SCN-CHX-A"-DTPA) is the preferred agent for attaching either 90Y or 213Bi to the antibody (Brechbiel, M.W. et al. J. Chem. Soc. Chem. Commun. 1169-1170, 1991). A form of this chelate has previously been tested in more than 70 doses in humans in ongoing trials at Memorial-Sloan Kettering Cancer Center (McDevitt, M.R. et al. J. Nucl. Med. 40:1722-1727, 1999). For 225AC, our initial studies will examine a novel bifunctional chelating agent termed p-SCN-Bz-
- 70 -HEHA (1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N",N'",N"",N""'-hexaacetic acid) (Deal, K.A. et al. J. Med. Chem. 42:2988-2992, 1999). The objective is to optimize the antibody conjugation and chelation ratios to maximize labeling yield and activity while maintaining suitable stability for in vivo utilization. Additional chelating agents also are used as they become available from the N.I.H. and other sources.
Initially, the antibody is rendered metal-free by incubation with a large molar excess of EDTA at pH=5. The EDTA and any metals scavenged from the antibody preparation are removed via continuous buffer exchange/dialysis so as to replace the pH=5 buffer with the conjugation buffer (Nikula, T.K. et al. Nucl. Med. Biol. 22:387-390, 1995).
Conditions that yield optimal chelator to antibody ratio but still remain immunoreactive are identified by systematically varying the chelator:antibody ratio, reaction time, temperature, and/or buffer systems about initial conditions that employ a 40-fold molar excess of chelator to antibody in HEPES buffer, pH 8.5. The number of chelates bound per antibody is determined using an established spectrophotometric method (Pippin, C.G. et al. Bioconjugate Chemistiy 3: 342-345, 1992).
For 90Y and 225AC constructs, labeling efficiency is measured directly. For 213Bi, initial antibody constructs are tested for chelation efficiency using 111m which has similar chelation chemistry as 213Bi but possesses the advantages of a longer half life (ti/2=3 days), ready availability, and traceable 7-emission. Once optimized using 1111n, labeling efficiency is determined for 213Bi.
Radiolabeled mAb is purified over a BioRad 10DG desalting column using 1% HSA
as the mobile phase and evaluated by instant thin layer liquid chromatography (ITLC) and/or high performance liquid chromatography (HPLC) to determine the percent incorporation of radionuclide (Zamora, P.O. et al. Biotechniques 16: 306-311, 1994). ITLC and HPLC
provide a means of establishing purity and identifying the percent of low molecular weight radiochemical impurities (i.e., metal chelates, colloids, and free metal).
Duplicate ITLC
strips for each mobile phase are developed, dried, and cut at the Rf of 0.5 mark and counted in a gamma counter. The HPLC system is equipped with both an online UV
absorption detector and radioactivity detector. The HPLC elution profile directly correlates radioactivity with protein and low molecular weight species as a function of the elution time. A TSK
SW3000xL column (TosoHaas, Montgomeryville, PA) is used and calibrated using a range of protein molecular weight standards.
- 71 -Example 11: Affinity and immunoreactivity of radiolabeled mAbs Once radiolabeled constructs are obtained, purified, and assessed for biochemical and radiochemical purity, biological activity is determined. Binding activity of the radioconstruct is performed by Scatchard analysis of binding data obtained using whole LNCaP
and 3T3-PSMA cells and/or membrane fractions as previously described (Scheinberg, D.A.
et al.
Leukemia 3: 440-445 (1991).
The immunoreactivity of the synthetic constructs is evaluated in order to correlate the chelate:antibody molar ratio with the biological activity. Briefly, 2 ng of labeled mAb is incubated with a ¨25-fold excess of PSMA as expressed on 3T3-PSMA cells. After a 30 min incubation at 0 C, the cells are collected by centrifugation and the supernatant containing unbound mAb is added to fresh 3T3-PSMA cells for an additional 30 min at 0 C.
Both sets of cells are centrifuged and washed twice with cold PBS. The cell pellets, supernatant and wash fractions are counted for radioactivity. Immunoreactivity is defined as the amount of radioactivity in the cell pellets divided by the total radioactivity in the cell pellets, supernatant and wash fractions.
Example 12: mAb internalization The activity of radiolabeled mAbs can be significantly modulated by their internalization rates. Internalization of the cell surface antibody-antigen complex is measured using Win radiolabeled antibody constructs (Caron, P.C. et al. Cancer Res 52:
6761-6767, 1992). Briefly, 5x105 3T3-PSMA cells are incubated at 37 C with 111In radiolabeled antibody for varying time periods. Cells are washed with PBS and cell-surface bound radiolabeled constructs are stripped with lml of 50mM glycine/150mM NaC1, pH=2.8. Total cell-associated radioactivity and acid-resistant (internalized) radioactivity are determined by y-counting to ascertain the rate of internalization. Parental 3T3 cells that do not express PSMA are used as a control to determine non-specific binding. Based upon previous results by other groups (Smith-Jones P.M. et al. Cancer Res 60: 5237-5243, 2000), significant internalization of PSMA after binding with one or more of the mAb constructs is expected.
Example 13: In vitro cytotoxicity studies
- 72 -Assessment of in vitro cytotoxicity of a-labeled inAbs was undertaken once the inununoreactivity of the radioimmunoconjugate was established. Approximately 50,000 target cells (either LNCaP or 3T3-PSMA cells) were treated in 96 well plates and analyzed 24-96 hours later. Quantification of cell death due to 225Ac-labeled constructs (or 213Bi) was accomplished by determining the uptake of3H-thymidine by surviving cells (Nikula, T.K. et al. J. NucL Med. 40: 166-176, 1999). Specificity was determined by use of control cells (PSMA-negative human prostate cell lines PC-3 and DU-145, as well as control 3T3 cells), blocking with excess unlabeled antibody, and control radioconjugates.
The cytotoxic effects of antibody conjugate concentration, specific activity, and time of exposure were then assessed. Cytotoxicity was expressed relative to that seen with 1M
HC1 (100% cell death) and media (background cell death). LD50 values were calculated by plotting cell viability as a function of the number of225Ac atoms bound on the cells (McDevitt, M.R. et al. (1998) Eur. NucL Med. 25: 1341-1351 (1998).
Multicellular spheroids of LNCaP-FGC cells had been established and were used to investigate the potential of radioimmunotherapy (RIT) to eradicate minimal disease in vitro.
These three-dimensional spheroids mimic tissue structures more accurately than monolayer cultures and thus provide a more relevant model of solid tumors (O'Connor, K.C. Pharm.
Res. 16: 486-493, 1999). LNCaP-FGC is a fast growing clone of the original LNCaP cell line, and the cells were grown using a liquid overlay technique to a size of 200-6001_tm (Ballangrud, A.M. et al. ain. Cancer Res. 5: 3171s-3176s, 1999). In larger spheroids, the inner mass of cells becomes necrotic, while the outer rim consists of proliferating tumor cells.
Antibody penetration was measured by confocal microscopy, and prior results suggested that an anti-PSMA antibody should penetrate to a depth of 40-501.un (Ballangrud, A.M. et al. 7th Conference on Radioimmunodetection and Radioimmunotherapy of Cancer, Princeton NJ, 1998). The in vitro cytotoxicity of225Ac-3.9 on LNCaP target cells is shown in Figure 23.
The percentage of viable PSMA+ LNCaP cells was plotted as a function of activity of the radioconjugate. Addition of a 100-fold excess of unlabeled antibody was used as a control for specificity.
Example 14: Evaluation of the in vivo efficacy of unlabeled and radiolabeled mAbs in mouse xenograft models of human prostate cancer
- 73 -Antibodies that are successful in the foregoing assays demonstrate significant specificity and functional properties that suggest they will be useful for therapeutic use. The most promising of these radiolabeled and "naked" mAb constructs are evaluated in the best available mouse models of prostate cancer. The studies employ an established xenograft model in which the LNCaP human prostate tumor cell line is injected into immunocompromised nude mice and allowed to form solid tumors (Ellis, W.J. et al. Clin Cancer Res 2: 1039-1048 (1996), which then are treated with both radiolabeled and unlabeled anti-PSMA mAb constructs. Follow-on studies also utilize a mouse xenograft model, CWR22, which reproduces many of the key biological features of human prostate cancer.
LNCaP tumor cell xenograft model A construct showing high affinity and high specificity is taken into the LNCaP
tumor cell xenograft in vivo model for biodistribution and pharmacokinetic analysis.
1111n-labeled anti-PSMA antibody is used for these studies due to its favorable chelation chemistry, radioactive half-life and traceable gamma emission. Timepoints are evaluated as appropriate for the half-lives of213Bi, , 225 c 177 a Lu and 90Y, which are the nuclides of therapeutic interest.
Labeled radioconstructs (1-5m) are injected i.v. into nude mice (normal and tumor bearing) and the mice are sacrificed at 5 mm, 15 min, 30 mm, 60 mm, 2 hrs, 4 hrs, 18hrs, and 24hrs post-injection. Blood and major organs are taken from animals, weighed, and the percent radioactivity injected per gram of tissue is determined (Nikula, T.K. et al.
J. Nucl. Med. 40:
166-176, 1999). Specificity is addressed by pre-injection with excess unlabeled construct.
Macroscopic tumor volume and animal survival rates is recorded throughout the experiments.
A dose-ranging study is also conducted to determine the toxicity of the constructs when administered via i.v. or i.p. injection to normal and tumor-bearing mice.
These animals are routinely examined for toxic side effects during the course of the studies by blood chemistry and physical examination. Animals are sacrificed during and at the conclusion of the study in order to collect blood and body tissues for further evaluation.
Previous data has demonstrated an approximate maximum tolerated dose of 250 Ci/mouse, so total doses are kept below that level.
Once i.v. biodistribution and toxicity is documented, radiotherapy of tumors is assessed. Groups of five mice are injected with <111g radiolabeled anti-PSMA
mAb construct both pre- and post-tumor challenge to assess anti-tumor activity.
Antigen negative
- 74 -(RAJI or RAMOS) xenografted tumors are also used as a control. Other controls include (1) treatment with unlabeled anti-PSMA mAb only and (2) excess unlabeled anti-PSMA
mAb pretreatment before 213Bi, 225Ac, 177Lu and/or 90Y-labeled anti-PSMA to block specific targeting.
Groups of tumor bearing mice are injected with unlabeled anti-PSMA mAbs (at equimolar concentrations) and several dose levels of radiolabeled anti-PSMA or a similarly labeled isotype control antibody. The effect on tumor growth is assessed over time.
Statistical differences between therapy groups is determined using an analysis of variance (ANOVA) method and animal survival is illustrated using Kaplan-Meier plots.
The efficacy of 213Bi, 225Ac, 177Lu and/or 90Y-labeled anti-PSMA constructs is correlated to the data obtained in vitro. Success in these experiments is defined as the ability to significantly (p <0.05) increase life-span and/or decrease tumor volume as compared to a radiolabeled isotype control mAb.
Furthermore, the tumor models are used to test whether predosing with unlabeled antibody prior to injection of radiolabeled antibody improves delivery of the radiolabeled antibody to the tumor. The tumor-bearing mice are injected with <1 pg radiolabeled anti-PSMA antibody with or without a prior single injection of 5-100pg of unlabeled antibody.
After several days, animals are sacrificed for evaluation of the distribution of radioactivity in the tumor, normal tissue, and blood. If predosing with unlabeled antibody improves delivery and targeting of radiolabeled antibody to the tumors, this approach is applied and optimized in toxicity and therapeutic studies.
In addition to overall survival, the role of timing of the injection after tumor transplantation (Day 1 vs 3 vs 7), the role of dosage (dose-response curves using 3-4 dose levels), the role of schedule (single vs multiple divided daily injections) and the specificity of the treatment (pre-treatment with unlabeled anti-PSMA to block targeting) is examined.
These in vivo studies are designed to address the maximum tolerated dose of radiolabeled antibody, the activity of the antibody, the optimal dosing schedule (single or multiple injections), and the effect on tumor size. Successful completion of this work enables determination of the feasibility of PSMA-targeted alpha particle radioimmunotherapy GRIT) of prostate cancer and identifies the optimal 213Bi and/or 225Ac-labeled constructs to enter into clinical development.
-75 -CWR22 mouse xenograft model The most promising anti-PSMA mAbs in unlabeled, toxin-labeled and/or radiolabeled form are tested in the CWR22 human prostate cancer xenograft mouse model, (Wainstein, M.A. et al. Cancer Res 54:6049-6052 (1994); Nagabhushan, M. et al. Cancer Res 56:3042-3046 (1996); Pretlow, T.G. et al. J Natl Cancer Inst 85:394-398 (1993)). This model has many features of the human condition including a dependence on androgens, a correlation between measured levels of PSA in serum and tumor size, and high-level expression of PSMA. Following androgen withdrawal, PSA levels decrease to nearly undetectable levels and tumor volume decreases. Later, the tumor regrows as an androgen-independent neoplasm, manifest initially by a rise in PSA and later, measurable tumor growth. After androgen withdrawal, tumors regrow at variable time periods.
Four to six week old nude athymic BALB/c male mice are obtained from the National Cancer Institute-Frederick Cancer Center and maintained in pressurized ventilated caging.
While immunodeficient in many respects, these mice mediate wild-type levels of ADCC and CML. The CWR22 tumor line is propagated in the animals by the injection of minced tumor tissue from an established tumor into the subcutaneous tissue of the flanks of athymic nude mice together with reconstituted basement membrane (Matrigel, Collaborative Research, Bedford, MA). To maintain serum androgen levels, the mice are administered 12.5-mg sustained-release testosterone pellets (Innovative Research of America, Sarasota, FL) subcutaneously before receiving tumors. Three to four weeks after inoculation, tumors of approximately 1.5 x 1.0 x 1.0 cm are measured. Androgens are withdrawn by surgical castration under pentobarbital anesthesia and removal of the sustained-release testosterone pellets. Tumor size is determined by caliper measurements of height, width and depth. PSA
values are performed on the serum of the mice after tail bleeding using a Tandem-R PSA
immuno-radiometric assay (Hybritech, San Diego, CA).
Groups of five mice are injected with anti-PSMA mAb or a similar isotype control mAb at dosages from 5-100 g to assess anti-tumor activity. The effect of scheduling single doses vs. multiple divided daily injections is also examined. Macroscopic tumor volume and animal survival rates are recorded throughout the experiments. Statistical differences between therapy groups are determined using an analysis of variance (ANOVA) method and animal survival are illustrated using Kaplan-Meier plots, with success defined as a difference
- 76 -of p<0.05. Similarly, the efficacy of "naked" mAbs is compared to that seen with 90Y, 177Lu, 213Bi and/or 225Ac-labeled anti-PSMA constructs.
