WO2000075348A1 - Recombinant anti-cd40 antibody and uses thereof - Google Patents

Recombinant anti-cd40 antibody and uses thereof Download PDF

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Publication number
WO2000075348A1
WO2000075348A1 PCT/US2000/015749 US0015749W WO0075348A1 WO 2000075348 A1 WO2000075348 A1 WO 2000075348A1 US 0015749 W US0015749 W US 0015749W WO 0075348 A1 WO0075348 A1 WO 0075348A1
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Prior art keywords
seq
protein
binding
atcc
monoclonal antibody
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PCT/US2000/015749
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French (fr)
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WO2000075348A9 (en
Inventor
Clay B. Siegall
Alan F. Wahl
Joseph A. Francisco
Henry P. Fell, Jr.
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Seattle Genetics, Inc.
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Priority to EP00939679A priority Critical patent/EP1190074B1/en
Priority to IL14695000A priority patent/IL146950A0/en
Priority to CA2376485A priority patent/CA2376485C/en
Priority to JP2001501630A priority patent/JP2003519470A/en
Application filed by Seattle Genetics, Inc. filed Critical Seattle Genetics, Inc.
Priority to HU0301789A priority patent/HUP0301789A2/en
Priority to AU54731/00A priority patent/AU784313B2/en
Priority to MXPA01012614A priority patent/MXPA01012614A/en
Priority to AT00939679T priority patent/ATE519501T1/en
Priority to BR0011394-8A priority patent/BR0011394A/en
Priority to NZ516250A priority patent/NZ516250A/en
Publication of WO2000075348A1 publication Critical patent/WO2000075348A1/en
Priority to IL146950A priority patent/IL146950A/en
Priority to NO20016007A priority patent/NO20016007L/en
Publication of WO2000075348A9 publication Critical patent/WO2000075348A9/en
Priority to HK03101596.3A priority patent/HK1049500A1/en

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    • 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
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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)

Definitions

  • the present invention relates to methods and compositions for the treatment of diseases and disorders, including cancer, inflammatory diseases or disorders and diseases or disorders of the immune system, comprising administering a CD40 binding protein which enhances binding of CD40 ligand to CD40.
  • CD40 binding proteins include recombmant/variant forms of monoclonal antibody S2C6 and derivatives thereof.
  • CD40 is a cell surface phosphorylated glycoprotem that is expressed on a variety of cell types, including B cells, B cell malignancies, follicular dendritic cells, basal epithelial cells, and carcinomas.
  • CD40 binds CD40 ligand ("CD40L") .
  • CD40L is expressed on activated T cells during inflammation and cancer (Younes et al . , 1998, Br. J. Haematol . 100:135-141; for a review see Grewal and Flavell, 1998, Annu. Rev. Immunol. 16:111-135).
  • CD40-med ated signaling in B cell -derived tumor lines can result in activation- induced cell death.
  • the strength of the activation signal is key to activation- induced tumor cell death (Grafton et al . , 1997, Cell. Immunol. 182:45-56). Therefore, compositions and methods for increasing receptor- ligand interaction and strength of activation signal between CD40 and CD40L would be of great value in treating disease.
  • CD40 AND CD40 LIGAND CD40 is a member of the TNF receptor superfamily. This family includes TNFrll, CD40, CD30, LMP-1, LTBr, ATAR, OX-40 and 4 -IBB receptors.
  • CD40 is constitutively expressed on B- lymphocytes, macrophages and dendritic cells and is induced by cytokme activation on fibroblasts, endothelial cells and epithelial cells (Van Kooten and Banchereau, 1997, Curr . Opm. Imunol . , 9: 330-337) .
  • CD40 has also been shown to be highly expressed on many human carcinomas including lung, bladder, gastric, breast and ovarian cancers (Stamenkovic et al., 1989, EMBO J. 8:1403-1410).
  • the ligand for CD40 is a membrane protein that is expressed on activated T cells. Receptor binding of CD40L results m CD40 multimerization, the generation of activation signals (for antigen presenting cells such as dendritic cells, monocytes and B cells) and the generation of growth and differentiation signals (for cytokme-activated fibroblasts and epithelial cells) .
  • CD40 signals are transduced from the multime ⁇ zed receptor via recruitment of a series of TNF receptor-associated factors ("TRAFs") (Kehry, 1996, J. Immunol. 156:2345-2348). Subsets of TRAFs interact differentially with TNF family members, including CD40, to provide stimuli to a wide variety of downstream pathways.
  • TNF receptor-associated factors TNF receptor-associated factors
  • TRAF1 and TRAF2 are implicated m the modulation of apoptosis (Speiser et al . , 1997, J. Ex . Med. 185:1777-1783; Yeh et al . , 1997, Immunity 7:715-725).
  • TRAFs 2, 5, and 6 participate m proliferation and activation events, including NF-kB and c-Jun N-terminal kmase activation.
  • binding of CD40 recruits TRAF2 and TRAF3 to the receptor complex and induces down-regulation of other TRAFs (Kuhune et al . , 1997, J. Exp. Med.186: 337-342).
  • CD40 binding is also dependent on membrane density (De Paoli et al . , 1997, Cytometry 30:33-38). Importantly, unlike the proliferative response seen with normal primary B cells, CD40 binding on neoplastic B cells can result m growth inhibition and activation-induced cell death (Funakoshi et al . , 1994, Blood 83:2787-2794). Thus, CD40 activation m the context of different cell types, transformation, resident TRAFs and co-stimuli can induce responses ranging from activation and proliferation to growth inhibition and apoptosis . 2.2 ANTI-CD40 ANTIBODIES
  • the ant ⁇ -CD40 monoclonal antibodies (“mAbs”) described to date are of three general classes: (1) those that block CD40/CD40L interaction by at least 90% and have anti-neoplastic properties (Armitage et al . , U.S. Patent No. 5,674,492; Fanslow et al . , 1995, Leukocyte Typing V, Schlossman et al . , eds . , 1:555-556); (2) those that antagonize signaling through CD40 (deBoer et al . , U.S. Patent No.
  • CD40.4 (PharMmgen, San Diego, California) , has been shown to increase the interaction between CD40 and CD40L by approximately 30-40% (Schlossman et al . , eds., 1995, Leukocyte Typing V: White Cell Differentiation Antigens 1:547-556).
  • Armitage et al . (U.S. Patent No. 5,674,492) describes methods using CD40 binding proteins, including mAb HuCD40-M2, that are capable of binding CD40 and inhibiting the binding of CD40 to CD40L, for preventing or treating disease characterized by neoplastic cells expressing CD40.
  • U.S. Patent No. 5,677,165 describes ant ⁇ -CD40 mAbs that, being free of significant agonistic activity, bind to CD40 on the surface of B-cells, and block B-cell activation.
  • An essential feature of U.S. Patent No. 5,677,165 is that upon binding of the ant ⁇ -CD40 mAb to human CD40 on the surface of normal human B cells, the growth or differentiation of normal human B cells is inhibited.
  • G28-5 binds to the B cell surface antigen Bp50 (now designated CD40) and stimulates activated B cells to traverse the cell cycle such that B cell proliferation is augmented.
  • Bp50 now designated CD40
  • G28-5 does not enhance activation of B cells m the presence of CD40L, and does not potentiate CD40/CD40L interaction.
  • S2C6 is an ant ⁇ -CD40 mAb that was prepared against a human bladder carcinoma (Paulie et al . , 1984, Cancer Immunol. Immunother. 17:165-179).
  • S2C6 binds to the CD40 receptor expressed on a variety of cell types including B- lymphocytes, endothelial and epithelial cells. S2C6 has been shown to have specificity toward neoplastic urothelium and B cell- de ⁇ ved malignant lymphocytes. Reactivity with a prostatic carcinoma cell line, HS, and weak reactivity with a melanoma has also been shown (Paulie et al . , 1984, Cancer Immunol. Immunother. 17:165-179). Studies have suggested the utility of S2C6 as a diagnostic marker for B cell malignancies (Paulie et al . , 1984, Cancer Immunol. Immunother. 17:165-179; Paulie et al .
  • S2C6 has agonistic activity on human peripheral B cells as demonstrated by its ability to stimulate primary B cell proliferation in a dose dependent manner (Paulie et al . , 1989, J. Immunol. 142:590-595).
  • Applicants have made the unexpected discovery of a new class of ant ⁇ -CD40 antibodies that, addition to delivering a stimulatory signal, enhances the interaction between CD40 and CD40L, enhances CD40L-med ⁇ ated stimulation and has m vivo anti-neoplastic activity. Production and use of these antibodies and related molecules are facilitated by the inventors ' cloning and sequencing of the variable region of mAb S2C6, and identification of the CDR and framework regions therein.
  • the present invention relates to molecules comprising the variable domain of mAb S2C6 or one or more of the complementarity-determining regions (CDRs) thereof having novel sequences (SEQ ID NO: 3, 4, 8, 9 or 10), which molecules (a) lmmunospecifically bind CD40 and (b) comprise one or more substitutions or insertions in primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hyb ⁇ doma deposited with the ATCC and assigned accession number PTA-110, or are not monoclonal antibody S2C6 as secreted by the hyb ⁇ doma deposited with the ATCC and assigned accession number PTA-110 and do not result from cleavage of S2C6 with papain or pepsin.
  • the molecules are not native monoclonal antibody S2C6 and do not comprise the native heavy or light chain of said monoclonal antibody S2C6.
  • the molecule is an antibody.
  • the antibody is not isotype IgGl .
  • the molecule comprises a light chain variable domain, the ammo acid sequence of SEQ ID NO: 2, or a heavy chain variable domain, the ammo acid sequence of SEQ ID NO: 7.
  • the invention further relates to chime ⁇ c/fusion proteins comprising a fragment of mAb S2C6 fused to an ammo acid sequence of a second protein, as well as to molecules wherein a fragment of mAb S2C6 is covalently bound (e.g., by use of a crosslinking agent) to another chemical structure.
  • a molecule is provided that immunospecifically binds CD40, which molecule comprises the heavy and/or light chain variable domain of mAb S2C6 fused to a second protein comprising the ammo acid sequence of bryodin 1 (BD1) .
  • the invention further relates to proteins comprising an ammo acid sequence that has at least 95% identity to SEQ ID NO: 2, SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which proteins (a) immunospecifically bind CD40 and (b) comprise one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hyb ⁇ doma deposited with the ATCC and assigned accession number PTA-110.
  • the proteins are not native monoclonal antibody S2C6 and do not comprise the native heavy or light chain of said monoclonal antibody S2C6.
  • the invention further relates to purified proteins, which proteins (a) compete for binding to CD40 with monoclonal antibody S2C6 as secreted by the hyb ⁇ doma deposited with the ATCC and assigned accession number PTA- 110, (b) increase the binding of CD40 ligand to CD40 by at least 45%, and (c) comprise one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hyb ⁇ doma deposited with the ATCC and assigned accession number PTA- 110, or are not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110 and do not result from cleavage of S2C6 with papam or pepsin.
  • the proteins are not native monoclonal antibody S2C6 and do not comprise the native heavy or light chain of said monoclonal antibody S2C6.
  • the invention further relates to nucleic acids encoding such molecules and proteins or which hybridize to a DNA consisting of the nucleotide sequence encoding such proteins; recombmant cells comprising such molecules and proteins, and methods of producing such proteins.
  • the isolated nucleic acid comprises a nucleotide sequence encoding a protein comprising (a) a heavy chain variable domain of monoclonal antibody S2C6 as secreted by the hyb ⁇ doma deposited with the ATCC and assigned accession number PTA-110, and (b) a human constant region.
  • the isolated nucleic acid comprises a nucleotide sequence encoding a protein comprising (a) a light chain variable domain of monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and (b) a human constant region.
  • the invention further relates to recombmant cells containing a recombmant nucleic acid vector comprising a nucleotide sequence encoding a protein, which protein competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and which protein increases the binding of CD40 ligand to CD40 by at least 45%.
  • the invention also provides methods of producm ⁇ such proteins comprising growing such cells such that the protein is expressed by the cell, and recovering the expressed protein.
  • the invention further relates to recombmant cells containing a recombmant nucleic acid vector comprising SEQ
  • SEQ ID NO: 13 SEQ ID NO: 14, or SEQ ID NO: 15 and methods of producing proteins comprising growing such cells, such that a protein encoded by the nucleotide sequence is expressed by the cell, and recovering the expressed protein.
  • compositions containing the molecules and antibodies of the invention are also provided.
  • the invention relates to pharmaceutical compositions comprising a molecule comprising SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO: 7, SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (I) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40, and (m) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110; and a pharmaceutically acceptable carrier.
  • the molecule is not native monoclonal antibody S2C6 and does not comprise the native heavy or light chain of said monoclonal antibody
  • the invention further relates to pharmaceutical compositions comprising a purified protein, which protein (1) competes for binding to CD40 with mAb S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (11) increases the binding of CD40 ligand to
  • CD40 by at least 45%, and (m) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as deposited with the ATCC and assigned accession number PTA-110, or is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110 and does not result from cleavage of S2C6 with papam or pepsin; and a pharmaceutically acceptable carrier.
  • the protein is not native monoclonal antibody S2C6 and does not comprise the native heavy or light chain of said monoclonal antibody S2C6.
  • compositions of the invention contain the molecules or antibodies of the invention m an amount effective for the treatment or prevention of cancer, or an amount effective for activating or augmenting an immune response, or an amount such that the immune response of the subject is activated or augmented.
  • the pharmaceutical compositions of the invention further comprise CD40 ligand.
  • the pharmaceutical composition comprises m an amount effective for the treatment or prevention of cancer or an immune disorder, or for activating or augmenting an immune response: (a) a molecule that immunospecifically binds CD40, which molecule increases the binding of CD40 ligand to CD40; (b) CD40 ligand; and (c) a pharmaceutically acceptable carrier.
  • the molecule can be native mAb S2C6 or native mAb 5C3 or an S2C6 derivative as
  • the invention further relates to methods for the treatment or prevention of cancer a subject, for activating or augmenting an immune response a subject, or for the treatment or prevention of an immune deficiency or
  • r disorder a subject comprising administering to the subject a therapeutically effective amount of the molecules or antibodies of the invention, e.g., an amount of a molecule comprising SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO: 2 , SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO: 2 , SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO
  • SEQ ID NO: 7 SEQ ID NO: 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (l) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and comprises one or more substitutions or insertions primary ammo acid sequence relative to native monoclonal antibody
  • the molecule is not native monoclonal antibody S2C6 and does not comprise the native heavy or light chain of said monoclonal antibody S2C6.
  • the invention further relates to methods for the treatment or prevention of cancer a subject, for 5 activating or augmenting an immune response a subject, or for the treatment or prevention of an immune deficiency or disorder a subject comprising administering to the subject a purified protein, which protein d) competes for binding to
  • CD40 with monoclonal antibody S2C6 as secreted by the 0 hybridoma deposited with the ATCC and assigned accession number PTA-110 increases the binding of CD40 ligand to CD40 by at least 45%, and (m) comprises one or more substitutions or insertions m the primary ammo acid sequence relative to native monoclonal antibody S2C6 as 5 deposited with the ATCC and assigned accession number PTA- 110, or is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110 and does not result from cleavage of S2C6 with papain or pepsin.
  • the protein is not native monoclonal antibody S2C6 and does not comprise the 5 native heavy or light chain of said monoclonal antibody S2C6.
  • the methods of the invention further comprise administering CD40 ligand to the subject.
  • the invention further relates to a method for the treatment or prevention of cancer or an immune disorder in a 1 ⁇ subject comprising administering to the subject, in an amount effective for said treatment or prevention: (a) a molecule that immunospecifically binds CD40, which molecule increases the binding of CD40 ligand to CD40; and (b) CD40 ligand, in which the molecule can be native mAb S2C6 or native mAb 5C3 or any of the S2C6 derivatives described herein.
  • the subject is a human.
  • the invention further relates to a transgenic non-human animal, plant, or an isolated cell containing one or more transgenes encoding a protein, which protein competes for binding to CD40 with monoclonal antibody S2C6 as secreted by 20 the hybridoma deposited with the ATCC and assigned accession number PTA-110, and which protein increases the binding of
  • FIG. 1 Structure of the light chain variable region of S2C6.
  • the nucleotide (SEQ ID NO:l) and amino acid (SEQ ID NO:l) are nucleotide and amino acid (SEQ ID NO:l).
  • V L variable region
  • FIGS 3A-3B Structure of variable regions of S2C6.
  • A The amino acid sequence (SEQ ID NO: 2) of S2C6 V L is shown.
  • B The amino acid sequence (SEQ ID NO: 7) of S2C6 V H is shown.
  • Complementarity-determining regions are underlined. The sequences of the four framework regions, adjacent to the CDRs , are shown.
  • the ammo acid sequences of V L CDRs 1-3 correspond to SEQ ID NOS:3-5, respectively.
  • the ammo acid sequences of V H CDRs 1-3 correspond to SEQ ID NOS:8-10, respectively.
  • FIG. 4 S2C6 mAb augments CD40-Ig binding to CD40L- expressmg Jurkat T cells.
  • CD40-Ig a soluble fusion protein of CD40 and human lmmunoglobulm binding to surface CD40L was done m the presence of increasing concentrations of ant ⁇ -CD40 monoclonal antibody ("mAb") .
  • mAbs were pre- mcubated for 1 hour with CD40-Ig followed by incubation for
  • CD40-Ig binding to target cells was detected by flow cytometry using a fluorescem isothiocyanate ( "FITC” ) -labeled anti-human Ig.
  • FITC fluorescem isothiocyanate
  • MFI log mean fluorescent intensity
  • Ramos B cells a human B cell lymphoma, were incubated m the presence of increasing concentrations of an ant ⁇ -CD40 mAb: S2C6, G28-5, or M3 or an irrelevant control mAb, EXA2-1H8. The mAbs were pre-incubated for 1 hour with CD40-expressmg target cells. Binding of the FITC- labeled CD40L to B cells was then detected directly by flow cytometry. The extent of CD40/CD40L binding was then determined from log mean fluorescent intensity. MFI minus background of each population is shown.
  • EXA2-1H8 B cell proliferation was measured by J H-TdR incorporation at 72 h following addition of stimulus.
  • Figure 7. Comparative proliferative response of primary human peripheral B cells to an ant ⁇ -CD40 mAb m the presence or absence of CD40L. Peripheral B cells were combined with non-proliferative CD40L + stimulator cells at a fixed ratio of 4:1 and increasing concentrations of an ant ⁇ -CD40 mAb. S2C6 G28-5 or the control antibody, EXA2-1H8. B cell proliferation was measured by J H-TdR incorporation at 72 h following addition of stimulus.
  • Figure 7. Comparative proliferative response of primary human peripheral B cells to an ant ⁇ -CD40 mAb m the presence or absence of CD40L. Peripheral B cells were combined with non-proliferative CD40L + stimulator cells at a fixed ratio of 4:1 and increasing concentrations of an ant ⁇ -CD40 mAb. S2C6 G28-5 or the control antibody, EXA
  • FIGS 8A-8C Anti-tumor activity of mAb S2C6 m vivo . Anti-tumor activity of S2C6 against (A) Ramos human B cell non-Hodgkm's lymphoma, (B) HS Sultan multiple myeloma, or
  • Solid lines indicate the number of surviving mice over time.
  • BD1-S2C6 sFv specifically binds to immobilized CD40-Ig ELISA.
  • BD1-S2C6 sFv single-chain ant ⁇ -CD40 lmmunotoxm consisting of bryodin 1 (BD1) fused to the variable region of monoclonal antibody S2C6 was expressed m E. coll as inclusion bodies, denatured and refolded. The refolded protein was then isolated using Blue 0
  • BDl-specific rabbit antiserum followed by the addition of horseradish peroxidase conjugated goat antl -rabbit Ig.
  • the 0 binding of BD1-S2C6 sFv to CD40-Ig was completely inhibited by the addition of excess S2C6 mAb but not by the addition of the control mAb.
  • the present invention relates to proteins encoded by and nucleotide sequences of S2C6 genes.
  • the invention further relates to fragments and other derivatives and analogs of such S2C6 proteins and nucleic acids.
  • the molecules (e.g., antibodies) of the invention comprise all or a portion of mAb S2C6 (the light chain and/or heavy chain, or light chain CDR 1 (SEQ ID NO : 3 ) and/or 2 (SEQ ID NO : 4 ) , and/or heavy chain CDR 1 (SEQ ID NO: 8), 2 (SEQ ID NO: 9), and/or 3 (SEQ ID NO: 10), or light chain CDR3 (SEQ ID NO: 5) m combination with any of the other CDRs and/or one or more of the four heavy chain and four light chain framework regions, provided that such molecules
  • mAb S2C6 are not native mAb S2C6 as deposited with the ATCC and assigned accession number PTA-110 or the heavy or light chain thereof. Such molecules may differ from S2C6 m sequence and/or m post-translational modification (glycosylation, amidation, peptide bonding or cross-linking to a non-S2C6
  • a molecule of the invention immunospecifically binds CD40 (or when multime ⁇ zed immunospecifically binds CD40) , competes with native S2C6 for binding to CD40, and/or increases the binding of CD40 ligand to CD40 by at least 45%, 50%, 60% or 65%.
  • ⁇ or derivatives are also withm the scope of the invention, as well as nucleic acids encoding native mAb S2C6. Production of the foregoing proteins, e . g. , by recombmant methods, is provided.
  • the invention also relates to S2C6 proteins and
  • 25 derivatives including but not limited to fusion/chime ⁇ c proteins which are functionally active, i . e . , which are capable of displaying one or more known functional activities associated with a full-length S2C6 mAb.
  • Such functional activities include but are not limited to ability to bind CD40, delivery of a stimulatory signal to the CD40 signaling pathway (e.g., so as to cause B cell proliferation), potentiation of the interaction of CD40L with CD40; ability to inhibit tumor growth; and ability to induce an immune response .
  • Antibodies to CD40 comprising S2C6, its derivatives and 5 analogs including but not limited to humanized antibodies; single chain antibodies; bispecific antibodies; and antibodies conjugated to chemotherapeutic agents or biological response modifiers, are additionally provided.
  • the invention further relates to methods of treating or preventing cancer, inflammatory diseases and disorders of the immune system comprising administering a composition of the invention alone or combination with CD40L.
  • Sections 6-9 disclose, inter alia , the cloning and characterization of S2C6 genes; the potentiation of the CD40/CD40L interaction; inhibition of tumor growth; 10 and binding of a single-chain ant ⁇ -CD40 immunotoxm to CD40- Ig.
  • S2C6 nucleic acids comprise the cDNA sequences of SEQ ID NOS : 1 and 6, or nucleic acids encoding an S2C6 protein ⁇ e . g. , a protein
  • the invention provides purified nucleic acids consisting of at least 8 nucleotides (i.e., a hybridizable portion) of an S2C6 gene sequence; m other embodiments, the nucleic acids consist of at least 25 (contiguous) nucleotides, 50 nucleotides, 100, or
  • nucleic acids 25 200 nucleotides of an S2C6 sequence, or a full-length S2C6 variable region coding sequence.
  • the nucleic acids are smaller than 50, 75, 100, or 200 or 5000 nucleotides m length. Nucleic acids can be single or double stranded.
  • the invention also relates to nucleic acids hybridizable to or complementary to the 30 foregoing sequences or their reverse complements, and m particular, such nucleic acids that encode proteins that bind to CD40, compete with S2C6 for binding to CD40, and/or increase the binding of CD40 ligand to CD40 by at least 45%,
  • nucleic acids are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • 35 provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of an S2C6 variable region gene.
  • nucleic acids encoding derivatives and analogs of S2C6 proteins are additionally provided.
  • a "nucleic acid encoding a fragment or portion of an S2C6 protein” shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the S2C6 protein and not the other contiguous portions of the S2C6 protein as a continuous sequence.
  • RNA is isolated from a mAb S2C6-producmg hybridoma and polymerase chain reaction is used to amplify desired variable region sequences, using 5 primers based on the sequences disclosed herein.
  • polymerase chain reaction is used to amplify desired variable region sequences, using 5 primers based on the sequences disclosed herein.
  • mRNA is isolated from a mAb S2C6 -producing hybridoma, cDNA is made and ligated into an expression vector (e.g., a bacte ⁇ ophage derivative) such that it is capable of 0 being expressed by the host cell into which it is then introduced.
  • an expression vector e.g., a bacte ⁇ ophage derivative
  • selection is on the basis of hybridization to a labeled probe representing a portion of an S2C6 gene or its RNA or a 5 fragment thereof (Benton and Davis, 1977, Science 196:180; Grunstem and Hogness, 1975, Proc . Natl . Acad . Sci . U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion (s) 0 and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene.
  • restriction enzyme digestion s
  • the presence of the desired gene may be r detected by assays based on the physical, chemical, or immunological properties of its expressed product.
  • assays based on the physical, chemical, or immunological properties of its expressed product.
  • cDNA clones, or DNA clones which hybrid- select the proper mRNAs can be selected and expressed to produce a protein that has, e . g . , similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, or functional activity, as known for an S2C6 protein.
  • ability to bind CD40 can be detected m an ELISA (enzyme-linked immunosorbent assay) -type procedure .
  • An S2C6 gene can also be identified by mRNA selection using nucleic acid hybridization followed by m vi tro Q translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Functional assays ⁇ e . g. , binding to CD40, etc.) of the m vi tro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences.
  • the S2C6 cDNA can be chemically synthesized from the sequence disclosed herein.
  • Other methods of isolating S2C6 genes known to the skilled artisan can be employed.
  • the identified and isolated S2C6 gene/cDNA can then be 0 inserted into an appropriate cloning vector.
  • a large number of vector-host systems known m the art may be used.
  • Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used.
  • Such vectors include, but are not 5 limited to, bacte ⁇ ophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives or the
  • the insertion into a cloning vector can, for example, be accomplished by ligatmg the DNA fragment into a cloning vector which has complementary 0 cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligatmg nucleotide sequences (linkers) onto the DNA termini; 5 these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and an S2C6 gene may be modified by homopolymeric tailing, or by PCR with primers containing the appropriate sequences . Recombmant molecules can be
  • the desired gene may be identified and isolated after insertion into a suitable
  • cloning vector m a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector.
  • S2C6 gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
  • the gene may be obtained m large quantities by growing transformants, isolating the recombmant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombmant DNA.
  • the S2C6 sequences provided by the instant invention include those nucleotide sequences encoding substantially the same am o acid sequences as found m native S2C6 variable regions, and those encoded ammo acid sequences with functionally equivalent ammo acids, as well as those
  • an appropriate expression vector i . e .
  • the necessary JJ transcriptional and translational signals can also be supplied by the native S2C6 gene and/or its flanking regions.
  • host-vector systems may be utilized to express the protem-codmg sequence. These include but are not limited to mammalian cell systems infected with virus ( e . g .
  • vaccinia virus adenovirus
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacte ⁇ ophage , DNA, plasmid DNA, or cosmid DNA; transgenic plants or transgenic non-human animals.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chime ⁇ c gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vi tro recombmant DNA and synthetic techniques and m vivo recombmants (genetic recombination) . Expression of a nucleic acid sequence encoding an S2C6 protein or peptide fragment may be regulated by a second nucleic acid sequence so that the S2C6 protein or peptide is expressed m a host transformed with the recombmant DNA molecule. For example, expression of an S2C6 protein may be controlled by any promoter/enhancer element known m the art.
  • Promoters that are not native S2C6 gene promoters which may be used to control S2C6 gene expression include, but are not limited to, the SV40 early promoter region (Benoist and
  • the promoter contained m the 3 ' long terminal repeat of Rous sarcoma virus (Yamamoto et al . , 1980, Cell 22:787-797), the herpes thymid e kmase promoter (Wagner et al . , 1981, Proc . Natl . Acad. Sci . U.S.A. 78:1441-1445), the regulatory sequences of the metallothionem gene (Brmster et al . , 1982, Nature 296:39- 42); prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff et al .
  • promoter elements from yeast i Q or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kmase) promoter, alkaline phosphatase promoter, and the following animal transcriptlonal control regions, which exhibit tissue specificity and have been utilized m transgenic animals: ⁇ elastase I gene control region which is active m pancreatic acinar cells (Swift et al . , 1984, Cell 38:639-646; Ornitz et al . , 1986, Cold Spring Harbor Symp . Quant. Biol .
  • beta- globm gene control region which is active m myeloid cells (Mogram et al . , 1985, Nature 315:338-340; Kollias et al . , 1986, Cell 46:89-94; myelm basic protein gene control region
  • a vector is used that comprises a promoter operably linked to an S2C6 gene nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene) .
  • Expression vectors containing S2C6 gene inserts can be identified by three general approaches: (a) nucleic acid hybridization; (b) presence or absence of "marker" gene functions; and (c) expression of inserted sequences.
  • the presence of an S2C6 gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted S2C6 gene.
  • the recombmant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of an S2C6 gene in the vector.
  • certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
  • recombmant expression vectors can be identified by assaying the S2C6 product expressed by the recombmant . Such assays can be based, for example, on the physical or functional properties of the S2C6 protein in in vitro assay systems, e . g. , potentiation of CD40L binding with
  • CD40 stimulation of proliferation of normal B cells; inhibition of tumor growth.
  • recombmant expression vectors can be propagated and prepared m quantity.
  • the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacte ⁇ ophage vectors (e.g., lambda phage) , and plasmid and cosmid DNA vectors, to name but a few.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product m the specific fashion desired. Expression from certain promoters can be elevated m the presence of certain mducers; thus, expression of the genetically engineered S2C6 protein may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce a non-glycosylated core protein product. Expression m yeast will produce a glycosylated product . Expression mammalian cells can be used to ensure "native" glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect processing reactions to d.i r ff r erent extents.
  • the S2C6-related protein that is expressed is an antibody or fragment or derivative thereof.
  • the recombmant antibody may contain a recombmant light chain variable domain, a recombmant heavy chain variable domain, or both.
  • both light and heavy chains or derivatives thereof are recombmantly expressed by a cell (see e.g., U.S. Patent No. 4,816,397 dated March 28, 1989 by Boss et al . )
  • a variety of host-vector systems may be utilized to express the protem- coding sequence.
  • viruses include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.) ; insect cell systems infected with virus ⁇ e . g . , baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA; transgenic 5 plants or transgenic non-human animals.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA
  • transgenic 5 plants or transgenic non-human animals include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.)
  • the invention provides ammo acid sequences of S2C6 proteins and fragments and derivatives thereof which comprise a complementarity-determining region (CDR) or which are otherwise functionally active, as well as nucleic acid sequences encoding the foregoing.
  • “Functionally active" S2C6 material as used herein refers to that material displaying one or more functional activities associated with a full-length (native) S2C6 protein, e.g., binding to CD40;
  • the invention provides fragments of an S2C6 protein consisting of at least 6 ammo
  • ⁇ n acids 10 ammo acids, 20 ammo acids, 50 ammo acids, 75 ammo acids or of at least 100 ammo acids and nucleic acids encoding the foregoing.
  • the gene product can be analyzed. This is achieved by assays based on the physical or 5 functional properties of the product, including radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay; stimulation of proliferation of normal B cells; CD40 binding assays, promotion of the binding of CD40 ligand to CD40, inhibition of tumor growth, etc.
  • S2C6 protein may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography) , centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the functional properties may be evaluated using
  • the S2C6 protein or derivative thereof can be synthesized by standard chemical methods known m the art based on the sequence disclosed herein (e.g., see Hunkapiller et al . , 1984, Nature 310:105-111).
  • such S2C6 proteins include but are not limited to those containing, as a primary ammo acid sequence, all or part of the ammo acid sequence substantially as depicted m Figures 3A-3B 10 (SEQ ID NOS:2 and 7), as well as fragments and other derivatives, and analogs thereof, including proteins homologous thereto.
  • S2C6 genes and proteins of the invention can be analyzed by various methods known the art. Some examples of such methods are described below.
  • the cloned DNA or cDNA corresponding to an S2C6 gene can be obtained from any source.
  • this invention provides nucleic acid probes recognizing an S2C6 gene.
  • S2C6 polymerase chain reaction
  • PCR polymerase chain reaction
  • Southern hybridization with an S2C6 gene-specifIC probe can allow the detection of an S2C6 gene m DNA or cDNA from a cell (e.g., hybridoma) .
  • Methods of amplification other than 5 PCR are commonly known and can also be employed.
  • the stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific S2C6 gene probe used. Modifications of these methods and other methods commonly known m the art can be used.
  • Restriction endonuclease mapping can be used to roughly determine the genetic structure of an S2C6 gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.
  • DNA sequence analysis can be performed by any techniques
  • ammo acid sequence of an S2C6 protein can be derived by deduction from the DNA sequence, or alternatively,
  • An S2C6 protein sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824).
  • a hydrophilicity profile can be 25 used to identify the hydrophobic and hydrophilic (potentially lmmunogenic) regions of the S2C6 protein and the corresponding regions of the gene sequence which encode such regions .
  • Biochemistry 13:222 can also be done, to identify regions of
  • Manipulation, translation, and secondary structure prediction, open reading frame prediction and plotting, as well as determination of sequence homologies, can also be accomplished using computer software programs available m
  • S2C6 ANTIBODY DERIVATIVES Described herein are methods for the production of S2C6 antibody derivatives capable of immunospecifically binding CD40
  • Such antibodies include but are not limited to monoclonal, humanized, chime ⁇ c, single chain, bispecific, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-bmdmg fragments of any of the above.
  • the S2C6 derivative comprises one or more deletions, additions and/or substitutions primary ammo acid sequence relative to the primary ammo acid sequence of S2C6.
  • the S2C6 derivative does not result from cleavage of S2C6 with papam or pepsin
  • the S2C6 derivative comprises one or more deletions, additions and/or substitutions primary ammo acid sequence relative to the primary ammo acid sequence of
  • S2C6 and does not result from cleavage of S2C6 with papam or pepsin.
  • Guidance for selection of suitable deletions, additions and/or substitutions is provided m this section and in Section 5.7, infra .
  • any technique that provides for the production of antibody molecules by continuous cell lines m culture may be used. These include but are not limited to the hybridoma technique of Kohler and Milste , (1975, Nature 256, 495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kozbor et al . , 1983, Immunology Today 4, 72; Cole et al . , 1983, Proc. Natl. Acad. Sci. USA 80, 2026-2030), and the EBV-hyb ⁇ doma technique to produce human monoclonal antibodies (Cole et al . , 1985, Monoclonal Antibodies And
  • Such antibodies or other ant ⁇ -CD40 antibodies available the art may, e.g., be used as the basis from which to clone and thus supply a complementary light chain if a S2C6 heavy chain is to be recombmantly expressed (the two chains may be recombmantly expressed in the same cell or combined m vitro after separate expression and purification) ,- alternatively, a light chain from an antibody of any specificity may be used.