These in vivo studies are designed to address the maximum tolerated dose of mAb, the activity of the antibody, the optimal dosage and dosing schedule (single or multiple divided injections), and the effect of treatment on tumor size. Successful completion of this work will enable determination of the feasibility of PSMA-targeted immunotherapy of prostate cancer and identification of the optimal constructs to enter into clinical development.
Example 15: Investigation Of Native PSMA Protein Conformation Extraction of PSMA from the cell surface of LNCaP and 3T3 cells LNCaP or 3T3 cells were grown to confluency in a T150 cell culture flask, detached using cell dissociation solution (Mediatech, Herndon, VA) and transferred to a 15m1 conical tube. The cells were washed twice with PBS and resuspended with 2m1 of MPerTM
Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Following incubation for 10 min at 4 C, cell debris and insoluble aggregates were removed by centrifugation at 15,000 rpm for 30 min at 4 C. The supernatant was transferred to a cryogenic vial and stored at -80 C until further use.
Production and Purification of Recombinant, Soluble PSMA (rsPSMA) The extracellular domain of PSMA (amino acids 44-750 of the full-length protein, SEQ ID NO:1) was obtained as a secreted protein from a DXB11 Chinese hamster ovary (CHO) cell line, stably transfected with an rsPSMA expression vector. The cells were gown in a Celligen Plus 2.2L Packed Bed Bioreactor (New Brunswick Scientific, Edison, NJ) in protein-free media. The Bioreactor was operated in perfusion mode, and supernatant was collected aseptically into collection bags maintained at 4 C. The protease inhibitor aprotinin was added to the harvest supernatant, which was concentrated 25-fold prior to storage at -90 C. For purification, the concentrate was thawed and purified using subsequent steps of Concanavalin A lectin affinity chromatography and Butyl-Sepharose hydrophobic interaction chromatography. The purified protein was dialyzed against 10mM potassium phosphate, pH
7Ø The purified rsPSMA protein is dimeric, and possesses folate hydrolase enzymatic activity when tested according to published procedures (Pinto et al., Clinical Cancer
- 77 -Research 2:1445, 1996) and reacts with each of a panel of conformation-specific monoclonal antibodies, indicating that rsPSMA adopts a native conformation.
Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting of the different PSMA
proteins For each individual PAGE analysis, 15111 of each cell lysate and 51.11 of the purified rsPSMA were used.
SDS-PAGE was performed using standard procedures. Samples were prepared by boiling for 5 minutes in the presence of Laemmli sample buffer (with or without the reducing agent dithiothreitol [DTT]). Samples were then applied on a 4-15% Tris-Glycine gel (BioRad, Hercules, CA). After electrophoresis for lh at 200V, the proteins were transferred onto nitrocellulose (BioRad) and analyzed by Western blotting.
The oligomeric nature of the different PSMA proteins was analyzed using Blue Native PAGE (BN-PAGE). Each sample was diluted with an equal volume of 2x BN-PAGE
sample buffer (0.1M MOPS! 0.1M Tris /40% glycerol! 0.1% Coomassie G-250) prior to loading onto the gel. BN-PAGE was performed using 4-12% BisTris gels (Invitrogen, Carlsbad, CA) and 50m_M MOPS / 50mM Tris, pH 7.7 as running buffer. Coomassie Blue was omitted from the cathode buffer to avoid interference with protein binding during the transfer of the proteins onto nitrocellulose. Following electrophoresis for 2.5hrs at 125V, the proteins were transferred onto a nitrocellulose membrane (BioRad) and analyzed by Western blotting.
Western blotting was performed as follows: Subsequent to transfer, the nitrocellulose membrane was blocked with 5% milk in PBS / 0.1% Triton X-100 / 0.02% SDS, which was also used for the subsequent wash and antibody incubation steps. PSMA proteins were detected using the anti-PSMA mAbs 3.1 or 3.9 (Progenics Pharmaceuticals) as primary antibody and HRP-labeled anti-mouse IgG as secondary antibody and lh incubation at room temperature. The membranes were colorimetrically developed using chemiluminescence (NEN Plus, Perkin Elmer Life Sciences, Boston, MA).
Results Both full-length PSMA and recombinant, soluble PSMA (rsPSMA) migrate on reducing and non-reducing SDS-PAGE with a molecular weight of ¨100 kDa (Fig.
5). The
- 78 -result for full-length PSMA is in accordance with prior observations (Israeli et al., U.S. Patent 5,538,866; Murphy et al., U.S. Patent 6,158,508; Israeli, et al., Cancer Research 54:1807, 1994; Troyer et al. Int. J. Cancer 62:552, 1995; Troyer et al., The Prostate 30:233, 1997;
Grauer et al., Cancer Research 58:4787, 1998). In each of these reports, full-length PSMA
migrated as a major band of 100-120 kDa, with a minor (typically <5% of the total PSMA
protein) 180-200 kDa band observed in a subset of reports (U.S. Patent 6,158,508; Troyer et al., 1995; Troyer et al., 1997). Troyer et al. (1995) describe the 180-200 kDa species as being a noncovalently associated PSMA dimer that can be disrupted with increasing concentrations of SDS detergent.
rsPSMA contains 94% (707 of 750) of the amino acids present in full-length PSMA, and the two proteins are not clearly resolved in this analysis, as expected.
SDS-PAGE allows the analysis of denatured proteins only. In order to examine native proteins in their native state, other techniques have to be employed, such as Blue Native PAGE (BN-PAGE). BN-PAGE is used to determine the native molecular weight of proteins and their noncovalent complexes (Schagger & v. Jagow, Anal. Biochem.
199:223-231, 1991; Schagger et al., Anal. Biochenz. 217:220-230, 1994). The dye Coomassie Blue G-250 binds to the hydrophobic domains on the surface of most proteins, enhances solubility, and introduces a charge shift on the native proteins resulting in migration towards the anode at pH 7.5 irrespective of the isoelectic point of the protein. Although the migration velocity of proteins in BN-PAGE varies somewhat, the molecular mass of proteins can be determined by their respective end points of migration due to the decreasing pore size of the acrylamide gradient present in the gels.
When analyzed by BN-PAGE, full-length PSMA (extracted from LNCaP or 3T3 cells) as well as purified rsPSMA migrate with a molecular weight of ¨190kDa (Fig. 6). This surprising observation for full-length PSMA indicates that the predominant form of cell-surface PSMA is a noncovalently associated dimer. This unexpected result can be contrasted with that of previous reports (U.S. Patent 6,158,508; Troyer et al. 1995;
Troyer et al., 1997), where the PSMA dimer represents a minor species in SDS-PAGE analyses.
Presumably, the noncovalent PSMA dimer is largely dissociated by boiling in the presence of the denaturing detergent SDS.
Moreover, the result for the purified rsPSMA protein indicates that the dimer is stabilized via interactions between extracellular amino acids in addition to or exclusive of
- 79 -amino acids in the transmembrane or intracellular segments, which are not present in rsPSMA.
Example 16: Dissociation of PSMA Multimers PSMA is a putative zinc metalloprotease, and site-directed mutagenesis of amino acids implicated in zinc binding results in a profound loss of enzymatic activity (Speno et al., Molecular Pharmacology, 55:179, 1999). These amino acids include His-377, Asp-387, Glu-425, Asp-453 and His-553. Ethylenediaminetetraacetic acid (EDTA) is a strong chelating agent for Zn2+ and other divalent cations, and thus has the potential to remove Zn24-or other coordinate divalent cations from PSMA. We have determined that EDTA
treatment causes the PSMA homodimer to dissociate into monomeric subunits. Similar results can be expected for other agents that possess similar chelating properties, such as ethyleneglycol-bis(beta-aminoethyl ether) (EGTA).
The purified rsPSMA protein was incubated with or without 10mIvIEDTA for 16 hr at 4 C and then analyzed by BN-PAGE. Under these conditions, the EDTA-treated protein was monomeric, whereas rsPSMA remained dimeric in the absence of EDTA.
Although the dissociation of the PSMA dimer into monomer was essentially complete, any residual dimeric protein can be removed if desired by gel filtration, ultracentrifugation or other size-based separation methods that are well-known to those skilled in the art.
Example 17: Methods for identifying promoters of PSMA dissociation Compounds are screened for the ability to promote dissociation of PSMA dimers using a method that includes:
(a) contacting a PSMA dimer with a compound under conditions that do not promote dissociation of the PSMA dimer in the absence of the compound;
(b) measuring the amount of PSMA monomer; and (c) comparing the amount of PSMA monomer measured in the presence of the compound with that observed in the absence of the compound.
An increase in the amount of PSMA monomer measured in the presence of the compound indicates that the compound is capable of promoting dissociation of the PSMA
dimer.
- 80 -In a further embodiment, compounds are screened for the ability to promote dissociation of PSMA dimers using a method that includes:
(a) contacting a PSMA dimer with a compound under conditions that do not promote dissociation of the PSMA dimer in the absence of the compound;
(b) measuring the amount of PSMA dimer; and (c) comparing the amount of PSMA dimer measured in the presence of the compound with that observed in the absence of the compound.
A decrease in the amount of PSMA dimer measured in the presence of the compound indicates that the compound is capable of promoting dissociation of the PSMA
dimer.
In a further embodiment, compounds are screened for the ability to promote dissociation of PSMA dimers using a method that includes:
(a) contacting a PSMA dimer with a compound under conditions that do not promote dissociation of the PSMA dimer in the absence of the compound;
(b) measuring the amounts of PSMA monomer and PSMA dimer;
(c) calculating a ratio of PSMA monomer to PSMA dimer; and (d) comparing the ratio obtained in (c) with that obtained in the absence of the compound.
An increase in the ratio measured in the presence of the compound indicates that the compound is capable of promoting dissociation of the PSMA dimer.
Example 18: Cell surface PSMA binding studies Flow cytonietry Parent 3T3 cells or PSMA-expressing 3T3 cells (2 x 105 cells per condition) were washed in PBS and incubated with PBS containing goat serum (10% v/v) for 20 minutes on ice to block non-specific binding sites. Anti-PSMA monoclonal antibodies (unpurified form in supernatants or purified mAbs) were added in serial dilutions to cells in 100111 PBS and incubated on ice for 30 minutes. Control anti-human IgG (Caltag, Burlingame, CA) was used to establish background binding. After two washes in PBS, the cells were incubated with anti-human IgG (BD Pharmingen, San Diego, CA) for 30 minutes on ice. Cells were washed twice in PBS, resuspended in 250g1 PBS and analyzed by flow cytometry using a FACScan machine (Becton Dickinson, Franklin Lakes, NJ) and CellQuest software. Viable cells were
- 81 -gated by forward scatter and side scatter parameters, and binding was quantified using histogram plots of mean fluorescence intensity (MFI) levels.
Anti-PSMA mAbs XG-006 (PTA-4403 and PTA-4404, heavy and light chain plasmids), XG-051 (PTA-4407 and PTA-4408), 4.40.1 (PTA-4360; 4.40, 4.40.1 and 4.40.2 are the same antibody that represent different stages of subcloning the hybridoma), 4.49.1, 4.292.1 (PTA-4390) and 4.304.1 were found to avidly bind to cell surface PSMA
(Figure 24).
Maximal Binding Flow cytometry data (mean fluorescence intensity v. antibody concentration) were transposed and plotted using Excel software (Microsoft, Redmond, WA). Results from representative experiments of at least three determinations are depicted in Figures 25A-25C.
Binding was compared by calculation of 50% effective concentration (EC50) using the Forecast function in Excel. The EC50 value represents the concentration of antibody required for half-maximal binding.
Anti-PSMA mAbs 10.3 (PSMA 10.3) and XG-006 were found to bind to 3T3-PSMA
cells and not 3T3 cells (Figure 25A). Antibody (26nM) was added to cells, which were analyzed by flow cytometry. Binding to cell-surface PSMA using serial dilutions of anti-PSMA mAb-containing culture supernatants of XG-006, 4.304.1, XG-026 (PTA-4405 and PTA-4406) and 4.49.1 also was demonstrated (Figure 25B). Binding to cell-surface PSMA
using serial dilutions of purified anti-PSMA mAbs XG-006 and 10.3 is represented by Figure 25C.
Example 19: Cvtotoxicity of toxin-labeled antibody PSMA-3T3, LNCaP, and/or C4-2 cells (and control cell lines 3T3 and PC3 that do not express PSMA) were plated at 2,500 cells/100 pL/well in 96-well microplates (Falcon) and were incubated overnight at 37 C in the presence of 5% CO2. The media used for PSMA-3T3 (and 3T3) and LNCaP (and C4-2 and PC3) was DMEM or RMPI 1640, respectively, containing 2 mM L-glutamine, 10% FBS, and 1% penicillin-streptomycin. 50 ng (in 50 pi) of Mab-Zap or Hum-ZAP (Advanced Targeting Systems, San Diego, CA) in medium was added in each well. Mab-Zap and Hum-Zap are goat anti-mouse IgG antibody or goat anti-human IgG antibody covalently linked to saporin, the most potent of the plant ribosome-
- 82 -inactivating proteins (RIP) from the seeds of the plant Saponaria officinalis.
Saporin induces cell death by apoptosis (Bergamaschi, G., Perfetti, V., Tonon, L., Novella, A., Lucotti, C., Danova, M., Glennie, M.J., Merlini, G.,Cazzola, M. Saporin, a ribosome-inactivating protein used to prepare immunotoxins, induces cell death via apoptosis. Br J Haematol 93, 789-94.
(1996)). The Mab-Zap did not bind to or internalize in cells in the absence of an appropriate primary antibody.
Murine 3.9, 5.4, mJ591 (ATCC# HB-12126) and human 006,4.40, 4.304 anti-PSMA
antibodies (and control IgG antibodies) were added into plates at different concentrations to bring the total volume to 200 pi, in triplicate. The plates were kept cold on ice for at least 30 Min to maximize Map-Zap or Hum-Zap binding to PSMA antibodies before internalization.
The plates were incubated for 2 days and then the medium was changed and incubated for another 2 days. After 4 days incubation, the medium was withdrawn and fresh medium containing 10 % Alamar Blue (20 IaL, Bioscience, Camarillo, CA) was added into each well and incubated for 2 hrs. A CytoFlour plate reader was used to measure fluorescence in 96-well plates at wavelengths of 530 nm excitation and 590 nm emission.