  • Nucleic acids encoding a S2C6 heavy chain or encoding a molecule comprising a S2C6 heavy chain variable domain can be transfected into a cell expressing an antibody light chain or molecule comprising an antibody light chain, for expression of a multime ⁇ c protein; the antibody light chain can be recombmant or non-recombmant , and may or may not have ant ⁇ -CD40 specificity.
  • S2C6 heavy Q chains or molecules comprising the variable region thereof or a CDR thereof can optionally be expressed and used without the presence of a complementary light chain or light chain variable region.
  • the invention provides a S2C6 heavy chain with CD40 binding affinity, or a _. molecule consisting of or (alternatively) comprising one or more copies of heavy chain CDR 8, 9, and/or 10, or a protein
  • such a protein can be N or C-terminal modified, e.g., by C-terminal amidation or N-termmal 0 acetylation.
  • a chime ⁇ c antibody is a molecule m which different portions are derived from different animal species, 0 such as those having a variable region derived from a mu ⁇ ne mAb and a human immunoglobulin constant region.
  • the chime ⁇ c antibody comprises a variable domain of monoclonal J antibody S2C6 secreted by the hybridoma as deposited with the ATCC and assigned accession number PTA-110, and a human constant region.
  • the variable domain of the chimeric antibody comprises the S2C6 V L (SEQ ID NO : 2 ) as depicted m Figure 3A and/or the S2C6 V H (SEQ ID NO: 7) as depicted m Figure 3B.
  • techniques have been developed for the production of humanized antibodies. (See, e.g., Queen, U.S. Patent No. 5,585,089 and Winter, U.S. Patent No.
  • An lmmunoglobulm light or heavy chain variable region consists of a "framework" region interrupted by three hyperva ⁇ able regions, referred to as complementarity- determmmg regions (CDRs) .
  • CDRs complementarity- determmmg regions
  • the extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest", Rabat, E. et al . , U.S. Department of Health and Human Services (1983) .
  • humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and framework regions from a human lmmunoglobulm molecule .
  • the invention encompasses an antibody or derivative thereof comprising a heavy or light chain variable domain, said variable domain comprising (a) a set of three complementarity-determmmg regions (CDRs) , m which said set of CDRs are from monoclonal antibody S2C6, and (b) a set of four framework regions, m which said set of framework regions differs from the set of framework regions m monoclonal antibody S2C6, and in which said antibody or derivative thereof immunospecifically binds CD40.
  • CDRs complementarity-determmmg regions
  • the set of framework regions is from a human monoclonal antibody, e.g., a human monoclonal antibody that
  • the invention encompasses an antibody or derivative thereof comprising a light chain variable domain, said variable domain comprising (a) a set of three complementarity-determmmg regions (CDRs) , m which said set of CDRs comprises SEQ ID NO : 3 or SEQ ID NO : 4 , and (b) a set of four framework regions, m which said set of framework regions differs from the set of framework regions m the light chain of monoclonal antibody S2C6, and m which said antibody or derivative thereof immunospecifically binds CD40.
  • CDRs complementarity-determmmg regions
  • the invention encompasses an antibody or derivative thereof comprising a heavy chain variable domain, said variable domain comprising (a) a set of three complementarity-determmmg regions (CDRs) , m which said set of CDRs comprises SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, and (b) a set of four framework regions, m which said set of framework regions differs from the set of framework regions in the heavy chain of monoclonal antibody S2C6, and m which said antibody or derivative thereof immunospecifically binds CD40.
  • CDRs complementarity-determmmg regions
  • single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an am o acid bridge, resulting in a single chain polypeptide.
  • the single chain antibody comprises the ammo acid sequences as depicted in Figure 3A and 3B (SEQ ID NOS : 2 and 7, respectively) .
  • the antibody to a CD40 polypeptide, peptide or other derivative, or analog thereof comprising all or a portion of SEQ ID NO : 1 or SEQ ID NO : 6 is a bispecific antibody (see generally, e.g. Fanger and
  • Such a bispecific antibody is genetically engineered to recognize both (1) an epitope and (2) one of a variety of "trigger" molecules, e.g. Fc receptors on myeloid cells, and CD3 and CD2 on T cells, that have been identified as being able to cause a cytotoxic T-cell to destroy a particular target.
  • Trigger molecules e.g. Fc receptors on myeloid cells, and CD3 and CD2 on T cells, that have been identified as being able to cause a cytotoxic T-cell to destroy a particular target.
  • Such bispecific antibodies can be prepared either by chemical conjugation, hybridoma, or recombmant molecular biology techniques known to the skilled artisan.
  • the bispecific antibody contains a molecule comprising the S2C6 heavy or light chain variable domain or a CDR sequence thereof, which molecule has the structure of an antibody heavy or light chain but which differs from the native S2C6 heavy or light chain (e.g., by having ammo acid substitution (s) m the framework region or a human constant domain) .
  • Antibody fragments that retain the ability to recognize CD40 may be generated by known techniques.
  • fragments include but are not limited to: The F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and the F(ab') fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse, et al . , 1989, Science 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • the invention further relates to S2C6 proteins, derivatives (including but not limited to fragments), analogs, and molecules of S2C6 proteins.
  • Nucleic acids encoding S2C6 protein derivatives and protein analogs are also provided.
  • the S2C6 proteins are encoded by the nucleic acids described m Section 5.1 above.
  • proteins, derivatives, or analogs are encoded by the sequence of SEQ ID NO:l or SEQ ID NO : 6.
  • derivatives and analogs related to an S2C6 protein are withm the scope of the present invention.
  • the derivative or analog is functionally active, i . e . , capable of exhibiting one or more functional activities associated with a full- length, S2C6 protein.
  • such derivatives or analogs which have the desired binding specificity can be used m immunoassays, or therapeutically for inhibition of tumor growth, etc.
  • a specific embodiment relates to an S2C6 protein fragment that binds CD40 and potentiates binding of CD40L to CD40.
  • Derivatives or analogs of an S2C6 protein can be tested for the desired activity by various immunoassays known in the art, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays , enzyme linked immunosorbent assay (ELISA), "sandwich” immunoassays, Western blots, immunofluorescence assays, protein A assays, immunoelectrophoretic assays, etc.
  • ELISA enzyme linked immunosorbent assay
  • Western blots Western blots
  • immunofluorescence assays protein A assays
  • immunoelectrophoretic assays etc.
  • assays known in the art can be used to detect or measure the ability to inhibit cell proliferation (e.g., inhibition of tumor cell growth) or ability to stimulate cell proliferation (e.g., proliferation of B cells) in vivo or in vi tro .
  • S2C6 derivatives can be made by altering
  • S2C6 sequences by substitutions, additions ⁇ e . g. , insertions) or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as an S2C6 gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of an S2C6 gene which is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • the S2C6 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of an S2C6 protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more ammo acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucme, lsoleucme, valme, prolme, phenylalan e, tryptophan and methionme .
  • the polar neutral ammo acids include glyc e, ser e, threonme, cysterne, tyrosme, asparag e, and glutamme.
  • the positively charged (basic) ammo acids include argmme, lysine and histid e.
  • the negatively charged (acidic) ammo acids include aspartic acid and glutamic acid. Such substitutions are generally understood to be conservative substitutions.
  • proteins consisting of or comprising a fragment of an S2C6 protein consisting of at least 10 (continuous) ammo acids of the S2C6 protein is provided.
  • the fragment consists of at least 20 or at least 50 ammo acids of the S2C6 protein.
  • such fragments are not larger than 50, 75, 100, or 200 ammo acids.
  • Derivatives or analogs of S2C6 proteins include but are not limited to those molecules comprising regions that are substantially homologous to an S2C6 protein or fragment thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity over an ammo acid sequence of identical size with no insertions or deletions or when compared to an aligned sequence in which the alignment is done by a computer homology program known the art) or whose encoding nucleic acid is capable of hybridizing to a coding S2C6 gene sequence, under high stringency, moderate stringency, or low stringency conditions.
  • computer programs for determining homology may include but are not limited to
  • the BLASTP j 0 program compares an amino acid query sequence against a protein sequence database
  • the BLASTN program compares a nucleotide query sequence against a nucleotide sequence database
  • the BLASTX program compares the six- frame conceptual translation products of a nucleotide query ⁇ sequence (both strands) against a protein sequence database
  • the TBLASTN program compares a protein query sequence against a nucleotide sequence database translated in all six reading frames (both strands) ;
  • the TBLASTX program compares the six- frame translations of a nucleotide query sequence against the six- frame translations of a nucleotide sequence database.
  • the S2C6 derivatives and analogs of the invention can be produced by various methods known in the art .
  • the manipulations which result in their production can occur at 5 the gene or protein level.
  • a cloned S2C6 gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al . , 1989, Molecular Cloning, A Labora tory Manual , 2d ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) .
  • the sequence can be cleaved at appropriate sites with restriction endonuclease (s) , followed by further enzymatic modification if desired, isolated, and ligated in vi tro .
  • an S2C6 nucleic acid sequence can be mutated in vi tro or in vi vo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vi tro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vi tro site-directed 0 mutagenesis (Hutchinson et al . , 1978, J. Biol. Chem.
  • Manipulations of an S2C6 protein sequence may also be made at the protein level .
  • S2C6 protein fragments or other derivatives or 5 analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycm, etc.
  • analogs and derivatives of an S2C6 protein can be chemically synthesized. For example, a peptide corresponding to a portion of an S2C6 protein which comprises the desired domain, or which mediates the desired activity m
  • Non-classical ammo acids include but are not limited to the D-isomers of the common
  • ammo acid can be D
  • the S2C6 protein, 20 fragment, analog, or derivative may be expressed as a fusion, or chime ⁇ c protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence of a different protein) .
  • the heterologous protein sequence can comprise a biological
  • the heterologous protein sequence can comprise enzymes such as ⁇ -lactamase or carboxylesterases or toxins such as bryodin 1, Pseudomonas exotoxm A, or gelonm, or a functionally active portion thereof.
  • the S2C6 protein can be chemically linked to chemotherapeutic agents, including but not limited to alkylatmg agents (e.g. nitrogen mustards, nitrosoureas,
  • antimetabolites e.g. folic acid analogs, py ⁇ midme analogs, purme analogs
  • natural products e.g. antibiotics, enzymes, biological response modifiers
  • miscellaneous agents e.g. substituted urea, platinum coordination complexes
  • hormones and antagonists e.g. estrogens, androgens , antiandrogen, gonadotropin releasing hormone analog
  • Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product
  • heterologous protein sequence can be covalently bound to the S2C6 -related sequences by other than
  • an S2C6 protein derivative is a chimeric or fusion protein comprising an S2C6 protein or fragment thereof (preferably consisting of at least a domain
  • the S2C6 protein is a toxin, enzyme or biological response modifier.
  • the amino acid sequence of the different protein is at least 6, 10, 20 or 30 continuous amino acids of the different protein or a portion of the different protein that is functionally active.
  • such a chimeric protein is produced by recombmant expression of a nucleic acid encoding the protein 0
  • Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding 5 frame, and expressing the chimeric product by methods commonly known in the art.
  • a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Chimeric genes comprising portions of an S2C6 gene fused to any heterologous protein- encoding sequences may be constructed.
  • a specific embodiment relates to a chimeric protein comprising a fragment of an S2C6 protein of at least 6 or 15 or 50 ammo acids, or a fragment that displays one or more functional activities of the S2C6 protein (e.g., comprising copies of one or more CDRs) .
  • the S2C6 protein or derivative thereof is chemically linked to a chemotherapeutic drug including but not limited to doxorubic , paclitaxel or docetaxel .
  • a S2C6-drug conjugate can deliver the drug to cells expressing CD40.
  • One or more drug molecules can be linked to the S2C6 protein or derivative. Linkages include but are not limited to hydrazone, peptide or carbohydrate linkages .
  • the derivative is a molecule comprising a region of homology with an S2C6 protein.
  • a first protein region can be considered "homologous" to a second protein region when the ammo acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical, when compared to any sequence m the second region of an equal number of ammo acids as the number contained in the first region (without any insertions or deletions) or when compared to an aligned sequence of the second region that has been aligned by a computer homology program known m the art .
  • a nucleic acid which is hybridizable to an S2C6 nucleic acid e.g., having a sequence as set forth m SEQ ID NOS : 1 or 6) , or to its reverse complement, or to a nucleic acid encoding an S2C6 derivative, or to its reverse complement under conditions of low stringency is provided.
  • procedures using such conditions of low stringency are as follows ( see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792).
  • Filters containing DNA are pretreated for 6 h at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 32 P-labeled probe is used. Filters are incubated in hybridization mixture for
  • a nucleic acid which is hybridizable to an S2C6 nucleic acid e.g., having a sequence 0 as set forth in SEQ ID NOS : 1 or 6) , or to its reverse complement, or to a nucleic acid encoding an S2C6 derivative, or to its reverse complement under conditions of high stringency is provided.
  • procedures using such conditions of high 5 stringency are as follows.
  • Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 s cpm of 32 P-labeled probe.
  • the invention provides for treatment or prevention of various diseases or disorders by administration of a therapeutic compound (termed herein "Therapeutic") .
  • a therapeutic compound include but are not limited to: S2C6 antibodies and derivatives thereof (e.g., as described hereinabove) ; and
  • Treatment as used herein shall be deemed to include any clinically desirable or beneficial effect on the disease or disorder, including but not limited to alleviation of one or more symptoms,
  • the Therapeutic is administered alone or in combination with CD40L for the treatment or prevention of malignancies (including but not limited to carcinoma and hematologic
  • the Therapeutic and CD40L can, but need not be, contained within the same formulation, i.e, administration of the Therapeutic and CD40 can be performed separately but concurrently or during the same course of treatment.
  • the malignant cells express CD40.
  • the cells of the malignancy need not express CD40, since endothelial cells of the vasculature associated with a malignant tumor should express CD40 and thus the Therapeutic of the invention should provide treatment efficacy even for tumors that do not express CD40.
  • the Therapeutic potentiates the binding of CD40L to CD40 by at least 45%, 50%, 60%, or 65%.
  • the Therapeutic is used to increase the immune response of an immunosuppressed individual, such as a person suffering from acquired immunodeficiency syndrome, from malignancy, or an infant or elderly person.
  • the Therapeutic may be chemically modified so that cells that it binds to are killed.
  • Such cells include but are not limited to multiple myeloma cells, lymphoma cells or carcinomas. Since all B- cells express CD40, this approach can result in suppression of the immune response.
  • a cytotoxic drug linked to S2C6 sequences e.g., a fusion protein
  • these modified ligands may be used to control autoimmune diseases.
  • the Therapeutic may be used to promote the proliferation and/or differentiation of CD40- bearing cells that are not B cells, for example, lung carcinoma cells, as a means of directly treating malignancy or as an adjunct to chemotherapy.
  • Malignancies which may be treated or prevented using a Therapeutic of the invention include but are not limited to those in Table 1 :
  • Hodgkin ' s disease non-Hodgkin ' s disease
  • Inflammatory diseases and deficiencies or disorders of the immune system which may be treated or prevented using a 15 Therapeutic of the invention include but are not limited to those m Table 2 :
  • INFLAMMATORY DISEASES AND IMMUNE SYSTEM DISORDERS systemic lupus erythematosus (SLE)
  • Scleroderma e.g., CRST syndrome
  • inflammatory myositis ?c S ⁇ gren's syndrome (SS) mixed connective tissue disease e.g., MCTD, Sharp's syndrome
  • inflammatory bowel disease e . g. , ulcerative colitis, Crohn's disease
  • acute respiratory distress syndrome 30 pulmonary inflammation osteoporosis delayed type hypersensitivity asthma primary biliary cirrhosis (PBC) ldiopathic thrombocytopenic purpura (ITP)
  • EFFECTIVE DOSE Toxicity and therapeutic efficacy of such Therapeutics can be determined by standard pharmaceutical procedures m cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the
  • ED 50 the dose therapeutically effective m 50% of the population
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Therapeutics that exhibit large therapeutic indices are preferred. While Therapeutics that exhibit large therapeutic indices are preferred. While Therapeutics that exhibit large therapeutic indices are preferred. While Therapeutics that exhibit large therapeutic indices are preferred. While Therapeutics that exhibit large therapeutic indices are preferred. While Therapeutics that
  • JO exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unmfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and ,- animal studies can be used m formulating a range of dosage for use in humans. Exemplary doses include but are not limited to from 1 ng/kg to 100 mg/kg.
  • the dosage of such Therapeutics lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose may preferably be estimated initially from cell culture assays.
  • a dose may be formulated m animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined m cell culture. Such information can be used to more accurately determine useful doses in humans. Levels m plasma may be measured, for example, by high performance liquid chromatography .
  • compositions for use m accordance with the present invention may be formulated m conventional JJ manner using one or more physiologically acceptable carriers or excipients.
  • the Therapeutics and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., O pregelatmised maize starch, polyvmylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystallme cellulose or calcium hydrogen phosphate) lubricants (e.g., magnesium stearate, talc or silica); dismtegrants (e.g., potato starch or sodium starch ⁇ glycolate) ; or wetting agents ( e . g. , sodium lauryl sulphate) .
  • binding agents e.g., O pregelatmised maize starch, polyvmylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystallme cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium
  • the tablets may be coated by methods well known m the art.
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicles before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbic acid) .
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner .
  • the Therapeutics for 5 use according to the present invention are conveniently delivered m the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, t ⁇ chlorofluoromethane, dichlorotetrafluoroethane , carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, t ⁇ chlorofluoromethane, dichlorotetrafluoroethane , carbon dioxide or other suitable gas.
  • a pressurized propellant e.g., dichlorodifluoromethane, t ⁇ chlorofluoromethane, dichlorotetrafluoroethane , carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the Therapeutics may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented m unit dosage form, e.g., m ampoules or m multi- dose containers, with or without an added preservative.