Internalization of toxin was mediated by anti-PSMA antibodies. The cell kill is illustrated in Figure 26 on C4-2 cells and in Figure 27 on PSMA-3T3 cells.
Human 4.304 anti-PSMA antibody was directly conjugated with saporin (Wrenn et al., Brain Res. 740:175-184, 1996), and its cytotoxicity was demonstrated using a similar protocol as described above (see Figure 28).
Example 20: Immunoreactivity PSMA-3T3, LNCaP and C4-2 were used as PSMA expressing cell lines and 3T3 was used as a control cell line not expressing PSMA. The cells were blocked with 10% goat serum on ice to reduce non-specific binding in this assay.
A small amount (1-5 ng) of labeled mAb was added into a cell pellet of 10 million cells and incubated at 0 C (on ice) with gentle mixing. After a 1 hour incubation, the cells were collected by centrifugation and the supernatant containing unbound mAb was transferred to a fresh cell pellet for an additional 1 hour incubation at 0 C.
Both sets of cells were centrifuged and washed twice with cold PBS. The cell pellets, supernatant and wash fractions were counted for radioactivity. hnmunoreactivity is defined as the amount of
- 83 -radioactivity in the cell pellets divided by the total radioactivity in the cell pellets, supernatant and wash fractions. These data are shown below in Table 3.
Table 3. Immunoreactivity of In-111 radiolabeled antibody on PSMA expressing cells Radiolabeled rnAb Immunoreactivity (%) Cell line In-111 4.304 92.6 (1.4) PSMA-3T3 (3T3) 92.6 PSMA-3T3 91.4 (1.7) PSMA-3T3 (3T3) 89.1 LNCaP
92.4 C4-2 Average= 91.6 1.5 In-111 4.40 87.7 (0.5) PSMA-3T3 (3T3) 86.8 PSMA-3T3 89.4 (1.5) PSMA-3T3 (3T3) Average- 88.0 1.3 In-111 mJ591 58.5 PSMA-3T3 54.9 (1.1) PSMA-3T3 (3T3) Average= 56.7 2.5 In-111 3.9 88 LNCaP

89 (2) PSMA-3T3 (3T3) 95.3 (0.5) PSMA-3T3 (3T3) 88.6 PSMA-3T3
84.8 C4-2 89.3 PSMA-3T3 Average= 88.6 3.2 Antibodies 4.40, 4.304 and mJ591 were conjugated to the bifunctional chelate CHX-A"-DTPA and antibody 3.9 was conjugated to C-DOTA.

Example 21: Competitive binding assay to identify binding epitopes To identify whether a given group of mAbs recognize distinct or overlapping epitopes on PSMA, competition binding assays were performed with In-111 radiolabeled antibodies.
2 x 105 cells (100 L) of PSMA-3T3 were plated into 96-well microplates, and antibodies 4.40, 4.304 and mJ591 (100 L) at different concentrations (series dilution) were added. The cells were incubated at 0 C for 30 mm. 20 I, of In-111 radiolabeled CHX-A"-DTPA
antibody constructs were added into each well. After a 2 hour incubation on ice for competition binding, the cells were washed 5 times using cold PBS. The cells containing bound In-111 antibodies were recovered from microplates into test tubes and counted in a gamma counter.
Results detailed in Figures 29 show that mJ591 blocked In-111 4.40 binding to PSMA-3T3 cells and did not block In-111 4.304. In addition, 4.40 and 4.304 did not block each other. Unmodified antibodies 4.304 and mJ591 were also used to compete with In-111 radiolabeled mJ591. Human 4.304 did not compete with In-111 mJ591 for binding to PSMA-3T3 (Figure 30).
Example 22: Binding affinity using Biacore 3000 To determine the kinetics and affinity of the antibodies, the antibodies in crude supernatants, in purified form and in bifunctional chelate modified forms were analyzed using a Biacore 3000 instrument (Biacore Inc., Piscataway, NJ). Biacore 3000 is a fully automated surface plasmon resonance (SPR)-based biosensor system that is designed to provide real-time kinetic data from assay formats that require no tags or labeling of compounds for biomolecular interactions. It is ideal for screening crude supernatants.
The streptavidin-coated sensor chips (SA chips, Biacore) were used to capture biotinylated anti-human IgG antibody (Sigma, St. Louis, MO). The entire sensor chip surface was conditioned with five injections of conditioning solution (1 M NaC1, 50mM
NaOH) and equilibrated with PBS buffer containing 0.005% polysorbate 20. Two to three thousand resonance units (RU) of biotinylated anti-human IgG antibody (Sigma) were immobilized onto the SA chip followed by an injection of regeneration buffer (glycine-HC1, pH 2.2).
Antibodies in supernatants were diluted to 2 pg/mL in PBS buffer and captured onto one anti-human IgG flow cell, while isotype-matched control human antibody (Sigma) was similarly captured on a second flow cell. rsPSMA at different concentrations in PBS
buffer was
- 85 -flowed over the cells at 30 pL/min for 3 min in an "association phase"
followed by a "dissociation phase" for 10 min. SPR was monitored and displayed as a function of time.
For each antibody at one concentration, the chip was regenerated and equilibrated. An example of the analysis of antibody PRGX1-XG-006 in association phase and dissociation phase at different concentrations of rsPSMA from 100 nM to 6.25 nM is shown in Figure 31.
Thermodynamic and kinetic rate constants of binding were calculated using the Biacore Evaluation software. For example, the affinity of XG-006 antibodies in a supernatant to rsPSMA was determined to be 4.92x10-1 M with a Ka of 1.3x105 M-1 s-1 and a Kd of 6.4x10-5 s-1. Selective data for several human PSMA antibodies in crude supernatant, purified form, and modified with bifunctional chelate is listed in Table 4 for comparison.
Binding activity of In-111 radiolabeled antibodies was determined by Scatchard analysis of binding data obtained using PSMA-expressing cells (LNCaP, C4.2, and parental 3T3 as a control). The experimental procedures and methods of data analysis have been described previously (Scheinberg, D.A. et al. Leukemia 3: 440-445 (1991).
Table 4: Kinetic rate constants of antibodies in crude supernatant, purified, bifunctional chelate modified forms along with KD determined using 1n-111 radiolabeled Scatchard analysis.

= 64371-608
- 86 -Antibodies Ka (M-11s4) Kd (e) KD(M) Avg KD
006 Supernatant 1.30E+05 6.40E-05 4.92E-10 4.92E-Purified 006-1 2.94E+05 1.37E-04 4.66E-10 Purified 006-2 2.26E+05 1.27E-04 5.62E-10 5.14E-4.40 Supematant 2.10E+05 1.25E-04 5.95E-10 5.95E-Purified 4.40-1 2.54E+05 1.52E-04 5.98E-10 Purified 4.40-2 2.43E+05 2.37E-04 9.75E-10 7.87E-CHX-4.40-1 2.57E+05 1.60E-04 6.23E-10 CHX-4.40-2 2.47E+05 1.55E-04 6.28E-10 6.25E-IN-111CHX-4.40-1 4.44E-09 IN-IllCHX-4.40-2 4.95E-09 4.70E-09 4.304 Supernatant 1.40E+05 1.25E-04 8.93E-10 8.93E-Purified 4.304-1 8.31E+04 1.20E-04 1.44E-09 Purified 4.304-2 1.06E+05 6.33E-05 5.97E-10 1.02E-CHX-4.304-1 6.19E+04 1.21E-04 1.95E-09 CHX-4.304-2 6.79E+04 1.49E-04 2.19E-09 2.07E-IN-111CHX-4.304-1 9.63E-09 IN-IllCHX-4.304-2 5.97E-09 7.80E-09 10.3 Supematant 1.90E+05 3.63E-04 1.91E-09 1.91E-Purified 10.3-1 3.28E+05 6.32E-05 1.93E-10 Purified 10.3-2 2.96E+05 6.43E-05 2.17E-10 2.05E-A comparison of the fully human antibodies 4.40.1,4.49.1, 051 and 006 and the =
murine antibody 3.9 was performed by Biacore. For each antibody for comparison, response was normalized to 100 RU. The graph of time vs. response difference for these antibodies is given in Figure 32. The binding affinities for these antibodies were determined to be 6.1, 6.7, 5.8, 4.8 and 13.7x10-10M, respectively.
Example 23: Characterization of cell lines for in vitro and in vivo studies Results from a Scatchard analysis using In-111 labeled anti-PSMA antibody 3.9 are represented in Figure 33. Transfected murine 3T3 cells express >1 million copies of PSMA
per cell, LNCAP cells (androgen dependent human prostate cancer cell line) express 0.64 million copies, while C4-2 cells (androgen independent) express 0.25 million copies per cell.
The affinity of 3.9 for cell surface PSMA is 6.4 nM for PSMA-3T3, 4.0 nM for LNCAP and 3.3 nM for C4-2 (4.6 nM is the average of these data).
A summary of the analyses of crude supernatants for the human anti-PSMA
=
antibodies is given in Table 5 below.
- 87 -Table 5: Characterization of anti-PSMA monoclonal antibodies Ab Conc Binding to 313- Biacore studies (pg/mL) PSMA (FACS) Supernatant PGNX Lysate PGNX AVG AVG C4.2 Anti-PSMA KD, M-1 Ka, Kd, s-1 EIA FACS Max EC50 FACS Western (x 1040) M-1s-1 ( x 10-5) binding ( x 105) PRGX1-XG1- 4.7 ND 1 ND 148 2.4 ND ' Conf.2 2.0 1.5 2.9 4.4.1 4.7 0.08 7 8 ND 5.2 ' Conf.
4.2 2.3 9.7 PRGX1-X61- 1.8 0.39 114 183 3.4 9.5 Conf. 4.8 1.3 6.4 PRGX1-XG1- 3.5 0.48 83 202 2.0 9.9 Conf. 5.8 1.4 8.2 4.40.1 , 4.3 0.33 53 163 2.3 10.8 Conf. 6.1 2.1 12.5 4.49.1 2.6 0.36 362 162 0.9 16.2 Conf. 6.7 3.1 20.7 4.292.1 2.7 0.18 75 195 6.0 9.2 Conf. 6.8 1.2 8.5 4.304.1 4.1 0.39 92 184 9.1 8.4 Conf. 8.7 1.4 12.5 4.232.1 2.4 0.49 97 .138 2.7 6.0 Linear3 9.4 1.5 13.8 4.153.1 5.9 0.29 279 182 5.3 14.8 Conf. 9.5 1.2 11.8 4.333.1 2.9 0.18 82 168 3.1 6.6 Conf. 11 0.7 8.5 PRGX1-XG1- 3.9 0.45 392 227 6.0 12.4 Conf. 16 0.6 10.4 ' 4 10.3 8.5 1.06 ND ND ND ' ND ND ' 19 1.9 36.4 .
pure 10.3 0.44 130 181 7.5 ND Conf. ND
4.7 4.22.1 2.8 0.08 7 ND ND 4.7 ND
20 1.7 33 .
4.248.1 3.5 0.37 7 ND ND 4.1 Conf. 27 1.0 28 4.54.1 10 0.14 267 162 3.9 13.6 ND 30 1.9 56*
4.7.1 5 0.23 156 141 1.6 10.2 Conf. 32 1.7 56 4.78.1 5.3 0.00 205 118 1.0 7.9 Conf. 53 2.4 125 4.48.1 - 4.9 0.06 14 ND ND 7.7 ND 62 0.9 59 4.209.1 3.5 0.22 60 ND ND 6.7 ND 142 0.9 125 4.177.1 - 1.1 0.15 236 174 2.4 10.6 ND 155 0.6 93 4.152.1 3.4 0.38 81 85 4.0 7.5 ND 163 0.8 126 4.28.1 - 4.2 0.04 112 155 4.2 11.3 ND 167 1.2 192 4.16.1 5.3 0.00 8 ND ND 7.8 ND 177 1.8 313 4.360.1 1.5 0.02 112 130 2.2 7.9 ND 197 1.0 201 4.288.1 - 15.4 0.02 67 141 4.1 6.5 ND 198 1.3 257 _ ___________________________________________________________________________ 4.219.2 0.5 0.34 69 ND ND 5.9 ND ND
_ ___________________________________________________________________________ PRGX1-XG1- 6.5 ND ND 71 7.9 ND ND No Binding Murine 3.9 13.7 0.7 9.7 - _ Control 6.34 2.24 14.2 1 ND=not determined 2 conf.=confonnational epitope 3 linear=linear epitope
- 88 -Example 24: Cytotoxicity of radiolabeled antibody The in vitro cytotoxicity of Ac-225 labeled anti-PSMA antibody was determined using methodology similar to that used in Example 19. Prostate cancer cells (1001.11, of C4-2, LNCaP, and PC3 cells at a concentration of 2 x 104 cells/mL) were placed into separate well of a 96 well microplate. After overnight incubation, the cells were treated with Ac-225 labeled human anti-PSMA 4.40 antibody at different concentrations for over 4 days. Cell cytotoxicity was quantified using Alamar Blue (Biosource International, Camarillo, CA).
Figure 34 shows a plot of cell survival vs. Ac-225 activity concentration. The for PSMA expressing cells (C4-2 and LNCaP) was <2 nCi/mL. However, the EC50 was 420 nCi/mL for PC3 cells, which do not express PSMA on the cell surface.
Therefore, the Ac-225 labeled human anti-PSMA 4.40 antibody shows > 200-fold selectivity in killing PSMA
expressing prostate cancer cells (C4-2 and LNCaP) vs. control cells (PC3).
Example 25: In Vivo Radioimmunotherapv with Lu-177 Labeled Antibodies Athymic nude mice from the National Cancer Institute were implanted subcutaneously with 2 x 106 PSMA-3T3 cells. After measurable tumors appeared at day 7 post implantation, the mice were treated by injection with either a single 250 uCi dose human anti-PSMA antibody 4.40 or 4.304 labeled with Lu-177 (University of Missouri Research Reactor), or were injected with buffer only as control. The tumor size of individual animals was measured using an electronic caliper. Figure 35 shows a plot of the median tumor size in each group overtime. Tumor growths were substantially reduced in Lu-177 antibody treated groups compared to the control group.
Example 26: Immunization with rsPSMA dimer preparations Immunization BALB/c mice were immunized by subcutaneous injection at days 0, 7, 14, and 42 with either 5 ps clinical rsPSMA lot # 4019-0001 (75 % dimer/25 % monomer) or 5 lig rsPSMA batch # TD045-003 run 1/peak 2 (100 % monomer) and adjuvanted with 50 lig alhydrogel per dose. Serum was drawn 10 days after the fourth immunization and analyzed by enzyme-linked immunoassay (ETA) and flow cytometry.