  • compositions may take such forms as suspensions, solutions or emulsions oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the Therapeutics may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the Therapeutics may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the Therapeutics may be formulated with suitable
  • polymeric or hydrophobic materials for example as an emulsion an acceptable oil
  • ion exchange resms for example, as a sparingly soluble derivatives, for example, as a sparingly soluble sal .
  • compositions may, if desired, be presented in a pack
  • J " 3J* 5 or dispenser device that may contain one or more unit dosage forms containing the active ingredient .
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration preferably for administration to a human.
  • the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a Therapeutic m combination with CD40 ligand .
  • the S2C6 light chain and heavy chain variable regions were cloned using methods essentially as described m
  • RNA was isolated from the S2C6 hybridoma.
  • First strand complementary DNA (cDNA) was prepared for the mouse kappa light chain and heavy chain variable regions using reverse transcriptase and anti-sense primers that annealed approximately 100 base pairs downstream of the JC junction.
  • a poly-G tail was added to the cDNA strands using terminal transferase and then double stranded DNA was synthesized using the polymerase chain reaction (PCR) .
  • the PCR primers specific for the poly-G tail or a sequence approximately 50 bases inside the cDNA for the light chain or heavy chain, were designed to include unique restriction sites. After amplification, the PCR products were digested with EcoRI and
  • Figures 3A-3B illustrate the ammo acid sequence of S2C6 V L and S2C6 V H ( Figure 3A and 3B, respectively) .
  • the CDRs are underlined.
  • the ammo acid sequences of V L CDRs 1-3 correspond to SEQ ID NOS: 3 -5, respectively.
  • the ammo acid sequences of V H CDRs 1-3 correspond to SEQ ID NOS: 8-10, respectively.
  • the resulting DNA sequences were then compared to the light chain and heavy chain variable regions of other murme antibodies of the same isotype and the reading frame and corresponding ammo acid sequences for the genes isolated from S2C6 were determined. To confirm the ammo acid sequences, the light chain and heavy chain variable regions of S2C6 mAb were subjected to N-terminal ammo acid analysis.
  • Rabat database can be retrieved using the Accession number at http://immuno.bme.nwu.edu/database_.html and SEQHUNT II. The results of these searches are shown below:
  • S2C6 VL (SEQ ID NO: 2) : a BLASTP search of the NR database with S2C6 VL as the query yielded no hits with 100% identity and 6 hits with 94% (106/112) identity.
  • These 6 are shown below: p ⁇ r
  • VL CDR1 (SEQ ID NO: 3) : a BLASTP search with VL CDR1 as the query yielded no hits with 100% identity and numerous hits with 93% identity (15/16) .
  • the first 5 of these are shown ⁇ _- below: dbj I BAA03480 I (D14627) lmmunoglobulm gamma-3 kappa chain [Mus musculus] db
  • VL CDR2 (SEQ ID NO: 4) : a BLASTP search of the NR database with VL CDR2 as the query yielded no hits.
  • VL CDR3 (SEQ ID NO: 5) : a BLASTP search of the NR database 0 with VL CDR3 as the query yielded no hits.
  • S2C6 VH (SEQ ID NO: 7) : a BLASTP search of the NR database using S2C6 VH as a query yielded no hits with 100% identity and numerous hits with up to 88% identity the first 5 of 5 which are shown below: gi I 3561044 (AF083186) ant ⁇ -HIV-1 p24 antibody D2 heavy chain [Mus musculus] pdb
  • VH CDR1 (SEQ ID NO: 8) : a BLASTP search of the NR database with VH CDR1 as the query yielded no hits.
  • VH CDR2 (SEQ ID NO: 9) : a BLASTP search of the NR database with VH CDR2 as the query yielded no hits with 100% identity, 1 hit with 94% identity (16/17) and numerous hits with less than 94% identity.
  • the 1 hit with 94% identity is shown: gi I 3561044 (AF083186) ant ⁇ -HIV-1 p24 antibody D2 heavy chain [Mus musculus]
  • VH CDR3 (SEQ ID NO: 10) : a BLASTP search of the NR database with VH CDR3 as the query yielded no hits.
  • S2C6 VL (SEQ ID NO : 2 ) : a BLASTP search of the Rabat database using S2C6 VL as the query yielded no hits with 100% identity and numerous hits with 89-91% identity to the query.
  • the first 5 are shown:
  • VL CDRl (SEQ ID NO: 3) : a BLASTP search of the Rabat database with VL CDRl as the query yielded no hits with 100% identity and numerous hits with 93% identity (15/16) .
  • the first 5 are shown below: KADBID 005720, mouse IG KAPPA LIGHT CHAIN VARIABLE
  • VL CDR2 (SEQ ID NO : 4 ) : a BLASTP search of the Rabat database with VL CDR2 as the query yielded no hits.
  • VL CDR3 (SEQ ID NO: 5) : a BLASTP search of the Rabat database with VL CDR3 as the query yielded 1 hit with 100% identity to the query:
  • S2C6 VH (SEQ ID NO: 7) : a BLASTP search of the Rabat database using S2C6 VH as the query yielded no hits with 100% identity and numerous hits with 79-85% identity to the query. The first 5 of the hits are shown below:
  • KADBID 001498 mouse IG HEAVY CHAIN VARIABLE REGION (HDEX24) , KADBID 001494, mouse IG HEAVY CHAIN VARIABLE REGION (HDEX5) , KADBID 001529, mouse IG HEAVY CHAIN VARIABLE REGION (163.72 'CD ,
  • RADBID 001500 mouse IG HEAVY CHAIN VARIABLE REGION (HDEX37) , KADBID 001597, mouse IG HEAVY CHAIN VARIABLE REGION (BB128 'CD ,
  • VH CDRl (SEQ ID NO: 8) : a BLASTP search of the Rabat database with VH CDRl as the query yielded no hits 0
  • VH CDR2 (SEQ ID NO: 9) : a BLASTP search of the Rabat database with VH CDR as the query yielded no hits with 100% identity and 10 hits with 87-88% identity to the query. The first 5 are shown :
  • VH CDR3 (SEQ ID NO: 10): BLASTP search of the Rabat database with VH CDR3 as the query yielded no hits.
  • the S2C6 hybridoma was cultured at 37°C in complete IMDM (Gibco BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS) , 100 units/ml penicillin and 100 mg/ml 5 streptomycin. The culture was harvested by cent ⁇ fugation and the supernatant was collected by filtration using a 0.2 micron filter. Subsequently the supernatant was loaded onto a GammaBindTM Sepharose column (Pierce) , washed with phosphate buffered saline (PBS), and eluted with 0.1 M glycine pH 2.5.
  • FBS fetal bovine serum
  • the antibody was neutralized with 1 M Tris pH 8.0, dialyzed into PBS, and filter sterilized. MAb preparations were analyzed by size exclusion chromatography. Only samples of greater than 99% monomeric protein were used for the studies described herein.
  • mice Ninety female C.B.-17 SCID mice were obtained (Taconic Labs, Germantown, NY) at age 6 to 8 weeks and quarantined for 2 weeks. Control groups of mice were injected intravenously ⁇ (i.v.) with a human B cell tumor line: Ramos (non-Hodgkins lymphoma) , HS Sultan (multiple myeloma) or IM-9 (multiple myeloma) cells (Ixl0 6 -2xl0 6 cells) .
  • Ramos non-Hodgkins lymphoma
  • HS Sultan multiple myeloma
  • IM-9 multiple myeloma
  • mice were divided into two groups; half were treated with 200 ⁇ l of a 1:10 dilution of anti-asialo-GMl (Wako Chemicals, Richmond, VA) i.v., one day prior to the injection of tumor cells, to 0 remove host natural killer cells (Murphy et al . , 1992, Eur. J. Immunol. 22:241) . Mice in the two groups were injected i.v. with Ramos, HS Sultan or IM-9 cells (Ixl0 6 -2xl0 s cells) .
  • mice in the test groups were then injected intraperitoneally (i.p.) with 1 mg/kg of S2C6 IgG prepared as described in 5 Section 7.1.2 starting on day 1 or day 5 post tumor implant, according to the following schedule and were monitored for partial paralysis or other signs of disease.
  • Peripheral blood B cells were isolated by positive selection using immobilized antibodies against both CD19 and CD20. The final isolated cell population contained greater than 85% B cells as determined by flow cytometry. For storage, the cells were diluted to 4xl0 7 cells/ml m fetal bovine serum (FBS) containing 10% dimethyl sulfoxide and stored in a liquid nitrogen freezer.
  • FBS fetal bovine serum
  • the plates were incubated at 37°C for 3 days and then pulsed for 16 h with 0.5 mCi 3 H-thym ⁇ dme/well .
  • Cells were harvested onto 96-well glass fiber filters using a Filtermate 196 HarvesterTM (Packard Instruments) and combined with scintillation fluid.
  • the extent of 3 H-thym ⁇ dme incorporated into nascent DNA was measured by liquid scintillation counting using a Topcount LSCTM (Packard Instruments) .
  • a Jurkat cell line selected to express constitutive high levels of CD40L ( "Jurkat/CD40L” ) was used as CD40L stimulator cells (Malik et al . , 1996, J. Immunol. 156:3952- 60) . To eliminate proliferation of the stimulator cells, they were treated with mitomycm C (50 mg/ml) PBS for 20 mm at 37°C followed by 3 washes m PBS prior to combining with B cells. B cells (lxl0 5 /well) were combined with Jurkat/CD40L cells and assayed as above.
  • B cells and IL-4 were initially combined with stimulator cells (2.5xl0 4 /well) directly followed by addition of the ant ⁇ -CD40 mAbs.
  • Monoclonal antibodies were titrated with either a fixed concentration of stimulator cells or stimulator cells were titrated with a fixed concentration of mAb.
  • the Jurkat/CD40L cell line was used as a target cell line m these assays. Cells were adjusted to a density to 2xl0 7 /ml at 50 ⁇ l per sample. Binding was performed m RPMI 1640 media (Gibco) + 10% FBS. To determine receptor saturation, Jurkat/CD40L cells were incubated with increasing concentrations of CD40-Ig (a soluble fusion protein of CD40 and human lmmunoglobulm) (Noelle et al . , 1992, Proc. Natl. Acad. Sci.
  • CD40-Ig a soluble fusion protein of CD40 and human lmmunoglobulm
  • Dilutions of soluble CD40-Ig and ant ⁇ -CD40 mAbs were made at a 4-fold final concentration, pre-mcubated on ice for 1 h and then combined with Jurkat cells on ice for 1 h. Cells were washed and labeled with FITC-Goat anti-human F(ab') 2 ,
  • CD40L expression on selected CD40L + Jurkat T cells was initially verified by flow cytometry with FITC-labeled anti- CD40L (data not shown) .
  • CD40 binding to CD40L on these target cells was then determined by flow cytometry of the Jurkat/CD40L cells using FITC-goat anti-human Ig to detect the bound CD40-Ig.
  • CD40-Ig showed receptor saturation at approximately 25 ⁇ g/ml CD40-Ig.
  • S2C6 complexed with CD40 at ratios ranging from 0.25 to 2:1 (mass:mass) resulted m a dose-dependent increase in CD40 binding to CD40L (approximately 50%, 100%, 146% and 220% at concentrations of approximately 6 ⁇ g/ml, 13 ⁇ g/ml, 25 ⁇ g/ml, and 50 ⁇ g/ml, respectively) ( Figure 4) .
  • a similar titration with the inhibitory antibody M3 blocked CD40/CD40L binding in a dose dependent manner.
  • mAb G28-5 showed no effect of CD40/CD40L binding at concentrations up to 25 ⁇ g/ml and was only slightly stimulatory at the highest concentration tested (50 ⁇ g/ml) , relative to control EXA2-1H8 Ig.
  • S2C6 promotes CD40/CD40L interaction. Further, S2C6 differs from G28-5 and M3 in its ability to increase CD40/CD40L interaction.
  • mAb S2C6 complexed with CD40 -expressing cells resulted m a maximal increase m CD40L binding of approximately 51% to 68% at concentrations ranging from 0.04 to 2 ⁇ g/ml ( Figure 5) .
  • m which mAb G28-5 had little effect on CD40/CD40L interaction
  • G28-5 showed inhibition of soluble ligand binding to CD40 at all concentrations tested.
  • a similar titration with the inhibitory mAb M3 also blocked CD40L/CD40 binding m a dose dependent manner.
  • the growth response of primary peripheral B cells to CD40L-expressing cells was measured the presence of an ant ⁇ -CD40 mAb (S2C6, G28-5 or M3 ) .
  • B cells were combined with increasing numbers of non-proliferatmg, Jurkat/CD40L cells m the presence or absence of a fixed level (30 ng/ml) of the various mAbs.
  • B cell activation m response to treatment was then measured by 3 H-thym ⁇ dme incorporation at 72 h post-stimulus.
  • T cell titration the presence of mAb M3 resulted in B cell proliferation similar to that seen with control Ig ( Figure 6) .
  • B cells were either 5 titrated with an ant ⁇ -CD40 mAb or combined with non- proliferat g CD40L * T stimulator cells at a fixed ratio of 4:1 (B:T) and titrated with an ant ⁇ -CD40 mAb (Figure 7) .
  • mice 25 female C.B.-17 SCID mice were divided into two groups (20 mice/group) . Half of the mice of each group were treated with anti-asialo-GMl to blunt host natural killer cell activity
  • mice were injected i.v. with Ramos, HS Sultan
  • mice were then injected i.p. with 1 mg/kg of mAb S2C6 IgG, as described m Materials and
  • Ramos human B cell lymphoma ( Figure 8A) , HS Sultan multiple myeloma ( Figure 8B) , or IM-9 multiple myeloma ( Figure 8C) , resulted m significant reduction in tumor mass and subsequent tumor-related morbidity and mortality.
  • efficacy was sustained m the presence of anti-asialo-GMl , suggesting that the increased survival the presence of mAb S2C6 was not due to nonspecific NR activity.
  • the IM-9 cell line is an aggressive tumor model that, like multiple myeloma, secretes human Ig as a surrogate marker of disease.
  • BD1-S2C6 sFv single-chain ant ⁇ -CD40 immunotoxm, a fusion protein consisting of the ammo acid sequence of bryod 1 (BD1) (Francisco et al . , 1997, J. Biol. Chem.
  • the first strands were then poly-G tailed and amplified by PCR using a poly-C anchor primer, which is complementary to the poly-G tail, and a primer nested approximately 50 bases mside the one used for first strand synthesis.
  • the PCR primers were designed to generate unique restriction sites at the 5 ' and 3 ' ends of the PCR products.
  • the two PCR products, containing the sequences coding for the light chain and heavy chain variable regions, were digested with EcoRI and HINDIII and ligated into pUC19 which had been digested with the same enzymes.
  • the resulting plasmids, pSG5 and pSGlO contain the DNA coding for S2C6 VL and S2C6 VH, respectively.
  • the DNA of both plasmids was sequenced and verified to match the ammo-terminal acid sequence of the parental monoclonal antibody.
  • the VH and VL fragments of S2C6 were "sewn" together (overlap extension PCR) as described by Gilliland et al . m the VH-VL orientation and ligated into a cloning vector.
  • the sFv fragment of BD1-G28-5 sFv (Francisco et al . , 1997, J. Biol . Chem . 272:24165-24169) was removed from pSE151 by restriction digestion and S2C6 sFv was ligated m its place.
  • the resulting plasmid, pSG40 contains the gene coding for BD1-S2C6 sFv under the control of the mducible T7 promoter .
  • pSG40 was transformed into competent E. coli strain BL21 (DE3 ) pLysS cells and the cells were grown m
  • PBS containing 0.3M L-arginme and 2 mM DTT dialyzed against 20 mM sodium phosphate buffer, pH 7.4, for subsequent purification.
  • the refolded protein was isolated using Blue Sepharose followed by affinity chromatography over immobilized CD40-Ig.
  • the purified protein was then tested for binding to immobilized CD40-Ig in ELISA.
  • Microtiter plates were coated with CD40-Ig at 0.5 ⁇ g/ml followed by the addition of dilutions of purified BD1-S2C6 sFv in PBS (pH 7.4) with 1% bovme serum albumin and 0.05% Tween-20 m the presence of 25 ⁇ g/ml S2C6 mAb (A) , 25 ⁇ g/ml control antibody BR96 (•) , or no excess antibody ( ⁇ ) .
  • Binding of BD1-S2C6 sFv to the immobilized receptor was detected by the addition of BD1-specific rabbit antiseru (Seattle Genetics, Inc., Bothell, Washington) followed by the addition of horseradish peroxidase conjugated goat ant1-rabbit Ig.
  • CD40-pos ⁇ t ⁇ ve malignancies such as Non- 10 Hodgkin's Lymphoma, Multiple Myeloma, and colon or other carcinomas are injected with recombmant humanized S2C6-ant ⁇ - CD40 monoclonal antibody (with murine CDRs and human framework regions) or a recombmant chimeric antibody comprising the variable region of S2C6 and the constant 15 region of a human antibody.
  • the recombmant antibody is prepared m vi tro . Treatment can commence at any time during the course of the disease, m the presence or absence of concomitant chemotherapy.
  • the treatment regimen includes weekly injections of the 20 agent diluted in saline or other physiologically compatible solution.
  • the dosage used for recombmant S2C6 is m the range of 0.1 mg/m 2 (of body surface area of the patient) to 1000 mg/m 2 with the preferred dosage being 100-500 mg/m 2 . 25
  • the route of injection is intravenous through either a peripheral IV access line or a central IV access line.