- 89 -EIA
rsPSMA lot # 4019-0001 or rsPSMA batch # TD045-003 run 1/peak 2 was passively adsorbed to 96-well microtiter plates. Remaining binding sites on the plate were blocked with a PBS/Casein/Tween 20 buffer. Serially diluted mouse serum or controls were added and bound antibody was detected using a goat anti-mouse IgG antibody conjugated to alkaline phosphatase. The ETA was developed with the substrate pNPP which produces a color change that is directly proportional to the amount of anti-PSMA antibody bound.
Absorbance was read at 405 nm with a correction of 620 nm. Antibody titer was defined as the highest dilution of mouse serum yielding a blank corrected absorbance of 0.1. Immune mouse serum with a known anti-PSMA titer or normal mouse serum with no anti-PSMA
reactivity was used as controls.
Flow Cytonietry Analysis PSMA-3T3 cells were incubated with 200 IA, of immune serum at a dilution of in PBS with 0.1 % sodium azide on ice for 30 minutes. Immune mouse serum with known anti-PSMA titer or normal mouse serum with no anti-PSMA reactivity was used as controls.
The cells were washed twice with PBS with 0.1 % sodium azide and incubated for minutes on ice with FITC-conjugated goat anti-mouse IgG. Cells were washed once, resuspended in PBS with 0.1 % sodium azide and subjected to flow cytometric analysis on FACScaliber (Becton Dickinson).
Results 5/5 mice immunized with rsPSMA lot # 4019-0001 showed an anti-PSMA antibody response by ETA. Antibody titer was similar for assay plates coated with rsPSMA lot # 4019-C001 (75 % dimer/25 % monomer) and assay plates coated with rsPSMA batch #

run 1/peak 2 (100 % monomer). Median response for the group was 1/6400.
4/5 mice immunized with rsPSMA batch # TD045-003 run 1/peak 2 showed an anti-PSMA antibody response by ETA. One mouse was negative. Antibody titer was similar for assay plates coated with rsPSMA lot # 4019-0001 (75 % dimer/25 % monomer) and assay plates coated with rsPSMA batch # TD045-003 run 1/peak 2 (100 % monomer).
Median response for the group was 1/6400.
The results of the ETA analysis are provided in Table 6.
- 90 -Table 6: Specificity of the anti-PSMA antibody response in mice vaccinated 4 times with rsPSMA 5 pig/dose and 50 jig/dose Alhydrogel Mouse ID # Immunogen EIA Titer vs. ETA Titer vs.
Median RFI vs.
Lot 4019-0001 Batch TD045-003 PSMA-3T3 runl/peak 2 cells A1BIM151 4019-0001 Dimer 1/3200 1/3200 84 ABIM152 4019-0001 Dimer 1/3200 1/3200 41 , AUM153 I 4019-0001 Dimer 1/25600 1/25600 76 Ir¨ABIN41 54 I 4019-0001 Dimer 1/12800 1/12800 63 A1311.4155 4019-0001 Dimer 1/6400 1/6400 74 AB14156 Monomer I 1/1600 1/1600 5 ABIM157 Monomer 1/6400 1/12800 8 ABIM158 Monomer 0 0 6 I ABIIVI159 LMonomer 1/6400 1/6400 6 ABIM160 I Monomer 1/6400 I 1/6400 I 12 As shown in Fig. 36, anti-PSMA antibody in the serum of mice immunized with a dimer preparation of rsPSMA (lot # 4019-0001) showed strong binding to PSMA-3T3 cells.
Anti-PSMA antibody in the serum of mice immunized with a 100% monomer preparation of rsPSMA (batch # TD045-003 run 1/peak 2) showed no binding to PSMA-3T3 cells.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose and variations can be made by those skilled in the art without departing from the scope of the invention which is defined by the following claims.

SEQUENCE LISTING
<110> PSMA DEVELOPMENT COMPANY, L.L.C.
MADDON, Paul J.
DONOVAN, Gerald P.
OLSON, William C.
SCHULKE, Norbert GARDNER, Jason MA, Dangshe <120> PSMA ANTIBODIES AND PROTEIN MULTIMERS
<130> P00453.70005.WO
<150> US 60/335,215 <151> 2001-10-23 <150> US 60/362,747 <151> 2001-03-07 <150> US 60/412,618 <151> 2002-09-20 <160> 33 <170> PatentIn version 3.1 <210> 1 <211> 750 <212> PRT
<213> Homo sapiens <400> 1 Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala Val Ala Thr Ala Arg Arg Pro Arg Trp Leu Cys Ala Gly Ala Leu Val Leu Ala Gly Gly Phe Phe Leu Leu Gly Phe Leu Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu Ala Thr Asn Ile Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu Leu Lys Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gin Ile Pro His Leu Ala Gly Thr Glu Gin Asn Phe Gin Leu Ala Lys Gin Ile Gin Ser Gin Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala His Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn Tyr Ile Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn Thr Ser Leu Phe Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val Ser Asp Ile Val Pro Pro Phe Ser Ala Phe Ser Pro Gin Gly Met Pro Glu Gly Asp Leu Val Tyr Val Asn Tyr Ala Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met Lys Ile Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val Phe Arg Gly Asn Lys Val Lys Asn Ala Gin Leu Ala Gly Ala Lys Gly Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val Lys Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val Gin Arg Gly Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu Thr Pro Gly Tyr Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala Glu Ala Val Gly Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala Gln Lys Leu Leu Glu Lys Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg Gly Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn Phe Ser Thr Gin Lys Val Lys Met His Ile His Ser Thr Asn Glu Val Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu Pro Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp Val Phe Gly Gly Ile Asp Pro Gin Ser Gly Ala Ala Val Val His Glu Ile Val Arg Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr Ile Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr Glu Trp Ala Glu Glu Asn Ser Arg Leu Leu Gin Glu Arg Gly Val Ala Tyr Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu Ser Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg Ile Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gin Arg Leu Gly Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn Trp Glu Thr Asn Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu Leu Val Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val Ala Gin Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gin Glu Met Lys Thr Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe Thr Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gin Asp Phe Asp Lys Ser Asn Pro Ile Val Leu Arg Met Met Asn Asp Gin Leu Met Phe Leu Glu Arg Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg His Val Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gin Ile Tyr Val Ala Ala Phe Thr Val Gin Ala Ala Ala Glu Thr Leu Ser Glu Val Ala VIM) 03/034903 <210> 2 <211> 7570 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 2 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 ggtaccaagc ttggatctca ccatggagtt gggactgcgc tggggcttcc tcgttgctct 960 tttaagaggt gtccagtgtc aggtgcaatt ggtggagtct gggggaggcg tggtccagcc 1020 tgggaggtcc ctgagactct cctgtgcagc gtctggattc gccttcagta gatatggcat 1080 gcactgggtc cgccaggctc caggcaaggg gctggagtgg gtggcagtta tatggtatga 1140 tggaagtaat aaatactatg cagactccgt gaagggccga ttcaccatct ccagagacaa 1200 ttccaagaac acgcagtatc tgcaaatgaa cagcctgaga gccgaggaca cggctgtgta 1260 ttactgtgcg agaggcggtg acttcctcta ctactactat tacggtatgg acgtctgggg 1320 ccaagggacc acggtcaccg tctcctcagc ctccaccaag ggcccatcgg tcttccccct 1380 ggcaccctct agcaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga 1440 ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca 1500 VIM) 03/034903 caccttcccg gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt 1560 gccctccagc agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa 1620 caccaaggtg gacaagagag ttggtgagag gccagcacag ggagggaggg tgtctgctgg 1680 aagccaggct cagcgctcct gcctggacgc atcccggcta tgcagtccca gtccagggca 1740 gcaaggcagg ccccgtctgc ctcttcaccc ggaggcctct gcccgcccca ctcatgctca 1800 gggagagggt cttctggctt tttccccagg ctctgggcag gcacaggcta ggtgccccta 1860 acccaggccc tgcacacaaa ggggcaggtg ctgggctcag acctgccaag agccatatcc 1920 gggaggaccc tgcccctgac ctaagcccac cccaaaggcc aaactctcca ctccctcagc 1980 tcggacacct tctctcctcc cagattccag taactcccaa tcttctctct gcagagccca 2040 aatcttgtga caaaactcac acatgcccac cgtgcccagg taagccagcc caggcctcgc 2100 cctccagctc aaggcgggac aggtgcccta gagtagcctg catccaggga caggccccag 2160 ccgggtgctg acacgtccac ctccatctct tcctcagcac ctgaactcct ggggggaccg 2220 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 2280 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 2340 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 2400 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 2460 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 2520 gccaaaggtg ggacccgtgg ggtgcgaggg ccacatggac agaggccggc tcggcccacc 2580 ctctgccctg agagtgaccg ctgtaccaac ctctgtccct acagggcagc cccgagaacc 2640 acaggtgtac accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac 2700 ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca 2760 gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2820 ctatagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2880 cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtotccggg 2940 taaatgagaa ttcctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt 3000 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 3060 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 3120 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 3180 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 3240 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg 3300 tgtggtggtt acgcgcagcg tgaccgctac acttgccagC gccctagcgc ccgctccttt 3360 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 3420 gggcatccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 3480 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 3540 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 3600 tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct attggttaaa 3660 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta 3720 gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa 3780 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 3840 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 3900 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 3960 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 4020 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 4080 gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa 4140 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag 4200 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta 4260 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct 4320 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt 4380 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc 4440 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc 4500 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca 4560 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct 4620 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga 4680 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag 4740 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg 4800 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg 4860 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt 4920 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg 4980 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac 5040 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag 5100 ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt 5160 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 5220 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 5280 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 5340 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 5400 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 5460 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 5520 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 5580 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 5640 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 5700 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 5760 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 5820 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 5880 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 5940 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 6000 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 6060 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 6120 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 6180 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 6240 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 6300 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 6360 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 6420 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 6480 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 6540 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 6600 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 6660 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 6720 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6780 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6840 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6900 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6960 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 7020 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 7080 VIM) 03/034903 %%3 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 7140 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 7200 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 7260 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 7320 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 7380 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 7440 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 7500 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7560 acctgacgtc 7570 <210> 3 <211> 7597 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 3 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 VIM) 03/034903 PCT/US02/33944 lth(63 ggtaccaagc ttggatctca ccatggggtc aaccgccatc ctcaccatgg agttggggct 960 gcgctgggtt ctcctcgttg ctcttttaag aggtgtccag tgtcaggtgc agctggtgga 1020 gtctggggga ggcgtggtcc agcctgggag gtccctgaga ctctcctgtg cagcgtctgg 1080 attcaccttc agtaactatg tcatgcactg ggtccgccag gctccaggca aggggctgga 1140 gtgggtggca attatatggt atgatggaag taataaatac tatgcagact ccgtgaaggg 1200 ccgattcacc atctccagag acaattccaa gaacacgctg tatctgcaaa tgaacagcct 1260 gagagccgag gacacggctg tgtattactg tgcgggtgga tataactgga actacgagta 1320 ccactactac ggtatggacg tctggggcca agggaccacg gtcaccgtct cctcagcctc 1380 caccaagggc ccatcggtct tccccctggc accctctagc aagagcacct ctgggggcac 1440 agcggccctg ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa 1500 ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact 1560 ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ,1620 =
ctgcaacgtg aatcacaagc ccagcaacac caaggtggac aagagagttg gtgagaggcc 1680 agcacaggga gggagggtgt ctgctggaag ccaggctcag cgctcctgcc tggacgcatc ,1740 ccggctatgc agtcccagtc cagggcagca aggcaggccc cgtctgcctc ttcacccgga 1800 ggcctctgcc cgccccactc atgctcaggg agagggtctt ctggcttttt ccccaggctc 1860 tgggcaggca caggctaggt gcccctaacc caggccctgc acacaaaggg gcaggtgctg 1920 ggctcagacc tgccaagagc catatccggg aggaccctgc ccctgaccta agcccacccc 1980 aaaggccaaa ctctccactc cctcagctcg gacaccttct ctcctcccag attccagtaa 2040 ctcccaatct tctctctgca gagcccaaat cttgtgacaa aactcacaca tgcccaccgt 2100 gcccaggtaa gccagcccag gcctcgccct ccagctcaag gcgggacagg tgccctagag 2160 tagcctgcat ccagggacag gccccagccg ggtgctgaca cgtccacctc catctcttcc 2220 tcagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 2280 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 2340 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 2400 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 2460 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 2520 gcccccatcg agaaaaccat ctccaaagcc aaaggtggga cccgtggggt gcgagggcca 2580 catggacaga ggccggctcg gcccaccctc tgccctgaga gtgaccgctg taccaacctc 2640 tgtccctaca gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga 2700 gatgaccaag aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat 2760 cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt 2820 gctggactcc gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg 2880 gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac 2940 gcagaagagc ctctccctgt ctccgggtaa atgagaattc ctcgagtcta gagggcccgt 3000 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 3060 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 3120 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3180 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3240 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc 3300 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 3360 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 3420 cggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt 3480 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 3540 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactutt 3600 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 3660 tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 3720 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccaggcagg 3780 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 3840 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 3900 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 3960 tggctgacta atttttttta tttatgcaga'ggccgaggcc gcctctgcct ctgagctatt 4020 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tccogggagc 4080 ttgtatatcc attttcggat ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac 4140 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 4200.
ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 4260 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 4320 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta 4380 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 4440 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca 4500 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 4560 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 4620 1/%3 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 4680 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 4740 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt 4800 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 4860 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 4920 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 4980 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 5040 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 5100 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac gagatttcga 5160 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 5220 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 5280 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 5340 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 5400 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 5460 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 5520 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 5580 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 5640 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 5700 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 5760 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 5820 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 5880 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 5940 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 6000 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag 6060 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 6120 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 6180 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 6240 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 6300 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 6360 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 6420 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 6480 11%3 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 6540 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 6600 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 6660 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 6720 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 6780 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 6840 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 6900 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 6960 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 7020 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 7080 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 7140 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 7200 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 7260 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 7320 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 7380 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 7440 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 7500 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 7560 tccgcgcaca tttccccgaa aagtgccacc tgacgtc 7597 <210> 4 <211> 7579 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 4 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 ggtaccaagc ttggatctca ccatggagtt gggacttagc tgggttttcc tcgttgctct 960 tttaagaggt gtccagtgtc aggtccagct ggtggagtct gggggaggcg tggtccagcc 1020 tgggaggtcc ctgagactct cctgtgcagc gtctggattc accttcagta gctatggcat 1080 gcactgggtc cgccaggctc caggcaaggg gctggactgg gtggcaatta tttggcatga 1140 tggaagtaat aaatactatg cagactccgt gaagggccga ttcaccatct ccagagacaa 1200 ttccaagaag acgctgtacc tgcaaatgaa cagtttgaga gccgaggaca cggctgtgta 1260 ttactgtgcg agagcttggg cctatgacta cggtgactat gaatactact tcggtatgga 1320 cgtctggggc caagggacca cggtcaccgt ctcctcagcc tccaccaagg gcccatcggt 1380 cttccccctg gcaccctcta gcaagagcac ctctgggggc acagcggccc tgggctgcct 1440 ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag 1500 cggcgtgcac accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt 1560 ggtgaccgtg ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa 1620 gcccagcaac accaaggtgg acaagagagt tggtgagagg ccagcacagg gagggagggt 1680 gtctgctgga agccaggctc agcgctcctg cctggacgca tcccggctat gcagtcccag 1740 tccagggcag caaggcaggc cccgtctgcc tcttcacccg gaggcctctg cccgccccac 1800 tcatgctcag ggagagggtc ttctggcttt ttccccaggc tctgggcagg cacaggctag 1860 gtgcccctaa cccaggccct gcacacaaag gggcaggtgc tgggctcaga cctgccaaga 1920 gccatatccg ggaggaccct gcccctgacc taagcccacc ccaaaggcca aactctccac 1980 tccctcagct cggacacctt ctctcctccc agattccagt aactcccaat cttctctctg 2040 cagagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccaggt aagccagccc 2100 aggcctcgcc ctccagctca aggcgggaca ggtgccctag agtagcctgc atccagggac 2160 VIM) 03/034903 aggccccagc cgggtgctga cacgtccacc tccatctctt cctcagcacc tgaactcctg 2220 gggggaccgt cagtcttcct ctteccecca aaacccaagg acaccctcat gatctcccgg 2280 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 2340 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 2400 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 2460 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 2520 atctccaaag ccaaaggtgg gacccgtggg gtgcgagggc cacatggaca gaggccggct 2580 cggcccaccc tctgccctga gagtgaccgc tgtaccaacc tctgtcccta cagggcagcc 2640 ccgagaacca caggtgtaca ccctgccccc atcccgggag gagatgacca agaaccaggt 2700 cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag 2760 caatgggcag ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc 2820 cttcttcctc tatagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt 2880 ctcatgctcc gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct 2940 gtctccgggt aaatgagaat tcctcgagtc tagagggccc gtttaaaccc gctgatcagc 3000 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3060 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3120 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 3180 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 3240 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag 3300 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 3360 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 3420 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 3480 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 3540 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 3600 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta 3660 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg 3720 tgtcagttag ggtgtggaaa gtccccaggc tccccaggca ggcagaagta tgcaaagcat 3780 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 3840 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 3900 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 3960 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg 4020 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 4080 atctgatcag cacgtgatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct 4140 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg 4200 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga 4260 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc 4320 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc 4380 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt 4440 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc 4500 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc 4560 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc 4620 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt 4680 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa-cagcggtcat 4740 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg 4800 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga 4860 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta 4920 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc 4980 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc 5040 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac 5100 tcgtccgagg gcaaaggaat agcacgtgct acgagatttc gattccaccg ccgccttcta 5160 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 5220 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta 5280 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 5340 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag 5400 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 5460 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 5520 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5580 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 5640 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 5700 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 5760 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 5820 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 5880 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 5940 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 6000 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg 6060 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 6120 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 6180 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 6240 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 6300 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 6360 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 6420 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 6480 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 6540 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 6600 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 6660 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 6720 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 6780 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 6840 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 6900 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 6960 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 7020 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 7080 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 7140 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 7200 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 7260 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 7320 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 7380 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 7440 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 7500 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 7560 aaaagtgcca cctgacgtc 7579 <210> 5 VIM) 03/034903 PCT/US02/33944 <211> 7558 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 5 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 , cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 ' atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 ggtaccaagc ttggatccca ccatggggtc aaccgtcatc ctcgccctcc tcctggctgt 960 tctccaagga gtctgtgccg aggtgcagct ggtgcagtct ggagcagagg tgaaaaagcc 1020 cggggagtct ctgaagatct cctgtaaggg ttctggatac agctttacca gttactggat 1080 cggctgggtg cgccagatgc ccgggaaagg cctggagtgg atggggatca tctatcctgg 1140 tgactctgat accagataca gcccgtcctt ccaaggccag gtcaccatct cagccgacaa 1200 gtccatcagc accgcctacc tgcagtggag cagcctgaag gcctcggaca ccgccatgta 1260 ttactgtgcg agacggatgg cagcagctgg cccctttgac tactggggcc agggaaccct 1320 ggtcaccgtc tcctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctctag 1380 caagagcacc tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga 1440 accggtgacg gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc 1500 tgtcctacag tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag 1560 VIM) 03/034903 cttgggcacc cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga 1620 caagagagtt ggtgagaggc cagcacaggg agggagggtg tctgctggaa gccaggctca 1680 gcgctcctgc ctggacgcat cccggctatg cagtcccagt ccagggcagc aaggcaggcc 1740 ccgtctgcct cttcacccgg aggcctctgc ccgccccact catgctcagg gagagggtct 1800 tctggctttt tccccaggct ctgggcaggc acaggctagg tgcccctaac ccaggccctg 1860 cacacaaagg ggcaggtgct gggctcagac ctgccaagag ccatatccgg gaggaccctg 1920 cccctgacct aagcccaccc caaaggccaa actctccact ccctcagctc ggacaccttc 1980 tctcctccca gattccagta actcccaatc ttctctctgc agagcccaaa tcttgtgaca 2040 aaactcacac atgcccaccg tgcccaggta agccagccca ggcctcgccc tccagctcaa 2100 ggcgggacag gtgccctaga gtagcctgca tccagggaca ggccccagcc gggtgctgac 2160 acgtccacct ccatctcttc ctcagcacct gaactcctgg ggggaccgtc agtcttcctc 2220 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 2280 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 2340 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 2400 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 2460 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaaggtggg 2520 acccgtgggg tgcgagggcc acatggacag aggccggctc ggcccaccct ctgccctgag 2580 agtgaccgct gtaccaacct ctgtccctac agggcagccc cgagaaccac aggtgtacac 2640 cctgccccca tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa 2700 aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa 2760 ctacaagacc acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct 2820 caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga 2880 ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta aatgagaatt 2940 cctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg 3000 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 3060 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 3120 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 3180 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 3240 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 3300 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 3360 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt 3420 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 3480 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 3540 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 3600 ttcttttgat ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat 3660 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag 3720 tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 3780 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 3840 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 3900 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 3960 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 4020 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgatgaa 4080 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt 4140 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg 4200 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta 4260 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga 4320 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga 4380 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggCca tggatgcgat 4440 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg 4500 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg 4560 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat 4620 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa 4680 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt 4740 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat 4800 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct 4860 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa 4920 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg 4980 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt 5040 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata 5100 gcacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 5160 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 5220 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 5280 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 5340 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 5400 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 5460 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 5520 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 5580 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 5640 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 5700 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 5760 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 5820 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 5880 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 5940 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg 6000 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 6060 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 6120 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 6180 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 6240 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 6300 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 6360 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 6420 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 6480 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 6540 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 6600 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 6660 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 6720 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 6780 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 6840 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 6900 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 6960 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 7020 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 7080 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 7140 VIM) 0134903 2/%3 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 7200 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 7260 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 7320 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 7380 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 7440 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 7500 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc 7558 <210> 6 <211> 7576 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 6 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 ggtaccaagc ttggatctca ccatggagtt tgggctgtgc tggattttcc tcgttgctct 960 tttaagaggt gtccagtgtc aggtgcagct ggtggagtct gggggaggcg tggtccagcc 1020 VIM) 01%34903 21%3 tgggaggtcc ctgagactct cctgtgcagc ctctggattc accttcatta gctatggcat 1080 gcactgggtc cgccaggctc caggcaaggg gctggagtgg gtggcagtta tatcatatga 1140 tggaagtaat aaatactatg cagactccgt gaagggccga ttcaccatct ccagagacaa 1200 ttccaagaac acgctgtatc tgcaaatgaa cagcctgaga gctgaggaca cggctgtgta 1260 ttactgtgcg agagtattag tgggagcttt atattattat aactactacg ggatggacgt 1320 ctggggccaa gggaccacgg tcaccgtctc ctcagcctcc accaagggcc catcggtctt 1380 ccccctggca ccctctagca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1440 caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1500 cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 1560 gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc 1620 cagcaacacc aaggtggaca agagagttgg tgagaggcca gcacagggag ggagggtgtc 1680 tgctggaagc caggctcagc gctcctgcct ggacgcatcc cggctatgca gtcccagtcc 1740 agggcagcaa ggcaggcccc gtctgcctct tcacccggag gcctctgccc gccccactca 1800 tgctcaggga gagggtcttc tggctttttc cccaggctct gggcaggcac aggctaggtg 1860 cccctaaccc aggccctgca cacaaagggg caggtgctgg gctcagacct gccaagagcc 1920 atatccggga ggaccctgcc cctgacctaa gcccacccca aaggccaaac tctccactcc 1980 ctcagctcgg acaccttctc tcctcccaga ttccagtaac tcccaatctt ctctctgcag 2040 agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccaggtaag ccagcccagg 2100 cctcgccctc cagctcaagg cgggacaggt gccctagagt agcctgcatc cagggacagg 2160 ccccagccgg gtgctgacac gtccacctcc atctcttcct cagcacctga actcctgggg 2220 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 2280 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 2340 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 2400 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 2460 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 2520 tccaaagcca aaggtgggac ccgtggggtg cgagggccac atggacagag gccggctcgg 2580 cccaccctct gccctgagag tgaccgctgt accaacctct gtccctacag ggcagccccg 2640 agaaccacag gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag 2700 cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa 2760 tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt 2820 cttcctctat agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc 2880 atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc 2940 tccgggtaaa tgagaattcc tcgagtctag agggcccgtt taaacccgct gatcagcctc 3000 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 3060 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 3120 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 3180 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga 3240 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc 3300 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 3360 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 3420 aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 3480 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 3540 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 3600 caaccctatc tcggtctatt cttttgattt ataagggatt ttggggattt cggc\ctattg 3660 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt 3720 cagttagggt gtggaaagtc cccaggctcc ccaggcaggc agaagtatgc aaagcatgca 3780 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 3840 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 3900 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 3960 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt 4020 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc 4080 tgatcagcac gtgatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 4140 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4200 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4260 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4320 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4380 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4440 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4500 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4560 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4620 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4680 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4740 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4800 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4860 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4920 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4980 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 5040 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 5100 tccgagggca aaggaatagc acgtgctacg agatttcgat tccaccgccg ccttctatga 5160 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 5220 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa 5280 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 5340 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta 5400 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 5460 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 5520 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ,5580 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 5640 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 5700 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 5760 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 5820 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 5880 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 5940 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 6000 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 6060 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 6120 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 6180 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 6240 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 6300 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 6360 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 6420 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 6480 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 6540 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 6600 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 6660 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 6720 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 6780 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 6840 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 6900 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 6960 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 7020 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 7080 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 7140 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 7200 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 7260 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 7320 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 7380 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 7440 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 7500 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 7560 agtgccacct gacgtc 7576 <210> 7 <211> 7561 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 7 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 ggtaccggat ctcaccatgg agttggggct gagctgggtt ttcctcgttg ctcttttaag 960 aggtgtccag tgtcaggagc agctggtgga gtctggggga ggcgtggtcc agcctgggag 1020 gtccctgaga ctctcctgtg cagcgtctgg attcaccttc agtacctatg gcatgcactg 1080 ggtccgccag gctccaggca aggggctgga gtgggtggca gttacatggc atgatggaag 1140 taataaatac tatgcagact ccgtgaaggg ccgattcacc atctccagag acaactccaa 1200 gaacacgctg tatctgcaaa tgaacagcct gagagccgag gacacggctg tgtattactg 1260 tgcgagagga ggagtgggag caacttacta ctactactac ggtatggacg tctggggcca 1320 agggaccacg gtcaccgtct cctcagcctc caccaagggc ccatcggtct tccccctggc 1380 accctctagc aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta 1440 cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac 1500 cttcccggct gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc 1560 ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac 1620 caaggtggac aagagagttg gtgagaggcc agcacaggga gggagggtgt ctgctggaag 1680 ccaggctcag cgctcctgcc tggacgcatc ccggctatgc agtcccagtc cagggcagca 1740 aggcaggccc cgtctgcctc ttcacccgga ggcctctgcc cgccccactc atgctcaggg 1800 agagggtctt ctggcttttt ccccaggctc tgggcaggca caggctaggt gcccctaacc 1860 caggccctgc acacaaaggg gcaggtgctg ggctcagacc tgccaagagc catatccggg 1920 aggaccctgc ccctgaccta agcccacccc aaaggccaaa ctctccactc cctcagctcg 1980 gacaccttct ctcctcccag attccagtaa ctcccaatct tctctctgca gagcccaaat 2040 cttgtgacaa aactcacaca tgcccaccgt gcccaggtaa gccagcccag gcctcgccct 2100 ccagctcaag gcgggacagg tgccctagag tagcctgcat ccagggacag gccccagccg 2160 ggtgctgaca cgtccacctc catctcttcc tcagcacctg aactcctggg gggaccgtca 2220 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 2280 VIM) 03/034903 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 2340 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 2400 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 2460 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 2520 aaaggtggga cccgtggggt gcgagggcca catggacaga ggccggctcg gcccaccctc 2580 tgccctgaga gtgaccgctg taccaacctc tgtccctaca gggcagcccc gagaaccaca 2640 ggtgtacacc ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg 2700 cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc 2760 ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta 2820 tagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt 2880 gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa 2940 atgactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag 3000 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 3060 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 3120 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3180 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 3240 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 3300 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 3360 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc 3420 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 3480 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3540 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 3600 ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa aaaatgagct 3660 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 3720 aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca attagtcagc 3780 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3840 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 3900 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 3960 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 4020 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgat 4080 gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 4140 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 4200 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 4260 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 4320 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 4380 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc 4440 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 4500 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 4560 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 4620 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 4680 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 4740 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 4800 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 4860 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 4920 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 4980 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 5040 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 5100 atagcacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 5160 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 5220 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 5280 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 5340 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 5400 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 5460 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 5520 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 5580 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 5640 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 5700 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 5760 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 5820 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 5880 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 5940 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6000 atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6060 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6120 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6180 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6240 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6300 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6360 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6420 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6480 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 6540 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 6600 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 6660 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 6720 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 6780 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 6840 =
ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 6900 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 6960 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 7020 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 7080 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7140 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7200 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7260 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 7320 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 7380 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 7440 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 7500 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 7560 <210> 8 <211> 6082 <212> DNA

<213> Artificial Sequence <220>
<223> Plasmid <400> 8 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg GO
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 aagcttggat ctcaccatga gggtccctgc tcagctcctg ggactcctgc tgctctggct 960 cccagatacc agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt 1020 aggagacaga gtcaccatca cttgccgggc gagtcagggc attagcaatt atttagcctg 1080 gtatcagcag aaaacaggga aagttcctaa gttcctgatc tatgaagcat ccactttgca 1140 atcaggggtc ccatctcggt tcagtggcgg tggatctggg acagatttca ctctcaccat 1200 cagcagcctg cagcctgaag atgttgcaac ttattactgt caaaattata acagtgcccc 1260 attcactttc ggccctggga ccaaagtgga tatcaaacga actgtggctg caccctctgt 1320 cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgctagcg ttgtgtgcct 1380 gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 1440 atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 1500 cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 1560 agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta 1620 ggaattcgcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 1680 3/%3 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1740 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1800 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1860 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1920 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 1980 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2040 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2100 ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2160 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2220 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2280 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa 2340 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 2400 agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca 2460 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 2520 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 2580 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 2640 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg 2700 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc 2760 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 2820 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 2880 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 2940 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 3000 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 3060 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 3120 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 3180 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 3240 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 3300 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3360 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 3420 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 3480 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 3540 31%3 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 3600 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 3660 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 3720 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 3780 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 3840 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 3900 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4020 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 4080 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4140 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4200 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4260 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4320 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4380 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 4440 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 4500 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4560 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 4620 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 4680 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 4740 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 4800 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 4860 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 4920 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 4980 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5040 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5100 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 5160 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 5220 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 5280 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5340 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5400 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 5460 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 5520 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 5580 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 5640 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 5700 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 5820 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 5880 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 5940 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6000 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc .6060 ccgaaaagtg ccacctgacg tc 6082 <210> 9 <211> 6082 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 9 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 aagcttggat ctcaccatga gggtccccgc tcagctcctg gggctcctgc tgctctgttt 960 cccaggtgcc agatgtgaca tccagatgac ccagtctcca tcctcactgt ctgcatctgt 1020 aggagacaga gtcaccatca cttgtcgggc gagtcagggc attaccaatt atttagcctg 1080 gtttcagcag aaaccaggga aagcccctaa gtcccttatc tatgctgcat ccagtttgca 1140 aagtggggtc ccatcaaagt tcagcggcag tggatctggg acagatttca gtctcaccat 1200 cagcagcctg cagcctgaag attttgcaac ttattactgc caacagtata atagttaccc 1260 gatcaccttc ggccaaggga cacgactgga gattaaacga actgtggctg caccatctgt 1320 cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgctagcg ttgtgtgcct 1380 gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 1440 atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 1500 cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga ,1560 agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta 1620 ggaattcgcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 1680 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1740 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1800 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1860 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1920 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 1980 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2040 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2100 ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2160 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2220 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2280 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa 2340 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 2400 agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca 2460 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 2520 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 2580 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 2640 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg 2700 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc 2760 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 2820 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 2880 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 2940 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 3000 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 3060 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 3120 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 3180 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 3240 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 3300 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3360 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 3420 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 3480 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 3540 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 3600( aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 3660 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 3720 cggggatctc atgctggagt tettcgccca ccccaacttg tttattgcag cttataatgg 3780 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 3840 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 3900 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4020 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 4080 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4140 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4200 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg , 4260 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4320 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4380 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 4440 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 4500 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4560 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 4620 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 4680 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 4740 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 4800 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 4860 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 4920 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 4980 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5040 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5100 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 5160 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 5220 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 5280 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5340 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5400 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 5460 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 5520 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 5580 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 5640 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 5700 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 5820 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 5880 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 5940 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6000 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6060 ccgaaaagtg ccacctgacg tc 6082 <210> 10 <211> 6082 VIM) 03/034903 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 10 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 aagcttggat ctcaccatga gggtccctgc tcagctcctg gggctcctgc tgctctgttt 960 cccaggtgcc agatgtgaca tccagatgac ccagtctcca tcctcactgt ctgcatctgt 1020 aggagacaga gtcaccatca cttgtcgggc gagtcagggc attagccatt atttagcctg 1080 gtttcagcag aaaccaggga aagcccctaa gtccctgatc tatgctgcat ccagtttgca 1140 aagtggggtc ccatcaaagt tcagcggcag tggatctggg acagatttca ctctcaccat 1200 cagcagccta cagcctgaag attttgcaac ttattactgc caacagtata atagtttccc 1260 gctcactttc ggcggaggga ccaaggtgga gatcaaacga actgtggctg caccatctgt 1320 cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgctagcg ttgtgtgcct 1380 gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 1440 atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 1500 cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 1560 agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta 1620 ggaattcgcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 1680 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1740 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1800 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1860 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1920 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 1980 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2040 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2100 ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2160 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2220 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2280 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa . 2340 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 2400 agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca 2460 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 2520 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 2580 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 2640 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg 2700 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc 2760 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 2820 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 2880 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 2940 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 3000 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 3060 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga , 3120 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 3180 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 3240 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 3300 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3360 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 3420 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 3480 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 3540 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 3600 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 3660 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 3720 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 3780 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 3840 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 3900 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4020 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 4080 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4140 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4200 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4260 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4320 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca ,4380 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 4440 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 4500 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4560 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 4620 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 4680 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 4740 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 4800 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 4860 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 4920 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 4980 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5040 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5100 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 5160 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 5220 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 5280 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5340 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5400 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 5460 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 5520 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 5580 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 5640 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 5700 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 5820 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 5880 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 5940 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6000 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6060 ccgaaaagtg ccacctgacg tc 6082 <210> 11 <211> 6085 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 11 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 4/%3 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 aagcttggat ctcaccatga gggtccccgc tcagcttctc ttccttctgc tactctggct 960 cccagatacc actggaggaa tagtgatgac gcagtctcca gccaccctgt ctgtgtctcc 1020 aggggaaaga gccaccctct cctgcaggac cagtcagagt attggctgga acttagcctg 1080 gtaccaacag aaacctggcc aggctcccag gctcctcatc tatggtgcat cttccaggac 1140 cactggtatc ccagccaggt tcagtggcag tgggtctggg acagagttca ctctcaccat 1200 cagcagcctg cagtctgaag attctgcagt ttattactgt cagcattatg ataactggcc 1260 catgtgcagt tttggccagg ggaccgagct ggagatcaaa cgaactgtgg ctgcaccatc 1320 tgtcttcatc ttcccgccat ctgatgagca gttgaaatct ggaactgcta gcgttgtgtg 1380 cctgctgaat aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct 1440 ccaatcgggt aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag 1500 cctcagcagc accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg 1560 cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg 1620 ttaggaattc gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga 1680 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1740 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1800 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 1860 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 1920 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 1980 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 2040 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 2100 atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 2160 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 2220 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 2280 accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt 2340 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 2400 gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc 2460 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2520 VIM) 01%34903 41%3 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 2580 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 2640 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 2700 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 2760 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt 2820 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct 2880 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga 2940 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg 3000 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact 3060 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 3120 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct 3180 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg 3240 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt 3300 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg 3360 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc 3420 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag 3480 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt 3540 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt 3600 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc 3660 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 3720 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 3780 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 3840 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 3900 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 3960 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4020 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 4080 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 4140 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 4200 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 4260 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4320 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4380 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4440 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4500 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt 4560 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4620 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4680 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 4740 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 4800 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4860 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4920 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 4980 gattttggtc atgagattat caaaaaggat cttCacctag atccttttaa attaaaaatg 5040 aagttttaaa tcaatctaaa gtatatatga-gtaaacttgg tctgacagtt accaatgctt 5100 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 5160 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 5220 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 5280 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 5340 , ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 5400 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 5460 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 5520 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 5580 agcactgcat aattctctta Ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 5640 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 5700 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 5760 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 5820 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 5880 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 5940 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 6000 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 6060 tccccgaaaa gtgccacctg acgtc 6085 <210> 12 VIM) 03/034903 <211> 6097 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 12 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 aagcttggat ctcaccatga gggtccctgc tcagctcctg gggctgctaa tgctctggat 960 acctggatcc agtgcagata ttgtgatgac ccagactcca ctctctctgt ccgtcacccc 1020 tggacagccg gcctccatct cctgcaagtc tagtcagagc ctcctgcata gtgatggaaa 1080 gacctttttg tattggtatc tgcagaagcc aggccagcct ccacagctcc tgatctatga 1140 ggtttccaac cggttctctg gagtgccaga taggttcagt ggcagcgggt cagggacaga 1200 tttcacactg aaaatcagcc gggtggaggc tgaggatgtt gggctttatt actgcatgca 1260 aagtatacag cttccgctca ctttcggcgg agggaccaag gtggagatca aacgaactgt 1320 ggctgcacca tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc 1380 tagcgttgtg tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt 1440 ggataacgcc ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga 1500 cagcacctac agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa 1560 agtctacgcc tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa 1620 caggggagag tgttaggaat tcgcggccgc tcgagtctag agggcccgtt taaacccgct 