  • the agent is administered as an infusion and not an IV push.
  • the effect of therapy with recombmant S2C6 is monitored by measuring: a) total lymphocyte and T and B lymphocyte .
  • a) total lymphocyte and T and B lymphocyte . counts m the peripheral blood; b) activity of T lymphocytes (helper T4 lymphocytes and cytolytic T8 lymphocytes) in vi tro; and/or c) changes m the morphology of tumors using techniques such as a computed tomographic (CT) scan, magnetic resonance imaging (MRI) scan, x-radiographic imaging, bone scan imaging and tumor biopsy sampling including bone marrow aspiration (BMA) .
  • CT computed tomographic
  • MRI magnetic resonance imaging
  • BMA bone marrow aspiration
  • the therapeutic regimen is developed to optimally treat CD40-pos ⁇ t ⁇ ve malignancies with minimal impact on the competency of the immune system with the ultimate goal of achieving tumor regression and complete eradication of tumor cells.

Abstract

The present invention relates to methods and compositions for the prevention and treatment of cancer, inflammatory diseases and disorders or deficiencies of the immune system. The methods of the invention comprise administering a CD40 binding protein that potentiates the binding of CD40 to CD40 ligand.

Description

RECOMBINANT ANTI-CD40 ANTIBODY AND USES THEREOF
1. FIELD OF THE INVENTION The present invention relates to methods and compositions for the treatment of diseases and disorders, including cancer, inflammatory diseases or disorders and diseases or disorders of the immune system, comprising administering a CD40 binding protein which enhances binding of CD40 ligand to CD40. CD40 binding proteins include recombmant/variant forms of monoclonal antibody S2C6 and derivatives thereof.
2. BACKGROUND OF THE INVENTION CD40 is a cell surface phosphorylated glycoprotem that is expressed on a variety of cell types, including B cells, B cell malignancies, follicular dendritic cells, basal epithelial cells, and carcinomas. CD40 binds CD40 ligand ("CD40L") . CD40L is expressed on activated T cells during inflammation and cancer (Younes et al . , 1998, Br. J. Haematol . 100:135-141; for a review see Grewal and Flavell, 1998, Annu. Rev. Immunol. 16:111-135). The interaction of CD40 with CD40L results in B cell activation and proliferation of normal B cells; however CD40-med ated signaling in B cell -derived tumor lines can result in activation- induced cell death. The strength of the activation signal is key to activation- induced tumor cell death (Grafton et al . , 1997, Cell. Immunol. 182:45-56). Therefore, compositions and methods for increasing receptor- ligand interaction and strength of activation signal between CD40 and CD40L would be of great value in treating disease.
2.1 CD40 AND CD40 LIGAND CD40 is a member of the TNF receptor superfamily. This family includes TNFrll, CD40, CD30, LMP-1, LTBr, ATAR, OX-40 and 4 -IBB receptors. CD40 is constitutively expressed on B- lymphocytes, macrophages and dendritic cells and is induced by cytokme activation on fibroblasts, endothelial cells and epithelial cells (Van Kooten and Banchereau, 1997, Curr . Opm. Imunol . , 9: 330-337) . CD40 has also been shown to be highly expressed on many human carcinomas including lung, bladder, gastric, breast and ovarian cancers (Stamenkovic et al., 1989, EMBO J. 8:1403-1410).
The ligand for CD40 is a membrane protein that is expressed on activated T cells. Receptor binding of CD40L results m CD40 multimerization, the generation of activation signals (for antigen presenting cells such as dendritic cells, monocytes and B cells) and the generation of growth and differentiation signals (for cytokme-activated fibroblasts and epithelial cells) . CD40 signals are transduced from the multimeπzed receptor via recruitment of a series of TNF receptor-associated factors ("TRAFs") (Kehry, 1996, J. Immunol. 156:2345-2348). Subsets of TRAFs interact differentially with TNF family members, including CD40, to provide stimuli to a wide variety of downstream pathways. TRAF1 and TRAF2 are implicated m the modulation of apoptosis (Speiser et al . , 1997, J. Ex . Med. 185:1777-1783; Yeh et al . , 1997, Immunity 7:715-725). TRAFs 2, 5, and 6 participate m proliferation and activation events, including NF-kB and c-Jun N-terminal kmase activation. In normal B cells, binding of CD40 recruits TRAF2 and TRAF3 to the receptor complex and induces down-regulation of other TRAFs (Kuhune et al . , 1997, J. Exp. Med.186: 337-342). The effects of CD40 binding are also dependent on membrane density (De Paoli et al . , 1997, Cytometry 30:33-38). Importantly, unlike the proliferative response seen with normal primary B cells, CD40 binding on neoplastic B cells can result m growth inhibition and activation-induced cell death (Funakoshi et al . , 1994, Blood 83:2787-2794). Thus, CD40 activation m the context of different cell types, transformation, resident TRAFs and co-stimuli can induce responses ranging from activation and proliferation to growth inhibition and apoptosis . 2.2 ANTI-CD40 ANTIBODIES
With at least one exception, the antι-CD40 monoclonal antibodies ("mAbs") described to date are of three general classes: (1) those that block CD40/CD40L interaction by at least 90% and have anti-neoplastic properties (Armitage et al . , U.S. Patent No. 5,674,492; Fanslow et al . , 1995, Leukocyte Typing V, Schlossman et al . , eds . , 1:555-556); (2) those that antagonize signaling through CD40 (deBoer et al . , U.S. Patent No. 5,677,165); and (3) those that deliver a stimulatory signal through CD40 but do not increase the interaction between CD40 and CD40L, e . g. , G28-5, (Ledbetter et al . , U.S. Patent No. 5,182,368; PCT Publication WO 96/18413) .
One mAb, CD40.4 (5C3) (PharMmgen, San Diego, California) , has been shown to increase the interaction between CD40 and CD40L by approximately 30-40% (Schlossman et al . , eds., 1995, Leukocyte Typing V: White Cell Differentiation Antigens 1:547-556).
Armitage et al . (U.S. Patent No. 5,674,492) describes methods using CD40 binding proteins, including mAb HuCD40-M2, that are capable of binding CD40 and inhibiting the binding of CD40 to CD40L, for preventing or treating disease characterized by neoplastic cells expressing CD40.
DeBoer et al . (U.S. Patent No. 5,677,165) describes antι-CD40 mAbs that, being free of significant agonistic activity, bind to CD40 on the surface of B-cells, and block B-cell activation. An essential feature of U.S. Patent No. 5,677,165 is that upon binding of the antι-CD40 mAb to human CD40 on the surface of normal human B cells, the growth or differentiation of normal human B cells is inhibited.
Ledbetter et al . (U.S. Patent No. 5,182,368) describes a ligand, G28-5, that binds to the B cell surface antigen Bp50 (now designated CD40) and stimulates activated B cells to traverse the cell cycle such that B cell proliferation is augmented. However, G28-5 does not enhance activation of B cells m the presence of CD40L, and does not potentiate CD40/CD40L interaction. S2C6 is an antι-CD40 mAb that was prepared against a human bladder carcinoma (Paulie et al . , 1984, Cancer Immunol. Immunother. 17:165-179). S2C6 binds to the CD40 receptor expressed on a variety of cell types including B- lymphocytes, endothelial and epithelial cells. S2C6 has been shown to have specificity toward neoplastic urothelium and B cell- deπved malignant lymphocytes. Reactivity with a prostatic carcinoma cell line, HS, and weak reactivity with a melanoma has also been shown (Paulie et al . , 1984, Cancer Immunol. Immunother. 17:165-179). Studies have suggested the utility of S2C6 as a diagnostic marker for B cell malignancies (Paulie et al . , 1984, Cancer Immunol. Immunother. 17:165-179; Paulie et al . , 1985, Eur. J. Cancer. Clm. Oncol. 21:701- 710) . In addition to detecting B cell malignancies, S2C6 has been shown to deliver strong growth-promoting signals to B lymphocytes (Paulie et al . , 1989, J. Immunol. 142:590-595).
S2C6 has agonistic activity on human peripheral B cells as demonstrated by its ability to stimulate primary B cell proliferation in a dose dependent manner (Paulie et al . , 1989, J. Immunol. 142:590-595).
Although competition studies have shown that G28-5 and S2C6 bind the same or proximal epitopes, the antibodies have been determined to be functionally different based primarily on the stated magnitude of stimulation achieved by either mAb on previously stimulated tonsillar B cells (Clark and Ledbetter, 1986, Proc . Natl . Acad. Sci . USA 83:4494-4498; Ledbetter et al . , U.S. Pat. No. 5,182,368). One hundred times more S2C6 compared to G28-5 was required to achieve tonsillar B cell activation under the specific conditions tested (Ledbetter et al . , U.S. Patent No. 5,182,368).
There is a need m the art for therapeutics with increased efficacy to treat or prevent cancer, activate or augment the immune system or treat or prevent an immune deficiency or disorder, a need provided by the present invention. Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.
3. SUMMARY OF THE INVENTION Applicants have made the unexpected discovery of a new class of antι-CD40 antibodies that, addition to delivering a stimulatory signal, enhances the interaction between CD40 and CD40L, enhances CD40L-medιated stimulation and has m vivo anti-neoplastic activity. Production and use of these antibodies and related molecules are facilitated by the inventors ' cloning and sequencing of the variable region of mAb S2C6, and identification of the CDR and framework regions therein.
The present invention relates to molecules comprising the variable domain of mAb S2C6 or one or more of the complementarity-determining regions (CDRs) thereof having novel sequences (SEQ ID NO: 3, 4, 8, 9 or 10), which molecules (a) lmmunospecifically bind CD40 and (b) comprise one or more substitutions or insertions in primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybπdoma deposited with the ATCC and assigned accession number PTA-110, or are not monoclonal antibody S2C6 as secreted by the hybπdoma deposited with the ATCC and assigned accession number PTA-110 and do not result from cleavage of S2C6 with papain or pepsin. In a specific embodiment, the molecules are not native monoclonal antibody S2C6 and do not comprise the native heavy or light chain of said monoclonal antibody S2C6. In another specific embodiment, the molecule is an antibody. In another embodiment, the antibody is not isotype IgGl . In another specific embodiment, the molecule comprises a light chain variable domain, the ammo acid sequence of SEQ ID NO: 2, or a heavy chain variable domain, the ammo acid sequence of SEQ ID NO: 7.
The invention further relates to chimeπc/fusion proteins comprising a fragment of mAb S2C6 fused to an ammo acid sequence of a second protein, as well as to molecules wherein a fragment of mAb S2C6 is covalently bound (e.g., by use of a crosslinking agent) to another chemical structure. In a specific embodiment, a molecule is provided that immunospecifically binds CD40, which molecule comprises the heavy and/or light chain variable domain of mAb S2C6 fused to a second protein comprising the ammo acid sequence of bryodin 1 (BD1) .
The invention further relates to proteins comprising an ammo acid sequence that has at least 95% identity to SEQ ID NO: 2, SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which proteins (a) immunospecifically bind CD40 and (b) comprise one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybπdoma deposited with the ATCC and assigned accession number PTA-110. In a specific embodiment, the proteins are not native monoclonal antibody S2C6 and do not comprise the native heavy or light chain of said monoclonal antibody S2C6.
The invention further relates to purified proteins, which proteins (a) compete for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybπdoma deposited with the ATCC and assigned accession number PTA- 110, (b) increase the binding of CD40 ligand to CD40 by at least 45%, and (c) comprise one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybπdoma deposited with the ATCC and assigned accession number PTA- 110, or are not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110 and do not result from cleavage of S2C6 with papam or pepsin. In a specific embodiment, the proteins are not native monoclonal antibody S2C6 and do not comprise the native heavy or light chain of said monoclonal antibody S2C6. The invention further relates to nucleic acids encoding such molecules and proteins or which hybridize to a DNA consisting of the nucleotide sequence encoding such proteins; recombmant cells comprising such molecules and proteins, and methods of producing such proteins.
In an embodiment, the isolated nucleic acid comprises a nucleotide sequence encoding a protein comprising (a) a heavy chain variable domain of monoclonal antibody S2C6 as secreted by the hybπdoma deposited with the ATCC and assigned accession number PTA-110, and (b) a human constant region.
In an embodiment, the isolated nucleic acid comprises a nucleotide sequence encoding a protein comprising (a) a light chain variable domain of monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and (b) a human constant region.
The invention further relates to recombmant cells containing a recombmant nucleic acid vector comprising a nucleotide sequence encoding a protein, which protein competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and which protein increases the binding of CD40 ligand to CD40 by at least 45%.
The invention also provides methods of producmσ such proteins comprising growing such cells such that the protein is expressed by the cell, and recovering the expressed protein.
The invention further relates to recombmant cells containing a recombmant nucleic acid vector comprising SEQ
ID NO:l, SEQ ID NO : 6 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and methods of producing proteins comprising growing such cells, such that a protein encoded by the nucleotide sequence is expressed by the cell, and recovering the expressed protein.
Pharmaceutical compositions containing the molecules and antibodies of the invention, preferably purified form, are also provided. In particular embodiments, the invention relates to pharmaceutical compositions comprising a molecule comprising SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO: 7, SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (I) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40, and (m) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110; and a pharmaceutically acceptable carrier. In a specific embodiment, the molecule is not native monoclonal antibody S2C6 and does not comprise the native heavy or light chain of said monoclonal antibody
S2C6.
The invention further relates to pharmaceutical compositions comprising a purified protein, which protein (1) competes for binding to CD40 with mAb S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (11) increases the binding of CD40 ligand to
CD40 by at least 45%, and (m) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as deposited with the ATCC and assigned accession number PTA-110, or is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110 and does not result from cleavage of S2C6 with papam or pepsin; and a pharmaceutically acceptable carrier. In a specific embodiment, the protein is not native monoclonal antibody S2C6 and does not comprise the native heavy or light chain of said monoclonal antibody S2C6.
In specific embodiments, the pharmaceutical compositions of the invention contain the molecules or antibodies of the invention m an amount effective for the treatment or prevention of cancer, or an amount effective for activating or augmenting an immune response, or an amount such that the immune response of the subject is activated or augmented.
In specific embodiments, the pharmaceutical compositions of the invention further comprise CD40 ligand. In a specific embodiment, the pharmaceutical composition comprises m an amount effective for the treatment or prevention of cancer or an immune disorder, or for activating or augmenting an immune response: (a) a molecule that immunospecifically binds CD40, which molecule increases the binding of CD40 ligand to CD40; (b) CD40 ligand; and (c) a pharmaceutically acceptable carrier. In this embodiment, for example, the molecule can be native mAb S2C6 or native mAb 5C3 or an S2C6 derivative as
5 described herein.
The invention further relates to methods for the treatment or prevention of cancer a subject, for activating or augmenting an immune response a subject, or for the treatment or prevention of an immune deficiency or
, r disorder a subject comprising administering to the subject a therapeutically effective amount of the molecules or antibodies of the invention, e.g., an amount of a molecule comprising SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID
NO: 7, SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (l) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and comprises one or more substitutions or insertions primary ammo acid sequence relative to native monoclonal antibody
S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110. In a specific embodiment, 0 the molecule is not native monoclonal antibody S2C6 and does not comprise the native heavy or light chain of said monoclonal antibody S2C6.
The invention further relates to methods for the treatment or prevention of cancer a subject, for 5 activating or augmenting an immune response a subject, or for the treatment or prevention of an immune deficiency or disorder a subject comprising administering to the subject a purified protein, which protein d) competes for binding to
CD40 with monoclonal antibody S2C6 as secreted by the 0 hybridoma deposited with the ATCC and assigned accession number PTA-110, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (m) comprises one or more substitutions or insertions m the primary ammo acid sequence relative to native monoclonal antibody S2C6 as 5 deposited with the ATCC and assigned accession number PTA- 110, or is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110 and does not result from cleavage of S2C6 with papain or pepsin. In a specific embodiment, the protein is not native monoclonal antibody S2C6 and does not comprise the 5 native heavy or light chain of said monoclonal antibody S2C6.
In specific embodiments, the methods of the invention further comprise administering CD40 ligand to the subject.
The invention further relates to a method for the treatment or prevention of cancer or an immune disorder in a 1^ subject comprising administering to the subject, in an amount effective for said treatment or prevention: (a) a molecule that immunospecifically binds CD40, which molecule increases the binding of CD40 ligand to CD40; and (b) CD40 ligand, in which the molecule can be native mAb S2C6 or native mAb 5C3 or any of the S2C6 derivatives described herein.
In a preferred embodiment, the subject is a human.
The invention further relates to a transgenic non-human animal, plant, or an isolated cell containing one or more transgenes encoding a protein, which protein competes for binding to CD40 with monoclonal antibody S2C6 as secreted by 20 the hybridoma deposited with the ATCC and assigned accession number PTA-110, and which protein increases the binding of
CD40 ligand to CD40 by at least 45%.
4. BRIEF DESCRIPTION OF THE FIGURES
25
Figure 1. Structure of the light chain variable region of S2C6. The nucleotide (SEQ ID NO:l) and amino acid (SEQ ID
NO : 2 ) sequences of the light chain variable region ("VL")are shown .
Figure 2. Structure of the heavy chain variable region JU of S2C6. The nucleotide (SEQ ID NO: 6) and amino acid (SEQ ID
NO: 7) sequences of the heavy chain variable region ("VH" ) of
S2C6 are shown.
Figures 3A-3B. Structure of variable regions of S2C6.
(A) The amino acid sequence (SEQ ID NO: 2) of S2C6 VL is shown. 35 (B) The amino acid sequence (SEQ ID NO: 7) of S2C6 VH is shown.