1680 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 1740 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 1800 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 1860 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 1920 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg 1980 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2040 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 2100 gtcaagctct aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg 2160 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 2220 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 2280 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttggggattt 2340 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg 2400 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccaggcaggc agaagtatgc 2460 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 2520 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 2580 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 2640 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 2700 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca 2760 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat 2820 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac 2880 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 2940 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc 3000 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 3060 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc 3120 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg 3180 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc 3240 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc 3300 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga 3360 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 3420 VIM) 03/034903 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg 3480 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 3540 ccgctoccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg 3600 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga 3660 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 3720 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 3780 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 3840 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 3900 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 3960 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 4020 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 4080 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 4140 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 4200 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 4260 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 4320 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 4380 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 4440 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 4500 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag 4560 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 4620 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 4680 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 4740 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 4800 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 4860 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 4920 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 4980 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 5040 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 5100 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 5160 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 5220 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 5280 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 5340 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 5400 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 5460 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 5520 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 5580 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 5640 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 5700 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 5760 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 5820 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 5880 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 5940 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 6000 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 6060 tccgcgcaca tttccccgaa aagtgccacc tgacgtc 6097 <210> 13 <211> 6094 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid <400> 13 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900 aagcttggat ctcaccatgg tgttgcagac ccaggtcttc atttctctgt tactctggat 960 ctctggtgcc tacggggaca tcgtgatgac ccagtctcca gactccctgg ctgtgtctct 1020 gggcgagagg gccaccatca actgcaagtc caaccagagt gtcttacaca gctccaacaa 1080 taagaactat ttagcttggt accagcagaa accaggacag cctcctaaat tgctcattta 1140 ttgggcattc ctccgggaat ccggggtccc tgaccgcttc agtggcagcg ggtctgggac 1200 agatttcact ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca 1260 ccaatattat tctactttat atactttcgg cggagggacc aaggtagaga tcaaacgaac 1320 ygtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac 1380 tgctagcgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa 1440 ggtggataac gccctccaat cgggtaactc ccaggagagt gtcacagagc aggacagcaa 1500 ggacagcacc tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca 1560 caaagtctac gcctgcgaag tcacccatca gggcctgagc tcgcccgtca caaagagctt 1620 caacagggga gagtgttagg cggccgctcg agtctagagg gcccgtttaa acccgctgat 1680 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 1740 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 1800 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 1860 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 1920 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 1980 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 2040 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 2100 aagctctaaa tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc 2160 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 2220 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 2280 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg 2340 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 2400 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa 2460 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 2520 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 2580 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 2640 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag 2700 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt 2760 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc 2820 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga 2880 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 2940 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat 3000 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 3060 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg 3120 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc 3180 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga 3240 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag 3300 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 3360 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg 3420 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata 3480 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg 3540 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac 3600 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc 3660 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat 3720 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc 3780 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 3840 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat 3900 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 3960 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 4020 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 4080 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4140 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4200 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4260 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4320 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4380 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4440 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4500 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4560 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4620 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4680 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4740 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4800 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4860 caccgctggt agcggtggtt tttttgtttg caagcagcag attatgcgca gaaaaaaagg 4920 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4980 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5040 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5100 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5160 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5220 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5280 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5340 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5400 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5460 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5520 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5580 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5640 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5700 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 5760 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 5820 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 5880 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 5940 gaaatgttga atactcatac tettccettt tcaatattat tgaagcattt atcagggtta 6000 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6060 gcgcacattt ccccgaaaag tgccacctga cgtc 6094 5/%3 <210> 14 <211> 481 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/Bg1II cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction <400> 14 ggatctcacc atggagttgg gactgcgctg gggcttcctc gttgctcttt taagaggtgt 60 ccagtgtcag gtgcaattgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct 120 gagactctcc tgtgcagcgt ctggattcgc cttcagtaga tatggcatgc actgggtccg 180 ccaggctcca ggcaaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa 240 atactatgca gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac 300 gcagtatctg caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag 360 aggcggtgac ttcctctact actactatta cggtatggac gtctggggcc aagggaccac 420 ggtcaccgtc tcctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctctag 480 <210> 15 <211> 142 <212> PRT
<213> Homo sapiens <400> 15 Met Glu Leu Gly Leu Arg Trp Gly Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Arg Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn P
VIM) 03/034903 51(63 Thr Gin Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Asp Phe Leu Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser <210> 16 <211> 463 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/Bg1II cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction <400> 16 ggatctcacc atgagggtcc ctgctcagct cctgggactc ctgctgctct ggctcccaga 60 taccagatgt gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga 120 cagagtcacc atcacttgcc gggcgagtca gggcattagc aattatttag cctggtatca 180 gcagaaaaca gggaaagttc ctaagttcct gatctatgaa gcatccactt tgcaatcagg 240 ggtcccatct cggttcagtg geggtggatc tgggacagat ttcactctca ccatcagcag 300 cctgcagcct gaagatgttg caacttatta ctgtcaaaat tataacagtg ccccattcac 360 tttoggccct gggaccaaag tggatatcaa acgaactgtg gctgcaccct ctgtcttcat 420 cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 463 <210> 17 <211> 127 <212> PRT
<213> Homo sapiens <400> 17 Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro Asp Thr Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly Ile Ser Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Thr Gly Lys Val Pro Lys Phe Leu Ile Tyr Glu Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gin Asn Tyr Asn Ser Ala Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys <210> 18 <211> 508 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/Bg1II cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction <400> 18 ggatctcacc atggggtcaa ccgccatcct caccatggag ttggggctgc gctgggttct 60 cctcgttgct cttttaagag gtgtccagtg tcaggtgcag ctggtggagt ctgggggagg 120 cgtggtccag cctgggaggt ccctgagact ctcctgtgca gcgtctggat tcaccttcag 180 taactatgtc atgcactggg tccgccaggc tccaggcaag gggctggagt gggtggcaat 240 tatatggtat gatggaagta ataaatacta tgcagactcc gtgaagggcc gattcaccat 300 ctccagagac aattccaaga acacgctgta tctgcaaatg aacagcctga gagccgagga 360 cacggctgtg tattactgtg cgggtggata taactggaac tacgagtacc actactacgg 420 tatggacgtc tggggccaag ggaccacggt caccgtctcc tcagcctcca ccaagggccc 480 atcggtcttc cccctggcac cctctagc 508 <210> 19 <211> 143 <212> PRT
<213> Homo sapiens <400> 19 Met Glu Leu Gly Leu Arg Trp Val Leu Leu Val Ala Leu Leu Arg Gly Val Gin Cys Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Val Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ile Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Len Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gly Tyr Asn Trp Asn Tyr Glu Tyr His Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser <210> 20 <211> 463 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/BglII cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction <400> 20 ggatctcacc atgagggtcc ccgctcagct cctggggctc ctgctgctct gtttcccagg 60 tgccagatgt gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga 120 cagagtcacc atcacttgtc gggcgagtca gggcattacc aattatttag cctggtttca 180 gcagaaacca gggaaagccc ctaagtccct tatctatgct gcatccagtt tgcaaagtgg 240 ggtcccatca aagttcagcg gcagtggatc tgggacagat ttcagtctca ccatcagcag 300 cctgcagcct gaagattttg caacttatta ctgccaacag tataatagtt acccgatcac 360 cttcggccaa gggacacgac tggagattaa acgaactgtg gctgcaccat ctgtcttcat 420 cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 463 <210> 21 <211> 127 <212> PRT
<213> Homo sapiens <400> 21 Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Cys Phe Pro Gly Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly Ile Thr Asn Tyr Leu Ala Trp Phe Gin Gin Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Pro Ile Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys <210> 22 <211> 490 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/BglII cloning junction, signal peptide, V region, portion of C region and 31XbaI/NheI (heavy) or NheI (light) cloning junction <400> 22 ggatctcacc atggagttgg gacttagctg ggttttcctc gttgctcttt taagaggtgt 60 ccagtgtcag gtccagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct 120 gagactctcc tgtgcagcgt ctggattcac cttcagtagc tatggcatgc actgggtccg 180 ccaggctcca ggcaaggggc tggactgggt ggcaattatt tggcatgatg gaagtaataa 240 atactatgca gactccgtga agggccgatt caccatctcc agagacaatt ccaagaagac 300 gctgtacctg caaatgaaca gtttgagagc cgaggacacg gctgtgtatt actgtgcgag 360 agcttgggcc tatgactacg gtgactatga atactacttc ggtatggacg tctggggcca 420 agggaccacg gtcaccgtct cctcagcctc caccaagggc ccatcggtct tccccctggc 480 accctctagc 490 <210> 23 <211> 145 <212> PRT
<213> Homo sapiens <400> 23 Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gin Cys Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Asp Trp Val Ala Ile Ile Trp His Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Trp Ala Tyr Asp Tyr Gay Asp Tyr Glu Tyr Tyr Phe Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser <210> 24 <211> 463 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/Bg1II cloning junction, signal peptide, V region, portion of C region and 3'Xbal/NheI (heavy) or NheI (light) cloning junction <400> 24 ggatctcacc atgagggtcc ctgctcagct cctggggctc ctgctgctct gtttcccagg 60 tgccagatgt gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga 120 cagagtcacc atcacttgtc gggcgagtca gggcattagc cattatttag cctggtttca 180 gcagaaacca gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg 240 ggtcccatca aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag 300 cctacagcct gaagattttg caacttatta ctgccaacag tataatagtt tcccgctcac 360 tttcggcgga gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat 420 cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 463 <210> 25 <211> 127 <212> PRT
<213> Homo sapiens <400> 25 Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Cys Phe Pro Gly Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly Ile Ser His Tyr Leu Ala Trp Phe Gin Gin Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys <210> 26 <211> 469 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/Bg1II cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction <400> 26 ggatcccacc atggggtcaa ccgtcatcct cgccctcctc ctggctgttc tccaaggagt 60 ctgtgccgag gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct 120 gaagatctcc tgtaagggtt ctggatacag ctttaccagt tactggatcg gctgggtgcg 180 ccagatgccc gggaaaggcc tggagtggat ggggatcatc tatcctggtg actctgatac 240 cagatacagc ccgtccttcc aaggccaggt caccatctca gccgacaagt ccatcagcac 300 cgcctacctg cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag 360 acggatggca gcagctggcc cctttgacta ctggggccag ggaaccctgg tcaccgtctc 420 ctcagcctcc accaagggcc catcggtctt ccccctggca ccctctagc 469 <210> 27 <211> 138 <212> PRT
<213> Homo sapiens <400> 27 Met Gly Ser Thr Val Ile Leu Ala Leu Leu Leu Ala Val Leu Gin Gly Val Cys Ala Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gin Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gin Gly Gin Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gin Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Met Ala Ala Ala Gly Pro Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser <210> 28 <211> 466 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/BglII cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction VIM) 03/034903 <400> 28 ggatctcacc atgagggtcc ccgctcagct tctcttcctt ctgctactct ggctcccaga 60 taccactgga ggaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga 120 aagagccacc ctctcctgca ggaccagtca gagtattggc tggaacttag cctggtacca 180 acagaaacct ggccaggctc ccaggctcct catctatggt gcatcttcca ggaccactgg 240 tatcccagcc aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag 300 cctgcagtct gaagattctg cagtttatta ctgtcagcat tatgataact ggcccatgtg 360 cagttttggc caggggaccg agctggagat caaacgaact gtggctgcac catctgtctt 420 catcttcccg ccatctgatg agcagttgaa atctggaact gctagc 466 <210> 29 <211> 128 <212> PRT
<213> Homo sapiens <400> 29 Met Arg Val Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro Asp Thr Thr Gly Gly Ile Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gin Ser Ile Gly Trp Asn Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Thr Thr Gly Ile Pro Ala 65 70 75 . 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gin Ser Giu Asp Ser Ala Val Tyr Tyr Cys Gin His Tyr Asp Asn Trp Pro Met Cys Ser Phe Gly Gin Gly Thr Glu Leu Glu Ile Lys <210> 30 <211> 487 <212> DNA
<213> Artificial Sequence <220>

<223> Includes BamHI/Bg1II cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction <400> 30 ggatctcacc atggagtttg ggctgtgctg gattttcctc gttgctcttt taagaggtgt 60 ccagtgtcag gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct 120 gagactctcc tgtgcagcct ctggattcac cttcattagc tatggcatgc actgggtccg 180 ccaggctcca ggcaaggggc tggagtgggt ggcagttata tcatatgatg gaagtaataa 240 atactatgca gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac 300 gctgtatctg caaatgaaca gcctgagagc tgaggacacg gctgtgtatt actgtgcgag 360 agtattagtg ggagctttat attattataa ctactacggg atggacgtct ggggccaagg 420 gaccacggtc accgtctcct cagcctccac caagggccca tcggtcttcc ccctggcacc 480 ctctagc 487 <210> 31 <211> 144 <212> PRT
<213> Homo sapiens <400> 31 Met Glu Phe Gly Leu Cys Trp Ile Phe Leu Val Ala Leu Leu Arg Gly Val Gin Cys Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Leu Val Gly Ala Leu Tyr Tyr Tyr Asn Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser <210> 32 6/%3 <211> 478 <212> DNA
<213> Artificial Sequence <220>
<223> Includes BamHI/BglII cloning junction, signal peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction <400> 32 ggatctcacc atgagggtcc ctgctcagct cctggggctg ctaatgctct ggatacctgg 60 atccagtgca gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca 120 gccggcctcc atctcctgca agtctagtca gagcctcctg catagtgatg gaaagacctt 180 tttgtattgg tatctgcaga agccaggcca gcctccacag ctcctgatct atgaggtttc 240 caaccggttc tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac 300 actgaaaatc agccgggtgg aggctgagga tgttgggctt tattactgca tgcaaagtat 360 acagcttccg ctcactttcg gcggagggac caaggtggag atcaaacgaa ctgtggctgc 420 accatctgtc ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgctagc 478 <210> 33 <211> 132 <212> PRT
<213> Homo sapiens <400> 33 Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Ile Pro Gly Ser Ser Ala Asp Ile Val Met Thr Gin Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gin Pro Ala Ser Ile Ser Cys Lys Ser Ser Gin Ser Leu Leu His Ser Asp Gly Lys Thr Phe Leu Tyr Trp Tyr Leu Glu Lys Pro Gly Gin Pro Pro Gin Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Met Gin Ser Ile Gin Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

Claims (88)

CLAIMS:
1. An isolated antibody or antigen-binding fragment thereof that selectively binds a prostate specific membrane antigen (PSMA) protein dimer, wherein each of the PSMA
proteins forming the dimer comprises amino acids 44-750 of SEQ ID NO:1.
2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody or antigen-binding fragment thereof inhibits at least one enzymatic activity of the PSMA protein dimer.
3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the enzymatic activity is selected from the group consisting of folate hydrolase activity, NAALADase activity, dipeptidyl dipeptidase IV activity, .gamma.-glutamyl hydrolase activity and combinations thereof.
4. A composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3 and an immunostimulatory oligonucleotide.
5. A composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3 and a cytokine.
6. The composition of claim 5, wherein the cytokine is selected from the group consisting of 1L-2, 1L-12, 1L-18 and GM-CSF.
7. The composition of any one of claims 4-6, further comprising a pharmaceutically-acceptable carrier, excipient or stabilizer.
8. A composition comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3 and a pharmaceutically acceptable carrier, excipient or stabilizer.
9. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody is raised by immunizing an animal with a preparation comprising a PSMA protein dimer.
10. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of PSMA 3.9 (ATCC patent deposit designation PTA-3258), PSMA 5.4 (ATCC patent deposit designation PTA-3268) and antigen-binding fragments thereof.
11. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of antibodies comprising:
(a) a heavy chain encoded by a nucleic acid molecule comprising the heavy chain coding region or regions of a nucleotide sequence set forth as SEQ ID
NO: 2, and a light chain encoded by a nucleic acid molecule comprising the light chain coding region or regions of a nucleotide sequence set forth as SEQ ID NO: 8;
(b) a heavy chain encoded by a nucleic acid molecule comprising the heavy chain coding region or regions of a nucleotide sequence set forth as SEQ ID
NO: 3, and a light chain encoded by a nucleic acid molecule comprising the light chain coding region or regions of a nucleotide sequence set forth as SEQ ID NO: 9;
(c) a heavy chain encoded by a nucleic acid molecule comprising the heavy chain coding region or regions of a nucleotide sequence set forth as SEQ ID
NO: 4, and a light chain encoded by a nucleic acid molecule comprising the light chain coding region or regions of a nucleotide sequence set forth as SEQ ID NO: 10;
(d) a heavy chain encoded by a nucleic acid molecule comprising the heavy chain coding region or regions of a nucleotide sequence set forth as SEQ ID
NO: 6, and a light chain encoded by a nucleic acid molecule comprising the light chain coding region or regions of a nucleotide sequence set forth as SEQ ID NO: 12;
and antigen-binding fragments thereof.
12. The isolated antibody or antigen-binding fragment thereof of claim 11, wherein the antibody or antigen-binding fragment thereof comprises:
(a) a heavy chain encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as SEQ ID NO: 2 and (b) a light chain encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as SEQ ID NO: 8; or an antigen-binding fragment thereof.
13. An isolated antibody or antigen-binding fragment thereof that specifically binds a PSMA protein dimer, wherein each of the PSMA proteins forming the dimer comprises amino acids 44-750 of SEQ ID NO:1, wherein the antibody or antigen-binding fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence encoding the antibody or antigen-binding fragment thereof as claimed in claim 11, wherein the at most 10% sequence variation does not occur within the coding region(s) for the complementarity determining region(s) (CDR(s)) of the nucleotide sequence encoding the antibody or antigen-binding fragment thereof as claimed in claim 11.
14. The isolated antibody or antigen-binding fragment thereof of claim 13, wherein the antibody or antigen-binding fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence that is:
(a) at least 95% identical;
(b) at least 97% identical;

(c) at least 98% identical; or (d) at least 99% identical.
15. The antigen-binding fragment of claim 11, comprising:
(a) a heavy chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as:
SEQ ID
NO: 14, and (b) a light chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as:
SEQ ID
NO: 16; or (c) a heavy chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as:
SEQ ID
NO: 18, and (d) a light chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as:
SEQ ID
NO: 20; or (e) a heavy chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as:
SEQ ID
NO: 22, and (f) a light chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as: SEQ ID NO:
24; or (g) a heavy chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as:
SEQ ID
NO: 30, and (h) a light chain variable region encoded by a nucleic acid molecule comprising the coding region or regions of a nucleotide sequence set forth as:
SEQ ID
NO: 32.
16. The antigen-binding fragment of claim 11, comprising:
(a) a heavy chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 15, and (b) a light chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 17; or (c) a heavy chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 19, and (d) a light chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 21; or (e) a heavy chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 23, and (f) a light chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 25; or (g) a heavy chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 31, and (h) a light chain variable region comprising an amino acid sequence set forth as: SEQ ID NO: 33.
17. An isolated antigen-binding fragment which comprises the three complementarity determining regions (CDRs) of the heavy chain variable region and the three CDRs of the light chain variable region of the antigen-binding fragment as claimed in claim 15 or claim 16.