Complementarity-determining regions ("CDR") are underlined. The sequences of the four framework regions, adjacent to the CDRs , are shown. The ammo acid sequences of VL CDRs 1-3 correspond to SEQ ID NOS:3-5, respectively. The ammo acid sequences of VH CDRs 1-3 correspond to SEQ ID NOS:8-10, respectively.
Figure 4. S2C6 mAb augments CD40-Ig binding to CD40L- expressmg Jurkat T cells. CD40-Ig (a soluble fusion protein of CD40 and human lmmunoglobulm) binding to surface CD40L was done m the presence of increasing concentrations of antι-CD40 monoclonal antibody ("mAb") . mAbs were pre- mcubated for 1 hour with CD40-Ig followed by incubation for
1 hour with CD40L-expressmg target cells. CD40-Ig binding to target cells was detected by flow cytometry using a fluorescem isothiocyanate ( "FITC" ) -labeled anti-human Ig.
The extent of CD40/CD40L binding was then determined from log mean fluorescent intensity ("MFI") . MFI minus background of each population is shown.
Figure 5. S2C6 mAb augments binding of soluble CD40L to
B cell surface CD40. Ramos B cells, a human B cell lymphoma, were incubated m the presence of increasing concentrations of an antι-CD40 mAb: S2C6, G28-5, or M3 or an irrelevant control mAb, EXA2-1H8. The mAbs were pre-incubated for 1 hour with CD40-expressmg target cells. Binding of the FITC- labeled CD40L to B cells was then detected directly by flow cytometry. The extent of CD40/CD40L binding was then determined from log mean fluorescent intensity. MFI minus background of each population is shown.
Figure 6. S2C6 enhances proliferative response of primary human peripheral B cells in the presence of CD40L"" stimulator cells and an antι-CD40 mAb. Peripheral B cells
(lxl05/well) were combined with increasing numbers of non- proliferative CD40L" Jurkat T stimulator cells and 30 ng/ml of an antι-CD40 mAb: S2C6, G28-5, or M3 or the control mAb,
EXA2-1H8. B cell proliferation was measured by JH-TdR incorporation at 72 h following addition of stimulus. Figure 7. Comparative proliferative response of primary human peripheral B cells to an antι-CD40 mAb m the presence or absence of CD40L. Peripheral B cells were combined with non-proliferative CD40L+ stimulator cells at a fixed ratio of 4:1 and increasing concentrations of an antι-CD40 mAb. S2C6 G28-5 or the control antibody, EXA2-1H8. B cell
5 proliferation was measured by 3H-TdR incorporation at 72 h following addition of stimulus.
Figures 8A-8C. Anti-tumor activity of mAb S2C6 m vivo . Anti-tumor activity of S2C6 against (A) Ramos human B cell non-Hodgkm's lymphoma, (B) HS Sultan multiple myeloma, or
,(-, (C) IM-9 multiple myeloma was assessed. SCID mice (5/group) were pretreated or not with anti-asialo-GMl to inhibit natural killer ("NK") activity and treated with mAb on day 1 or day 5 following injection of Ixl06-2xl0c tumor cells.
Solid lines indicate the number of surviving mice over time.
Figure 9. BD1-S2C6 sFv specifically binds to immobilized CD40-Ig ELISA. BD1-S2C6 sFv (single-chain antι-CD40 lmmunotoxm consisting of bryodin 1 (BD1) fused to the variable region of monoclonal antibody S2C6) was expressed m E. coll as inclusion bodies, denatured and refolded. The refolded protein was then isolated using Blue 0
Sepharose followed by affinity chromatography over immobilized CD40-Ig. The purified protein was then tested for binding to immobilized CD40-Ig ELISA. Microtiter plates were coated with CD40-Ig at 0.5 μg/ml followed by the addition of dilutions of purified BD1-S2C6 sFv m the 5 presence of 25 μg/ml S2C6 mAb (A) , 25 μg/ml control antibody
BR96 (•) , or no excess antibody (■) . Binding of BD1-S2C6 sFv to the immobilized receptor was detected by the addition of
BDl-specific rabbit antiserum followed by the addition of horseradish peroxidase conjugated goat antl -rabbit Ig. The 0 binding of BD1-S2C6 sFv to CD40-Ig was completely inhibited by the addition of excess S2C6 mAb but not by the addition of the control mAb.
5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to proteins encoded by and nucleotide sequences of S2C6 genes. The invention further relates to fragments and other derivatives and analogs of such S2C6 proteins and nucleic acids. In various specific embodiments, the molecules (e.g., antibodies) of the invention comprise all or a portion of mAb S2C6 (the light chain and/or heavy chain, or light chain CDR 1 (SEQ ID NO : 3 ) and/or 2 (SEQ ID NO : 4 ) , and/or heavy chain CDR 1 (SEQ ID NO: 8), 2 (SEQ ID NO: 9), and/or 3 (SEQ ID NO: 10), or light chain CDR3 (SEQ ID NO: 5) m combination with any of the other CDRs and/or one or more of the four heavy chain and four light chain framework regions, provided that such molecules
10 are not native mAb S2C6 as deposited with the ATCC and assigned accession number PTA-110 or the heavy or light chain thereof. Such molecules may differ from S2C6 m sequence and/or m post-translational modification (glycosylation, amidation, peptide bonding or cross-linking to a non-S2C6
, _. sequence, etc.) . In various specific embodiments, a molecule of the invention immunospecifically binds CD40 (or when multimeπzed immunospecifically binds CD40) , competes with native S2C6 for binding to CD40, and/or increases the binding of CD40 ligand to CD40 by at least 45%, 50%, 60% or 65%. Nucleic acids encoding such molecules, e.g., S2C6 fragments
^ or derivatives, are also withm the scope of the invention, as well as nucleic acids encoding native mAb S2C6. Production of the foregoing proteins, e . g. , by recombmant methods, is provided.
The invention also relates to S2C6 proteins and
25 derivatives including but not limited to fusion/chimeπc proteins which are functionally active, i . e . , which are capable of displaying one or more known functional activities associated with a full-length S2C6 mAb. Such functional activities include but are not limited to ability to bind CD40, delivery of a stimulatory signal to the CD40 signaling pathway (e.g., so as to cause B cell proliferation), potentiation of the interaction of CD40L with CD40; ability to inhibit tumor growth; and ability to induce an immune response .
Antibodies to CD40 comprising S2C6, its derivatives and 5 analogs including but not limited to humanized antibodies; single chain antibodies; bispecific antibodies; and antibodies conjugated to chemotherapeutic agents or biological response modifiers, are additionally provided.
The invention further relates to methods of treating or preventing cancer, inflammatory diseases and disorders of the immune system comprising administering a composition of the invention alone or combination with CD40L.
The invention is illustrated by way of examples set forth Sections 6-9 below which disclose, inter alia , the cloning and characterization of S2C6 genes; the potentiation of the CD40/CD40L interaction; inhibition of tumor growth; 10 and binding of a single-chain antι-CD40 immunotoxm to CD40- Ig.
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections which follow.
15
5.1 ISOLATION OF S2C6 GENES
The invention relates to the nucleotide sequences of S2C6 nucleic acids. In specific embodiments, S2C6 nucleic acids comprise the cDNA sequences of SEQ ID NOS : 1 and 6, or nucleic acids encoding an S2C6 protein { e . g. , a protein
20 having the sequence of SEQ ID NOS : 2 and 7) . The invention provides purified nucleic acids consisting of at least 8 nucleotides (i.e., a hybridizable portion) of an S2C6 gene sequence; m other embodiments, the nucleic acids consist of at least 25 (contiguous) nucleotides, 50 nucleotides, 100, or
25 200 nucleotides of an S2C6 sequence, or a full-length S2C6 variable region coding sequence. In the same or other embodiments, the nucleic acids are smaller than 50, 75, 100, or 200 or 5000 nucleotides m length. Nucleic acids can be single or double stranded. The invention also relates to nucleic acids hybridizable to or complementary to the 30 foregoing sequences or their reverse complements, and m particular, such nucleic acids that encode proteins that bind to CD40, compete with S2C6 for binding to CD40, and/or increase the binding of CD40 ligand to CD40 by at least 45%,
50%, 60%, or 65%. In specific aspects, nucleic acids are
35 provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of an S2C6 variable region gene.
Nucleic acids encoding derivatives and analogs of S2C6 proteins are additionally provided. As is readily apparent, as used herein, a "nucleic acid encoding a fragment or portion of an S2C6 protein" shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the S2C6 protein and not the other contiguous portions of the S2C6 protein as a continuous sequence.
0 5.2 CLONING PROCEDURES
Specific embodiments for the cloning of an S2C6 gene follow. In a specific embodiment, total RNA is isolated from a mAb S2C6-producmg hybridoma and polymerase chain reaction is used to amplify desired variable region sequences, using 5 primers based on the sequences disclosed herein. For an illustrative example, see Section 6, infra. By way of another example, mRNA is isolated from a mAb S2C6 -producing hybridoma, cDNA is made and ligated into an expression vector (e.g., a bacteπophage derivative) such that it is capable of 0 being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed product. In one embodiment, selection is on the basis of hybridization to a labeled probe representing a portion of an S2C6 gene or its RNA or a 5 fragment thereof (Benton and Davis, 1977, Science 196:180; Grunstem and Hogness, 1975, Proc . Natl . Acad . Sci . U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion (s) 0 and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene.
Alternatively, the presence of the desired gene may be r detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid- select the proper mRNAs , can be selected and expressed to produce a protein that has, e . g . , similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, or functional activity, as known for an S2C6 protein. For example, ability to bind CD40 can be detected m an ELISA (enzyme-linked immunosorbent assay) -type procedure .
An S2C6 gene can also be identified by mRNA selection using nucleic acid hybridization followed by m vi tro Q translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Functional assays { e . g. , binding to CD40, etc.) of the m vi tro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences.
In another embodiment, the S2C6 cDNA can be chemically synthesized from the sequence disclosed herein. Other methods of isolating S2C6 genes known to the skilled artisan can be employed.
The identified and isolated S2C6 gene/cDNA can then be 0 inserted into an appropriate cloning vector. A large number of vector-host systems known m the art may be used.
Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not 5 limited to, bacteπophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives or the
Bluescript vector (Stratagene) . The insertion into a cloning vector can, for example, be accomplished by ligatmg the DNA fragment into a cloning vector which has complementary 0 cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligatmg nucleotide sequences (linkers) onto the DNA termini; 5 these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and an S2C6 gene may be modified by homopolymeric tailing, or by PCR with primers containing the appropriate sequences . Recombmant molecules can be
5 introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
In an alternative method, the desired gene may be identified and isolated after insertion into a suitable
20 cloning vector m a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector.
In specific embodiments, transformation of host cells with recombmant DNA molecules that incorporate an isolated
S2C6 gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained m large quantities by growing transformants, isolating the recombmant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombmant DNA. 20
The S2C6 sequences provided by the instant invention include those nucleotide sequences encoding substantially the same am o acid sequences as found m native S2C6 variable regions, and those encoded ammo acid sequences with functionally equivalent ammo acids, as well as those
25 encoding other S2C6 derivatives or analogs, as described below for S2C6 derivatives and analogs.
5.3 EXPRESSION OF S2C6 GENES
The nucleotide sequence coding for an S2C6 protein or a
30 functionally active analog or fragment or other derivative thereof (see Section 5.6), can be inserted into an appropriate expression vector, i . e . , a vector which contains the necessary elements for the transcription and translation of the inserted protem-cod g sequence. The necessary JJ transcriptional and translational signals can also be supplied by the native S2C6 gene and/or its flanking regions. A variety of host-vector systems may be utilized to express the protem-codmg sequence. These include but are not limited to mammalian cell systems infected with virus ( e . g . , vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteπophage , DNA, plasmid DNA, or cosmid DNA; transgenic plants or transgenic non-human animals. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeπc gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vi tro recombmant DNA and synthetic techniques and m vivo recombmants (genetic recombination) . Expression of a nucleic acid sequence encoding an S2C6 protein or peptide fragment may be regulated by a second nucleic acid sequence so that the S2C6 protein or peptide is expressed m a host transformed with the recombmant DNA molecule. For example, expression of an S2C6 protein may be controlled by any promoter/enhancer element known m the art.
Promoters that are not native S2C6 gene promoters which may be used to control S2C6 gene expression include, but are not limited to, the SV40 early promoter region (Benoist and
Chambon, 1981, Nature 290:304-310), the promoter contained m the 3 ' long terminal repeat of Rous sarcoma virus (Yamamoto et al . , 1980, Cell 22:787-797), the herpes thymid e kmase promoter (Wagner et al . , 1981, Proc . Natl . Acad. Sci . U.S.A. 78:1441-1445), the regulatory sequences of the metallothionem gene (Brmster et al . , 1982, Nature 296:39- 42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff et al . , 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the lac promoter (DeBoer et al . , 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful proteins from recombmant bacteria" m Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopalme synthetase promoter region (Herrera-
5 Estrella et al . , Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al . , 1981, Nucl . Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al . , 1984, Nature 310:115-120); promoter elements from yeast iQ or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kmase) promoter, alkaline phosphatase promoter, and the following animal transcriptlonal control regions, which exhibit tissue specificity and have been utilized m transgenic animals: ις elastase I gene control region which is active m pancreatic acinar cells (Swift et al . , 1984, Cell 38:639-646; Ornitz et al . , 1986, Cold Spring Harbor Symp . Quant. Biol . 50:399-409; MacDonald, 1987, Hepatology 7:425-515); a gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), an immunoglobulm gene control region which is active m lymphoid cells (Grosschedl et al . , 1984, Cell 38:647-658; Adames et al . , 1985, Nature 318:533-538; Alexander et al . , 1987, Mol . Cell. Biol . 7:1436-1444), mouse mammary tumor virus control region which is active m testicular, breast, lymphoid and mast cells (Leder et al . ,
25
1986, Cell 45:485-495), albumin gene control region which is active in liver (Pmkert et al . , 1987, Genes and Devel .
1:268-276), alpha-fetoprotem gene control region which is active in liver (Krumlauf et al . , 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al . , 1987, Science 235:53-58; alpha 1-
30 antitrypsm gene control region which is active m the liver
(Kelsey et al . , 1987, Genes and Devel. 1:161-171), beta- globm gene control region which is active m myeloid cells (Mogram et al . , 1985, Nature 315:338-340; Kollias et al . , 1986, Cell 46:89-94; myelm basic protein gene control region
"3--:
JJ which is active m oligodendrocyte cells m the brain
(Readhead et al . , 1987, Cell 48:703-712); rayosin light cham- 2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al . , 1986, Science 234:1372-1378). In a specific embodiment, a vector is used that comprises a promoter operably linked to an S2C6 gene nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene) . Expression vectors containing S2C6 gene inserts can be identified by three general approaches: (a) nucleic acid hybridization; (b) presence or absence of "marker" gene functions; and (c) expression of inserted sequences. In the first approach, the presence of an S2C6 gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted S2C6 gene. In the second approach, the recombmant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of an S2C6 gene in the vector. For example, if the S2C6 gene is inserted within the marker gene sequence of the vector, recombinants containing the S2C6 insert can be identified by the absence of the marker gene function. In the third approach, recombmant expression vectors can be identified by assaying the S2C6 product expressed by the recombmant . Such assays can be based, for example, on the physical or functional properties of the S2C6 protein in in vitro assay systems, e . g. , potentiation of CD40L binding with
CD40; stimulation of proliferation of normal B cells; inhibition of tumor growth.
Once a particular recombmant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it . Once a suitable host system and growth conditions are established, recombmant expression vectors can be propagated and prepared m quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteπophage vectors (e.g., lambda phage) , and plasmid and cosmid DNA vectors, to name but a few.
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product m the specific fashion desired. Expression from certain promoters can be elevated m the presence of certain mducers; thus, expression of the genetically engineered S2C6 protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce a non-glycosylated core protein product. Expression m yeast will produce a glycosylated product . Expression mammalian cells can be used to ensure "native" glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect processing reactions to d.irffrerent extents.
In specific embodiments, the S2C6-related protein that is expressed is an antibody or fragment or derivative thereof. The recombmant antibody may contain a recombmant light chain variable domain, a recombmant heavy chain variable domain, or both. In a specific embodiment, both light and heavy chains or derivatives thereof are recombmantly expressed by a cell (see e.g., U.S. Patent No. 4,816,397 dated March 28, 1989 by Boss et al . ) A variety of host-vector systems may be utilized to express the protem- coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.) ; insect cell systems infected with virus { e . g . , baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA; transgenic 5 plants or transgenic non-human animals.
5.4 IDENTIFICATION AND PURIFICATION OF GENE PRODUCTS In particular aspects, the invention provides ammo acid sequences of S2C6 proteins and fragments and derivatives thereof which comprise a complementarity-determining region (CDR) or which are otherwise functionally active, as well as nucleic acid sequences encoding the foregoing. "Functionally active" S2C6 material as used herein refers to that material displaying one or more functional activities associated with a full-length (native) S2C6 protein, e.g., binding to CD40;
15 stimulation of proliferation of normal B cells; inhibition of tumor growth; increase the binding of CD40 ligand to CD40 by at least 45%.
In specific embodiments, the invention provides fragments of an S2C6 protein consisting of at least 6 ammo
~n acids, 10 ammo acids, 20 ammo acids, 50 ammo acids, 75 ammo acids or of at least 100 ammo acids and nucleic acids encoding the foregoing.