18. An expression vector comprising an isolated nucleic acid molecule encoding the isolated antibody or antigen-binding fragment as claimed in any one of claims 11-16.
19. A host cell transformed or transfected by the expression vector as claimed in claim 18.
20. The isolated antibody or antigen-binding fragment thereof of any one of claims 1, 10-12, 15 and 16, wherein the antibody or antigen-binding fragment thereof binds live cells expressing PSMA protein dimer.
21. The isolated antibody or antigen-binding fragment thereof of claim 20, wherein the cells are endothelial cells of tumor neovasculature.
22. The isolated antibody or antigen-binding fragment thereof of claim 20, wherein the cells are prostate tumor cells.
23. The isolated antibody or antigen-binding fragment thereof of claim 22, wherein the prostate tumor cells are LNCaP cells.
24. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 11, 12, 15 and 16, wherein the antibody is a recombinant antibody.
25. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 11, 12, 15 and 16, wherein the antibody is a humanized antibody.
26. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16, wherein the antibody is a chimeric antibody.
27. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16, wherein the antibody is a human antibody.
28. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15, 16 and 24-27, wherein the antibody is a monoclonal antibody.
29. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16, wherein the isolated antigen-binding fragment is selected from the group consisting of a Fab fragment, a F(ab')2 fragment and a Fv fragment.
30. A composition comprising:
the antibody or antigen-binding fragment thereof of any one of claims 10-17 and 20-29 and a pharmaceutically acceptable carrier, excipient or stabilizer.
31. A composition comprising:
a combination of two or more antibodies or antigen-binding fragments thereof as claimed in any one of claims 1-3, 10-17 and 20-29, and a pharmaceutically acceptable carrier, excipient or stabilizer.
32. The composition as claimed in any one of claims 7, 8, 30 or 31, further comprising an antitumor agent, an immunostimulatory agent, an immunomodulator or a combination thereof.
33. The composition of claim 32, wherein the antitumor agent is a cytotoxic agent, an agent that acts on tumor neovasculature or a combination thereof.
34. The composition of claim 32, wherein the immunomodulator is .alpha.-interferon, .gamma.-interferon, tumor necrosis factor-.alpha. or a combination thereof.
35. The composition of claim 32, wherein the immunostimulatory agent is interleukin-2, an immunostimulatory oligonucleotide or a combination thereof.
36. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16, conjugated to a label.
37. The isolated antibody or antigen-binding fragment thereof of claim 36, wherein the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label and a chromophore label.
38. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16 conjugated to at least one therapeutic moiety.
39. The isolated antibody or antigen-binding fragment thereof of claim 38, wherein the therapeutic moiety is:
(a) a drug;
(b) a toxin or a fragment thereof;
(c) an enzyme or a fragment thereof; and/or (d) an immunostimulatory or immunomodulating agent.
40. The isolated antibody or antigen-binding fragment thereof of claim 39, wherein the drug is a cytotoxic drug.
41. The isolated antibody or antigen-binding fragment thereof of claim 40, wherein the cytotoxic drug is selected from the group consisting of calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ABA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, paclitaxel, doeetaxel, dolastatin 10, auristatin E and auristatin PHE.
42. The isolated antibody or antigen-binding fragment thereof of claim 39, wherein the immunostimulatory or immunomodulating agent is a cytokine, chemokine or adjuvant.
43. The isolated antibody or antigen-binding fragment thereof of claim 38, wherein the therapeutic moiety is an anti-tumor agent.
44. The isolated antibody or antigen-binding fragment thereof of claim 43, wherein the anti-tumor agent is an agent that acts on tumor neovasculature.
45. The isolated antibody or antigen-binding fragment thereof of claim 44, wherein the agent that acts on tumor neovasculature is a tubulin-binding agent or antiangiogenic agent.
46. The isolated antibody or antigen-binding fragment thereof of claim 45, wherein the tubulin-binding agent is combrestatin A4, angiostatin, endostatin or interferon inducible protein 10.
47. The isolated antibody or antigen-binding fragment thereof of claim 45, wherein the antiangiogenic agent is 2ME2, Angiostatin, Angiozyme, Anti-VEGF RhuMAb, Apra (CT-2584), Avicine, Benefin, BMS275291, Carboxyamidotriazole, 25 CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC 412), CM101, Combretastatin A-4 Prodrug, EMD
121974, Endostatin, Flavopiridol, Genistein (GCP), IM-862, ImmTher, Interleukin-12, Iressa (ZD1839), Marimastat, Metastat (Col-3), Neovastat, Octreotide, Penicillamine, Photofrin, Photopoint, PI-88, Prinomastat (AG-3340), PTK787 (ZK22584), R0317453, 30 Solimastat, Squalamine, SU 101, SU 5416, SU-6668, Suradista (FCE 26644), Suramin (Metaret), Tetrathiomolybdate, Thalidomide, TNP-470 or Vitaxin.
48. The isolated antibody or antigen-binding fragment thereof of claim 39, wherein the immunomodulating agent is .alpha.-interferon, .gamma.-interferon or tumor necrosis factor alpha (TNF.alpha.).
49. A composition comprising:
the antibody or antigen-binding fragment as claimed in claim 36 or 38 and a pharmaceutically acceptable carrier, excipient, or stabilizer.
50. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16, conjugated to a radioisotope.
51. The isolated antibody or antigen-binding fragment thereof of claim 50, wherein the radioisotope:
(a) emits a radiations, .beta. radiations and/or .gamma. radiations; and/or (b) is selected from the group consisting of 225AC, 211At, 212Bi, 213Bi, 186Rh, 188Rh, 177Lu, 90Y, 131I, 67Cu, 125I, 123I, 77Br, 153Sm, 166Ho, 64Cu, 212Pb, 224Ra and 223Ra.
52. A composition comprising the isolated antibody or antigen-binding fragment thereof as claimed in claim 50 or 51 and a pharmaceutically acceptable carrier, excipient, or stabilizer.
53. A kit for detecting PSMA-expressing cancer for diagnosis, prognosis or monitoring comprising:
the isolated labeled antibody or antigen-binding fragment thereof of claim 36, and one or more compounds for detecting the label.
54. The kit of claim 53, wherein the PSMA-expressing cancer is prostate cancer.
55. The kit of claim 53, wherein the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label and a chromophore label.
56. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16 packaged in lyophilized form or in an aqueous medium.
57. A method for detecting the presence of PSMA protein dimers, or a cell expressing PSMA protein dimer, in a sample comprising:
contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16 for a time sufficient to allow the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the PSMA-antibody complex or PSMA-antigen-binding fragment complex, wherein the presence of a complex in the sample is indicative of the presence in the sample of PSMA
protein dimers or a cell expressing PSMA protein dimer.
58. A method for diagnosing a PSMA-expressing cancer in a subject, said method comprising:
administering to a subject suspected of having, or previously diagnosed with, PSMA-expressing cancer an amount of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16;
allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the PSMA-antibody complex or PSMA-antigen-binding fragment complex, wherein the presence of a complex in the subject suspected of having, or previously diagnosed with, PSMA-expressing cancer is indicative of the presence of a PSMA-expressing cancer.
59. A method for assessing the prognosis of a subject with a PSMA-expressing cancer, said method comprising:
administering the antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16 to a subject suspected of having or previously diagnosed with PSMA-expressing cancer, allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the complex, wherein the amount of the complex in the subject suspected of having or previously diagnosed with PSMA-expressing cancer is indicative of the prognosis.
60. A method for assessing the effectiveness of a treatment of a subject with a PSMA-expressing cancer, said method comprising:

administering the antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16 to a subject treated for a PSMA-expressing cancer, allowing the formation of a complex between the antibody or antigen-binding fragment thereof and PSMA, and detecting the formation of the complex, wherein the amount of the complex in the subject suspected of having or previously diagnosed with PSMA-expressing cancer is indicative of the effectiveness of the treatment.
61. The method of any one of claims 58-60, wherein the PSMA-expressing cancer is prostate cancer.
62. The method of any one of claims 57-60, wherein the antibody or antigen-binding fragment thereof is labeled, and/or wherein a second antibody is administered to detect the first antibody or antigen-binding fragment thereof.
63. Use of an effective amount of the antibody or antigen-binding fragment thereof of claim 38 for inhibiting the growth of a cell expressing PSMA protein dimer.
64. Use an effective amount of the antibody or antigen-binding fragment thereof of claim 38 for inducing cytolysis of a cell expressing PSMA protein dimer.
65. Use of the antibody or antigen-binding fragment thereof of claim 38 for treating or preventing a PSMA-expressing cancer in a subject having a PSMA-expressing cancer or at risk of having a PSMA-expressing cancer.
66. The use of claim 65, wherein the PSMA-expressing cancer is prostate cancer.
67. The use of claim 65, wherein another therapeutic agent to treat or prevent the PSMA-expressing cancer is used at any time before, during or after the antibody or antigen-binding fragment thereof.
68. The use of claim 67, wherein:
(a) the therapeutic agent is a vaccine which immunizes the subject against PSMA;
(b) the antibody or antigen-binding fragment thereof is conjugated to at least one therapeutic moiety, and/or (c) the antibody or antigen-binding fragment thereof is conjugated to a radioisotope.
69. The use of claim 68, wherein the therapeutic moiety is a cytotoxic drug, a drug which acts on the tumor neovasculature or a combination thereof.
70. The use of claim 69, wherein the cytotoxic drug is selected from the group consisting of calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, paclitaxel, docetaxel, dolastatin, 10, auristatin E and auristatin PHE.
71. The use of claim 68, wherein the radioisotope:
(a) emits .alpha. radiations, .beta. radiations and/or .gamma. radiations;
and/or (b) is selected from the group consisting of 225AC, 211At, 212Bi, 213B/, 186Rh, 188Rh, 177Lo, 90Y, 131I, 67Cu, 125I, 123I, 77Br, 153Sm, 166Ho, 64Cu, 212PD , 224Ra and 223Ra.
72. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16 for inhibiting folate hydrolase, N-acetylated .alpha.-linked acidic dipeptidase (NAALADase), dipeptidyl dipeptidase IV or .gamma.-glutamyl hydrolase activity, under conditions wherein the isolated antibody or antigen-binding fragment thereof inhibits the folate hydrolase, NAALADase, dipeptidyl dipeptidase IV or .gamma.-glutamyl hydrolase activity.
73. The use of claim 72, wherein the folate hydrolase, N-acetylated .alpha.-linked acidic dipeptidase (NAALADase), dipeptidyl dipeptidase IV or .gamma.-glutamyl hydrolase polypeptide is isolated and/or is contained in a sample selected from the group consisting of a cell, a cell homogenate, a tissue and a tissue homogenate.
74. The use of claim 72, wherein the folate hydrolase, N-acetylated .alpha.-linked acidic dipeptidase (NAALADase), dipeptidyl dipeptidase IV or .gamma.-glutamyl hydrolase polypeptide is contained in a mammal.
75. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-3, 10-12, 15 and 16 conjugated to at least one therapeutic agent for specific delivery of said at least one therapeutic agent to PSMA-expressing cells.
76. The use of claim 75, wherein the therapeutic agent is:
(a) an antitumor drug;
(b) a toxin or a fragment thereof;
(c) an enzyme or a fragment thereof; and/or (d) an immunostimulatory or immunomodulating agent.
77. The use of claim 76, wherein the antitumor drug is a cytotoxic drug, a drug which acts on the tumor neovasculature or a combination thereof.
78. The use of claim 77, wherein the cytotoxic drug is selected from the group consisting of calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C; vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin PHE.
79. The use of claim 76, wherein the immunostimulatory or immunomodulating agent is a cytokine, chemokine or adjuvant.
80. The use of claim 77, wherein the drug that acts on the tumor neovasculature is a tubulin-binding agent or antiangiogenic agent.
81. The use of claim 80, wherein the tubulin-binding agent is combrestatin A4, angiostatin, endostatin or interferon inducible protein 10.
82. The use of claim 80, wherein the antiangiogenic agent is 2ME2, Angiostatin, Angiozyme, Anti-VEGFRhuMAb, Apra (CT-2584), Avicine, Benefin, BMS275291, Carboxyamidotriazole, 25 CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC 412), CM101, Combretastatin A-4 Prodrug, EMD 121974, Endostatin, Flavopiridol, Genistein (GCP), IM-862, ImmTher, Interleukin-12, Iressa (ZD1839), Marimastat, Metastat (Col-3), Neovastat, Octreotide, Penicillamine, Photofrin, Photopoint, PI-88, Prinomastat (AG-3340), PTK787 (ZK22584), R0317453, 30 Solimastat, Squalamine, SU 101, SU 5416, SU-6668, Suradista (FCE 26644), Suramin (Metaret), Tetrathiomolybdate, Thalidomide, TNP-470 or Vitaxin.
83. The use of claim 79, wherein the immunomodulating agent is .alpha.-interferon, .gamma.-interferon or tumor necrosis factor alpha (TNF.alpha.).
84. A plasmid which encodes the antibody or antigen-binding fragment thereof of any one of claims 11-16.
85. A plasmid selected from the group consisting of AB-PG1-XG1-006 Heavy Chain (SEQ ID NO: 2) (ATCC patent deposit designation PTA-4403), AB-PG1-XG1-Light Chain (SEQ ID NO: 8) (ATCC patent deposit designation PTA-4404), AB-PG1-XG1-026 Heavy Chain (SEQ ID NO: 3) (ATCC patent deposit designation PTA-4405), AB-PG1-XG1-026 Light Chain (SEQ ID NO: 9) (ATCC patent deposit designation PTA-4406), AB-PG1-XG1-051 Heavy Chain (SEQ ID NO: 4) (ATCC patent deposit designation PTA-4407), AB-PG1-XG1-051 Light Chain (SEQ ID NO: 10) (ATCC patent deposit designation PTA-4408), AB-PG1- XG1-077 Heavy Chain (SEQ
ID
NO: 6) (ATCC patent deposit designation PTA-4411), and AB-PG1-XG1-077 Light Chain (SEQ ID NO: 12) (ATCC patent deposit designation PTA-4412).
86. A host cell transformed or transfected by the plasmid as claimed in claim 84 or 85.
87. A hybridoma cell line that produces an antibody selected from the group consisting of PSMA 3.9 (ATCC patent deposit designation PTA-3258) and PSMA 5.4 (ATCC patent deposit designation PTA-3268).
88. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein each of the PSMA proteins forming the dimer is a recombinant soluble PSMA
(rsPSMA) polypeptide consisting of amino acids 44-750 of SEQ ID NO: 1.
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