Once a recombmant which expresses the S2C6 gene sequence is identified, the gene product can be analyzed. This is achieved by assays based on the physical or 5 functional properties of the product, including radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay; stimulation of proliferation of normal B cells; CD40 binding assays, promotion of the binding of CD40 ligand to CD40, inhibition of tumor growth, etc.
30 Once the S2C6 protein is identified, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography) , centrifugation, differential solubility, or by any other standard technique for the purification of proteins. The functional properties may be evaluated using
35 any suitable assay (see Section 5.7) . Alternatively, the S2C6 protein or derivative thereof can be synthesized by standard chemical methods known m the art based on the sequence disclosed herein (e.g., see Hunkapiller et al . , 1984, Nature 310:105-111).
In a specific embodiment of the present invention, such S2C6 proteins, whether produced by recombmant DNA techniques or by chemical synthetic methods or by purification of native proteins, include but are not limited to those containing, as a primary ammo acid sequence, all or part of the ammo acid sequence substantially as depicted m Figures 3A-3B 10 (SEQ ID NOS:2 and 7), as well as fragments and other derivatives, and analogs thereof, including proteins homologous thereto.
5.5 STRUCTURE OF S2C6 GENES AND PROTEINS i The structure of S2C6 genes and proteins of the invention can be analyzed by various methods known the art. Some examples of such methods are described below.
5.5.1 GENETIC ANALYSIS
The cloned DNA or cDNA corresponding to an S2C6 gene can
20 be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517),
Northern hybridization (see e.g., Freeman et al . , 1983, Proc.
Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, 1982, Molecular Cloning, A 5 Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York), and DNA sequence analysis.
Accordingly, this invention provides nucleic acid probes recognizing an S2C6 gene. For example, polymerase chain reaction (PCR; U.S. Patent Nos . 4,683,202, 4,683,195 and
4,889,818; Gyllenstem et al . , 1988, Proc. Natl. Acad. Sci. 0
U.S.A. 85:7652-7656; Ochman et al . , 1988, Genetics 120:621-
623; Loh et al . , 1989, Science 243:217-220) followed by
Southern hybridization with an S2C6 gene-specifIC probe can allow the detection of an S2C6 gene m DNA or cDNA from a cell (e.g., hybridoma) . Methods of amplification other than 5 PCR are commonly known and can also be employed. The stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific S2C6 gene probe used. Modifications of these methods and other methods commonly known m the art can be used.
Restriction endonuclease mapping can be used to roughly determine the genetic structure of an S2C6 gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.
DNA sequence analysis can be performed by any techniques
10 known m the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol . 65:499-560), the Sanger dideoxy method (Sanger et al . , 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699), or use of an
Λ Γ automated DNA sequenator (e.g., Applied Biosystems, Foster City, California) .
5.5.2 PROTEIN ANALYSIS The ammo acid sequence of an S2C6 protein can be derived by deduction from the DNA sequence, or alternatively,
20 by direct sequencing of the protein, e.g., with an automated ammo acid sequencer.
An S2C6 protein sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824). A hydrophilicity profile can be 25 used to identify the hydrophobic and hydrophilic (potentially lmmunogenic) regions of the S2C6 protein and the corresponding regions of the gene sequence which encode such regions .
Secondary, structural analysis (Chou and Fasman, 1974,
Biochemistry 13:222) can also be done, to identify regions of
30 , c an S2C6 protein that assume specific secondary structures.
Manipulation, translation, and secondary structure prediction, open reading frame prediction and plotting, as well as determination of sequence homologies, can also be accomplished using computer software programs available m
35 the art . 5 6 mAb S2C6 ANTIBODY DERIVATIVES Described herein are methods for the production of S2C6 antibody derivatives capable of immunospecifically binding CD40 Such antibodies include but are not limited to monoclonal, humanized, chimeπc, single chain, bispecific, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-bmdmg fragments of any of the above. In one embodiment, the S2C6 derivative comprises one or more deletions, additions and/or substitutions primary ammo acid sequence relative to the primary ammo acid sequence of S2C6. In another embodiment, the S2C6 derivative does not result from cleavage of S2C6 with papam or pepsin In yet another embodiment, the S2C6 derivative comprises one or more deletions, additions and/or substitutions primary ammo acid sequence relative to the primary ammo acid sequence of
S2C6 and does not result from cleavage of S2C6 with papam or pepsin. Guidance for selection of suitable deletions, additions and/or substitutions is provided m this section and in Section 5.7, infra .
For preparation of additional monoclonal antibodies to
CD40, any technique that provides for the production of antibody molecules by continuous cell lines m culture may be used. These include but are not limited to the hybridoma technique of Kohler and Milste , (1975, Nature 256, 495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kozbor et al . , 1983, Immunology Today 4, 72; Cole et al . , 1983, Proc. Natl. Acad. Sci. USA 80, 2026-2030), and the EBV-hybπdoma technique to produce human monoclonal antibodies (Cole et al . , 1985, Monoclonal Antibodies And
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies or other antι-CD40 antibodies available the art may, e.g., be used as the basis from which to clone and thus supply a complementary light chain if a S2C6 heavy chain is to be recombmantly expressed (the two chains may be recombmantly expressed in the same cell or combined m vitro after separate expression and purification) ,- alternatively, a light chain from an antibody of any specificity may be used. Nucleic acids (e.g., a plasmid) encoding a S2C6 heavy chain or encoding a molecule comprising a S2C6 heavy chain variable domain can be transfected into a cell expressing an antibody light chain or molecule comprising an antibody light chain, for expression of a multimeπc protein; the antibody light chain can be recombmant or non-recombmant , and may or may not have antι-CD40 specificity. Alternatively, S2C6 heavy Q chains or molecules comprising the variable region thereof or a CDR thereof can optionally be expressed and used without the presence of a complementary light chain or light chain variable region. In various embodiments, the invention provides a S2C6 heavy chain with CD40 binding affinity, or a _. molecule consisting of or (alternatively) comprising one or more copies of heavy chain CDR 8, 9, and/or 10, or a protein
(peptide or polypeptide) the sequence of which consists of, or comprises, one or more copies of CDR 8, 9 or 10. In a specific embodiment, such a protein can be N or C-terminal modified, e.g., by C-terminal amidation or N-termmal 0 acetylation.
In addition, techniques developed for the production of
"chimeπc antibodies" (Morrison, et al . , 1984, Proc. Natl.
Acad. Sci., 81, 6851-6855; Neuberger, et al . , 1984, Nature
312, 604-608; Takeda, et al . , 1985, Nature 314, 452-454) by 5 splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeπc antibody is a molecule m which different portions are derived from different animal species, 0 such as those having a variable region derived from a muπne mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al . , U.S. Patent No. 4,816,567; and Boss et al . , U.S. Patent No. 5,816,397.) In a specific embodiment, the chimeπc antibody comprises a variable domain of monoclonal J antibody S2C6 secreted by the hybridoma as deposited with the ATCC and assigned accession number PTA-110, and a human constant region. In specific embodiments the variable domain of the chimeric antibody comprises the S2C6 VL (SEQ ID NO : 2 ) as depicted m Figure 3A and/or the S2C6 VH (SEQ ID NO: 7) as depicted m Figure 3B. In addition, techniques have been developed for the production of humanized antibodies. (See, e.g., Queen, U.S. Patent No. 5,585,089 and Winter, U.S. Patent No. 5,225,539.) An lmmunoglobulm light or heavy chain variable region consists of a "framework" region interrupted by three hypervaπable regions, referred to as complementarity- determmmg regions (CDRs) . The extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest", Rabat, E. et al . , U.S. Department of Health and Human Services (1983) . Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and framework regions from a human lmmunoglobulm molecule .
The invention encompasses an antibody or derivative thereof comprising a heavy or light chain variable domain, said variable domain comprising (a) a set of three complementarity-determmmg regions (CDRs) , m which said set of CDRs are from monoclonal antibody S2C6, and (b) a set of four framework regions, m which said set of framework regions differs from the set of framework regions m monoclonal antibody S2C6, and in which said antibody or derivative thereof immunospecifically binds CD40.
Preferably, the set of framework regions is from a human monoclonal antibody, e.g., a human monoclonal antibody that
Figure imgf000028_0001
In a specific embodiment, the invention encompasses an antibody or derivative thereof comprising a light chain variable domain, said variable domain comprising (a) a set of three complementarity-determmmg regions (CDRs) , m which said set of CDRs comprises SEQ ID NO : 3 or SEQ ID NO : 4 , and (b) a set of four framework regions, m which said set of framework regions differs from the set of framework regions m the light chain of monoclonal antibody S2C6, and m which said antibody or derivative thereof immunospecifically binds CD40.
In a specific embodiment, the invention encompasses an antibody or derivative thereof comprising a heavy chain variable domain, said variable domain comprising (a) a set of three complementarity-determmmg regions (CDRs) , m which said set of CDRs comprises SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, and (b) a set of four framework regions, m which said set of framework regions differs from the set of framework regions in the heavy chain of monoclonal antibody S2C6, and m which said antibody or derivative thereof immunospecifically binds CD40.
Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242, 423-426; Huston, et al . , 1988, Proc. Natl. Acad. Sci. USA 85, 5879-5883; and Ward, et al . , 1989, Nature 334, 544-546) can be adapted to produce single chain antibodies using S2C6 sequences. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an am o acid bridge, resulting in a single chain polypeptide. In a specific embodiment, the single chain antibody comprises the ammo acid sequences as depicted in Figure 3A and 3B (SEQ ID NOS : 2 and 7, respectively) .
In a specific embodiment, the antibody to a CD40 polypeptide, peptide or other derivative, or analog thereof comprising all or a portion of SEQ ID NO : 1 or SEQ ID NO : 6 is a bispecific antibody (see generally, e.g. Fanger and
Drakeman, 1995, Drug News and Perspecti ves 8_ 133-137) . Such a bispecific antibody is genetically engineered to recognize both (1) an epitope and (2) one of a variety of "trigger" molecules, e.g. Fc receptors on myeloid cells, and CD3 and CD2 on T cells, that have been identified as being able to cause a cytotoxic T-cell to destroy a particular target. Such bispecific antibodies can be prepared either by chemical conjugation, hybridoma, or recombmant molecular biology techniques known to the skilled artisan. In a specific embodiment, the bispecific antibody contains a molecule comprising the S2C6 heavy or light chain variable domain or a CDR sequence thereof, which molecule has the structure of an antibody heavy or light chain but which differs from the native S2C6 heavy or light chain (e.g., by having ammo acid substitution (s) m the framework region or a human constant domain) .
Antibody fragments that retain the ability to recognize CD40 may be generated by known techniques. For example, such fragments include but are not limited to: The F(ab')2 fragments, which can be produced by pepsin digestion of the antibody molecule and the F(ab') fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse, et al . , 1989, Science 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
5.7 S2C6 PROTEINS, DERIVATIVES AND ANALOGS
In addition to those antibody molecules/variants y described in Section 5.6 above, the invention further relates to S2C6 proteins, derivatives (including but not limited to fragments), analogs, and molecules of S2C6 proteins. Nucleic acids encoding S2C6 protein derivatives and protein analogs are also provided. In one embodiment, the S2C6 proteins are encoded by the nucleic acids described m Section 5.1 above.
In particular aspects, the proteins, derivatives, or analogs are encoded by the sequence of SEQ ID NO:l or SEQ ID NO : 6.
The production and use of derivatives and analogs related to an S2C6 protein are withm the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, i . e . , capable of exhibiting one or more functional activities associated with a full- length, S2C6 protein. As one example, such derivatives or analogs which have the desired binding specificity can be used m immunoassays, or therapeutically for inhibition of tumor growth, etc. A specific embodiment relates to an S2C6 protein fragment that binds CD40 and potentiates binding of CD40L to CD40. Derivatives or analogs of an S2C6 protein can be tested for the desired activity by various immunoassays known in the art, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays , enzyme linked immunosorbent assay (ELISA), "sandwich" immunoassays, Western blots, immunofluorescence assays, protein A assays, immunoelectrophoretic assays, etc.
In addition, assays known in the art can be used to detect or measure the ability to inhibit cell proliferation (e.g., inhibition of tumor cell growth) or ability to stimulate cell proliferation (e.g., proliferation of B cells) in vivo or in vi tro .
In particular, S2C6 derivatives can be made by altering
S2C6 sequences by substitutions, additions { e . g. , insertions) or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as an S2C6 gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of an S2C6 gene which is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the S2C6 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of an S2C6 protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more ammo acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucme, lsoleucme, valme, prolme, phenylalan e, tryptophan and methionme . The polar neutral ammo acids include glyc e, ser e, threonme, cysterne, tyrosme, asparag e, and glutamme. The positively charged (basic) ammo acids include argmme, lysine and histid e. The negatively charged (acidic) ammo acids include aspartic acid and glutamic acid. Such substitutions are generally understood to be conservative substitutions.
In a specific embodiment of the invention, proteins consisting of or comprising a fragment of an S2C6 protein consisting of at least 10 (continuous) ammo acids of the S2C6 protein is provided. In other embodiments, the fragment consists of at least 20 or at least 50 ammo acids of the S2C6 protein. In specific embodiments, such fragments are not larger than 50, 75, 100, or 200 ammo acids. Derivatives or analogs of S2C6 proteins include but are not limited to those molecules comprising regions that are substantially homologous to an S2C6 protein or fragment thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity over an ammo acid sequence of identical size with no insertions or deletions or when compared to an aligned sequence in which the alignment is done by a computer homology program known the art) or whose encoding nucleic acid is capable of hybridizing to a coding S2C6 gene sequence, under high stringency, moderate stringency, or low stringency conditions.
Specifically, by way of example computer programs for determining homology may include but are not limited to
TBLASTN, BLASTP, FASTA, TEASTA, and CLUSTALW (Pearson and
Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8) : 2444-8 ;
Altschul et al., 1990, J. Mol. Biol. 215 (3) : 403-10 ; Thompson, et al . , 1994, Nucleic Acids Res. 22 (22 ): 4673 -80 ; Higgms, et al . , 1996, Methods Enzymol 266:383-402; Altschul, et al . , 1990, J. Mol. Biol. 215 (3 ) : 403 - 10) . Default parameters for each of these computer programs are well known and can be utilized. Specifically, Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov) (Altschul et al . , 1990, J. of Molec . Biol., 215:403-410, "The BLAST Algorithm; Altschul et al . , 1997, Nuc. Acids Res. 25:3389-3402) is a heuristic search
5 algorithm tailored to searching for sequence similarity which ascribes significance using the statistical methods of Karlin and Altschul 1990, Proc. Natl Acad. Sci. USA, 87:2264-68; 1993, Proc. Nat ' 1 Acad. Sci. USA 90:5873-77. Five specific BLAST programs perform the following tasks: 1) The BLASTP j0 program compares an amino acid query sequence against a protein sequence database; 2) The BLASTN program compares a nucleotide query sequence against a nucleotide sequence database; 3) The BLASTX program compares the six- frame conceptual translation products of a nucleotide query ς sequence (both strands) against a protein sequence database; 4) The TBLASTN program compares a protein query sequence against a nucleotide sequence database translated in all six reading frames (both strands) ; 5) The TBLASTX program compares the six- frame translations of a nucleotide query sequence against the six- frame translations of a nucleotide sequence database.
Smith-Waterman (database: European Bioinformatics Institute wwwz.ebi.ac.uk/bic_sw/) (Smith-Waterman, 1981, J. of Molec. Biol., 147:195-197) is a mathematically rigorous algorithm for sequence alignments. 5
FASTA (see Pearson et al . , 1988, Proc. Nat ' 1 Acad. Sci.
USA, 85:2444-2448) is a heuristic approximation to the Smith- Waterman algorithm. For a general discussion of the procedure and benefits of the BLAST, Smith-Waterman and FASTA algorithms see Nicholas et al . , 1998, "A Tutorial on 0 Searching Sequence Databases and Sequence Scoring Methods"
(www.psc.edu) and references cited therein.
The S2C6 derivatives and analogs of the invention can be produced by various methods known in the art . The manipulations which result in their production can occur at 5 the gene or protein level. For example, a cloned S2C6 gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al . , 1989, Molecular Cloning, A Labora tory Manual , 2d ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) . The sequence can be cleaved at appropriate sites with restriction endonuclease (s) , followed by further enzymatic modification if desired, isolated, and ligated in vi tro . In the production of a modified gene encoding a derivative or analog of the S2C6 protein, care should be taken to ensure that the modified gene remains within the same translational reading O frame as the native protein, uninterrupted by translational stop signals, in the gene region where the desired S2C6 protein activity is encoded.
Additionally, an S2C6 nucleic acid sequence can be mutated in vi tro or in vi vo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vi tro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vi tro site-directed 0 mutagenesis (Hutchinson et al . , 1978, J. Biol. Chem.
253:6551), PCR with primers containing a mutation, etc.
Manipulations of an S2C6 protein sequence may also be made at the protein level . Included within the scope of the invention are S2C6 protein fragments or other derivatives or 5 analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4 , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycm, etc. In addition, analogs and derivatives of an S2C6 protein can be chemically synthesized. For example, a peptide corresponding to a portion of an S2C6 protein which comprises the desired domain, or which mediates the desired activity m
5 vi tro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical ammo acids or chemical am o acid analogs can be introduced as a substitution or addition into the S2C6 sequence. Non-classical ammo acids include but are not limited to the D-isomers of the common
JO am o acids, α-amino lsobutyπc acid, 4 -ammobutyπc acid,
Abu, 2 -ammo butyric acid, γ-Abu, e-Ahx, 6 -ammo hexanoic acid, Aib, 2 -ammo isobutyric acid, 3 -ammo propionic acid, ornithme, norleucme, norvalme, hydroxyprolme, sarcosme, citrullme, cysteic acid, tbutyiglycme , t-butylalanme,
^ phenyiglycme, cyclohexylalanme, β-alanme, fluoro-ammo acids, designer ammo acids such as β -methyl ammo acids, Cα- methyl ammo acids, N -methyl ammo acids, and ammo acid analogs in general. Furthermore, the ammo acid can be D
(dextrorotary) or L (levorotary) .
In other specific embodiments, the S2C6 protein, 20 fragment, analog, or derivative may be expressed as a fusion, or chimeπc protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence of a different protein) . The heterologous protein sequence can comprise a biological
25 response modifier, including but not limited to interferon-α, mterferon γ# mterleukm-2 , mterleukm-4 , mterleukm-6 , and tumor necrosis factor, or a functionally active portion thereof. Alternatively, the heterologous protein sequence can comprise enzymes such as β-lactamase or carboxylesterases or toxins such as bryodin 1, Pseudomonas exotoxm A, or gelonm, or a functionally active portion thereof. Additionally, the S2C6 protein can be chemically linked to chemotherapeutic agents, including but not limited to alkylatmg agents (e.g. nitrogen mustards, nitrosoureas,
35 tπazenes) ; antimetabolites (e.g. folic acid analogs, pyπmidme analogs, purme analogs); natural products (e.g. antibiotics, enzymes, biological response modifiers); miscellaneous agents ( e . g. substituted urea, platinum coordination complexes); and hormones and antagonists (e.g. estrogens, androgens , antiandrogen, gonadotropin releasing hormone analog); or functionally active portion thereof (see,
<. e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, pp. 1225-1287, 1996) . Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product
■O by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. In different embodiments, the heterologous protein sequence can be covalently bound to the S2C6 -related sequences by other than
15 a peptide bond, e.g., by use of chemical crosslinking agents well known in the art.
In a specific embodiment, an S2C6 protein derivative is a chimeric or fusion protein comprising an S2C6 protein or fragment thereof (preferably consisting of at least a domain
„ or motif of the S2C6 protein, or at least 10, 50 or 100 amino acids of the S2C6 protein) joined at its amino- or carboxy- terminus via a peptide bond to an amino acid sequence of a different protein. In a specific embodiment, the different protein is a toxin, enzyme or biological response modifier.
In specific embodiments, the amino acid sequence of the different protein is at least 6, 10, 20 or 30 continuous amino acids of the different protein or a portion of the different protein that is functionally active. In one embodiment, such a chimeric protein is produced by recombmant expression of a nucleic acid encoding the protein 0
(comprising an S2C6-coding sequence joined in-frame to a coding sequence for a different protein) . Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding 5 frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Chimeric genes comprising portions of an S2C6 gene fused to any heterologous protein- encoding sequences may be constructed. A specific embodiment relates to a chimeric protein comprising a fragment of an S2C6 protein of at least 6 or 15 or 50 ammo acids, or a fragment that displays one or more functional activities of the S2C6 protein (e.g., comprising copies of one or more CDRs) . In a specific embodiment, the S2C6 protein or derivative thereof is chemically linked to a chemotherapeutic drug including but not limited to doxorubic , paclitaxel or docetaxel . Such a S2C6-drug conjugate can deliver the drug to cells expressing CD40. One or more drug molecules can be linked to the S2C6 protein or derivative. Linkages include but are not limited to hydrazone, peptide or carbohydrate linkages .
In another specific embodiment, the derivative is a molecule comprising a region of homology with an S2C6 protein. By way of example, in various embodiments, a first protein region can be considered "homologous" to a second protein region when the ammo acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical, when compared to any sequence m the second region of an equal number of ammo acids as the number contained in the first region (without any insertions or deletions) or when compared to an aligned sequence of the second region that has been aligned by a computer homology program known m the art .
5.8 HYBRIDIZATION CONDITIONS
In a specific embodiment, a nucleic acid which is hybridizable to an S2C6 nucleic acid (e.g., having a sequence as set forth m SEQ ID NOS : 1 or 6) , or to its reverse complement, or to a nucleic acid encoding an S2C6 derivative, or to its reverse complement under conditions of low stringency is provided. By way of example and not limitation, procedures using such conditions of low stringency are as follows ( see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792). Filters containing DNA are pretreated for 6 h at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm 32P-labeled probe is used. Filters are incubated in hybridization mixture for
10 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS . The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography . If j,- necessary, filters are washed for a third time at 65-68°C and re-exposed to film. Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations) .
In a specific embodiment, a nucleic acid which is hybridizable to an S2C6 nucleic acid (e.g., having a sequence 0 as set forth in SEQ ID NOS : 1 or 6) , or to its reverse complement, or to a nucleic acid encoding an S2C6 derivative, or to its reverse complement under conditions of high stringency is provided. By way of example and not limitation, procedures using such conditions of high 5 stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 10s cpm of 32P-labeled probe. Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0. IX SSC at 50°C for 45 min before autoradiography. Other conditions of high 5 stringency which may be used are well known in the art . In a specific embodiment, a nucleic acid which is hybridizable to an S2C6 nucleic acid (e.g., having a sequence as set forth in SEQ ID NOS : 1 or 6), or to its reverse complement, or to a nucleic acid encoding an S2C6 derivative, or to its reverse complement under conditions of moderate stringency is provided. Selection of appropriate conditions for such stringencies is well known in the art (see e.g., Sambrook et al . , 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; see also, Ausubel et al . , eds., in ■O the Current Protocols in Molecular Biology series of laboratory technique manuals, 1987-1997 Current Protocols,® 1994-1997 John Wiley and Sons, Inc.).
5.9 THERAPEUTIC USES
15 The invention provides for treatment or prevention of various diseases or disorders by administration of a therapeutic compound (termed herein "Therapeutic") . Such Therapeutics include but are not limited to: S2C6 antibodies and derivatives thereof (e.g., as described hereinabove) ; and
20 nucleic acids encoding such S2C6 antibodies and derivatives ( e . g. , as described hereinabove). "Treatment" as used herein shall be deemed to include any clinically desirable or beneficial effect on the disease or disorder, including but not limited to alleviation of one or more symptoms,
25 regression, slowing or cessation of progression, etc. In specific embodiments of the invention, the Therapeutic is administered alone or in combination with CD40L for the treatment or prevention of malignancies (including but not limited to carcinoma and hematologic
,n malignancies), inflammatory diseases, and disorders of the immune system. The Therapeutic and CD40L can, but need not be, contained within the same formulation, i.e, administration of the Therapeutic and CD40 can be performed separately but concurrently or during the same course of treatment. In a specific embodiment, the malignant cells express CD40. Alternatively, the cells of the malignancy need not express CD40, since endothelial cells of the vasculature associated with a malignant tumor should express CD40 and thus the Therapeutic of the invention should provide treatment efficacy even for tumors that do not express CD40. In a preferred embodiment, the Therapeutic potentiates the binding of CD40L to CD40 by at least 45%, 50%, 60%, or 65%. In specific embodiments, the Therapeutic is used to increase the immune response of an immunosuppressed individual, such as a person suffering from acquired immunodeficiency syndrome, from malignancy, or an infant or elderly person.
In other embodiments of the invention, the Therapeutic may be chemically modified so that cells that it binds to are killed. Such cells include but are not limited to multiple myeloma cells, lymphoma cells or carcinomas. Since all B- cells express CD40, this approach can result in suppression of the immune response. For example, a cytotoxic drug linked to S2C6 sequences (e.g., a fusion protein) may be used in vivo to cause immunosuppression in order to cross histocompatibility barriers in transplant patients; alternatively, these modified ligands may be used to control autoimmune diseases.
In other embodiments, the Therapeutic may be used to promote the proliferation and/or differentiation of CD40- bearing cells that are not B cells, for example, lung carcinoma cells, as a means of directly treating malignancy or as an adjunct to chemotherapy. Malignancies which may be treated or prevented using a Therapeutic of the invention include but are not limited to those in Table 1 :
TABLE 1
MALIGNANCIES AND RELATED DISORDERS
Leukemia acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyelocytic mye1omonocytic monocytic erythroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia Polycythemia vera Lymphoma
Hodgkin ' s disease non-Hodgkin ' s disease
Multiple myeloma Waldenstrόm1 s macroglobulmemia Heavy chain disease Solid tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma osteosarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma
Ewing ' s tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma colorectal carcinoma pancreatic cancer breast cancer ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma
Wilms ' tumor cervical cancer uterine cancer testicular tumor lung carcinoma small cell lung carcinoma non small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma ependymoma pmealoma
5 hemangioblastoma acoustic neuroma oligodendrogiloma menangioma melanoma neuroblastoma retmoblastoma nasopharyngeal carcinoma
10 esophageal carcinoma
Inflammatory diseases and deficiencies or disorders of the immune system which may be treated or prevented using a 15 Therapeutic of the invention include but are not limited to those m Table 2 :
20 TABLE 2
INFLAMMATORY DISEASES AND IMMUNE SYSTEM DISORDERS systemic lupus erythematosus (SLE)
Scleroderma (e.g., CRST syndrome) inflammatory myositis ?c S όgren's syndrome (SS) mixed connective tissue disease (e.g., MCTD, Sharp's syndrome) rheumatoid arthritis multiple sclerosis inflammatory bowel disease ( e . g. , ulcerative colitis, Crohn's disease) acute respiratory distress syndrome 30 pulmonary inflammation osteoporosis delayed type hypersensitivity asthma primary biliary cirrhosis (PBC) ldiopathic thrombocytopenic purpura (ITP)
35
5.9.1 EFFECTIVE DOSE Toxicity and therapeutic efficacy of such Therapeutics can be determined by standard pharmaceutical procedures m cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the
5 ED50 (the dose therapeutically effective m 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapeutics that exhibit large therapeutic indices are preferred. While Therapeutics that
JO exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unmfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and ,- animal studies can be used m formulating a range of dosage for use in humans. Exemplary doses include but are not limited to from 1 ng/kg to 100 mg/kg. The dosage of such Therapeutics lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a Therapeutic, the therapeutically effective dose may preferably be estimated initially from cell culture assays. A dose may be formulated m animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined m cell culture. Such information can be used to more accurately determine useful doses in humans. Levels m plasma may be measured, for example, by high performance liquid chromatography .
5.9.2 FORMULATIONS Pharmaceutical compositions for use m accordance with the present invention may be formulated m conventional JJ manner using one or more physiologically acceptable carriers or excipients. Thus, the Therapeutics and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., O pregelatmised maize starch, polyvmylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystallme cellulose or calcium hydrogen phosphate) lubricants (e.g., magnesium stearate, talc or silica); dismtegrants (e.g., potato starch or sodium starch ^ glycolate) ; or wetting agents ( e . g. , sodium lauryl sulphate) .
The tablets may be coated by methods well known m the art.
Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbic acid) . The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner .
For administration by inhalation, the Therapeutics for 5 use according to the present invention are conveniently delivered m the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, tπchlorofluoromethane, dichlorotetrafluoroethane , carbon dioxide or other suitable gas. In the case of a pressurized
5 aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
JO The Therapeutics may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented m unit dosage form, e.g., m ampoules or m multi- dose containers, with or without an added preservative. The
- _. compositions may take such forms as suspensions, solutions or emulsions oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The Therapeutics may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, 25 the Therapeutics may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the Therapeutics may be formulated with suitable
30 polymeric or hydrophobic materials (for example as an emulsion an acceptable oil) or ion exchange resms, or as sparingly soluble derivatives, for example, as a sparingly soluble sal .
The compositions may, if desired, be presented in a pack
J "3J*5 or dispenser device that may contain one or more unit dosage forms containing the active ingredient . The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration preferably for administration to a human. In specific embodiments, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a Therapeutic m combination with CD40 ligand .
The invention is further described m the following examples which are no way intended to limit the scope of the invention.
6. EXAMPLE : CLONING OF SC26 VARIABLE REGIONS
6.1 MATERIALS AND METHODS
The S2C6 light chain and heavy chain variable regions were cloned using methods essentially as described m
Gilliland et al . , 1996, Tissue Antigens 47:1-20. Total RNA was isolated from the S2C6 hybridoma. First strand complementary DNA (cDNA) was prepared for the mouse kappa light chain and heavy chain variable regions using reverse transcriptase and anti-sense primers that annealed approximately 100 base pairs downstream of the JC junction.
A poly-G tail was added to the cDNA strands using terminal transferase and then double stranded DNA was synthesized using the polymerase chain reaction (PCR) . The PCR primers, specific for the poly-G tail or a sequence approximately 50 bases inside the cDNA for the light chain or heavy chain, were designed to include unique restriction sites. After amplification, the PCR products were digested with EcoRI and
Hindlll cloned into pUC19 that had been digested with the same restriction enzymes. These reactions were ligated, transformed into E. coli DH5α, and the resulting clones were screened by restriction analysis. Clones that were positive by restriction digestion analysis were sequenced by DNA sequencing on a Li-Cor fluorescence sequencer. The nucleotide (SEQ ID NO:l) and ammo acid (SEQ ID NO: 2) sequences of the light chain variable region (VL) are shown m Figure 1. The nucleotide (SEQ ID NO: 6) and ammo acid (SEQ ID NO: 7) sequences of the heavy chain variable region (VH) are shown m Figure 2. Figures 3A-3B illustrate the ammo acid sequence of S2C6 VL and S2C6 VH (Figure 3A and 3B, respectively) . The CDRs are underlined. The ammo acid sequences of VL CDRs 1-3 correspond to SEQ ID NOS: 3 -5, respectively. The ammo acid sequences of VH CDRs 1-3 correspond to SEQ ID NOS: 8-10, respectively.
The resulting DNA sequences were then compared to the light chain and heavy chain variable regions of other murme antibodies of the same isotype and the reading frame and corresponding ammo acid sequences for the genes isolated from S2C6 were determined. To confirm the ammo acid sequences, the light chain and heavy chain variable regions of S2C6 mAb were subjected to N-terminal ammo acid analysis.
The ammo acid sequences of S2C6 VL, S2C6 VH and the
CDRs of both the VL and VH were submitted for BLASTP searches on April 21, 1999 using both the NR database (All non- ^ a redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF) and the Rabat database (Rabat's database of sequences of immunological interest) . The sequences found using the NR database can be retrieved using the Accession number at http://www.ncbi.nlm.nih.gov. The sequences found using the
Rabat database can be retrieved using the Accession number at http://immuno.bme.nwu.edu/database_.html and SEQHUNT II. The results of these searches are shown below:
BLASTP SEARCHES USING NR DATABASE
S2C6 VL (SEQ ID NO: 2) : a BLASTP search of the NR database with S2C6 VL as the query yielded no hits with 100% identity and 6 hits with 94% (106/112) identity. These 6 are shown below: pιr||PT0359 IgG kappa chain V region (R4A.12) - mouse (fragment) gi I 196660 (M59949) lmmunoglobul kappa-chain VJ region [Mus musculus] gi I 196954 (M12183) kappa-cham V-region [Mus musculus] >gι I 2247 [Mus musculus] pir I |B34904 Ig kappa chain precursor V region (12-40 and 5-14) ... emb| CAA80076 | (Z22102) lmmunoglobulm variable region [Mus musculus] dbj |BAA22172 I (AB006833) anti-pseudouπdme monoclonal JO antibody...
VL CDR1 (SEQ ID NO: 3) : a BLASTP search with VL CDR1 as the query yielded no hits with 100% identity and numerous hits with 93% identity (15/16) . The first 5 of these are shown ^ _- below: dbj I BAA03480 I (D14627) lmmunoglobulm gamma-3 kappa chain [Mus musculus] db |BAA22172 I (AB006833) anti-pseudouπdme monoclonal antibody... gi 14101647 (AF005352) lmmunoglobulm V-region light chain [Mus musculus] gi 13377681 (AF078800) single chain antι-HIV-1 Rev variable fragment... gi I 1870366 (U55625) anti-DNA lmmunoglobulm light chain IgM [Mus musculus] 5
VL CDR2 (SEQ ID NO: 4) : a BLASTP search of the NR database with VL CDR2 as the query yielded no hits.
VL CDR3 (SEQ ID NO: 5) : a BLASTP search of the NR database 0 with VL CDR3 as the query yielded no hits.
S2C6 VH (SEQ ID NO: 7) : a BLASTP search of the NR database using S2C6 VH as a query yielded no hits with 100% identity and numerous hits with up to 88% identity the first 5 of 5 which are shown below: gi I 3561044 (AF083186) antι-HIV-1 p24 antibody D2 heavy chain [Mus musculus] pdb|lA6T|B Cham B, Fab Fragment Of Mabl-Ia Monoclonal Antibody gi 12895955 (AF045895) IgGl heavy chain mABl-IA [Mus musculus] emb I CAA80023 I (Z22049) lmmunoglobul variable region [Mus musculus] gi I 194510 (M91695) lmmunoglobulm gamma-1 chain [Mus musculus]
VH CDR1 (SEQ ID NO: 8) : a BLASTP search of the NR database with VH CDR1 as the query yielded no hits.
VH CDR2 (SEQ ID NO: 9) : a BLASTP search of the NR database with VH CDR2 as the query yielded no hits with 100% identity, 1 hit with 94% identity (16/17) and numerous hits with less than 94% identity. The 1 hit with 94% identity is shown: gi I 3561044 (AF083186) antι-HIV-1 p24 antibody D2 heavy chain [Mus musculus]
VH CDR3 (SEQ ID NO: 10) : a BLASTP search of the NR database with VH CDR3 as the query yielded no hits.
BLAST SEARCHES USING KABAT DATABASE
S2C6 VL (SEQ ID NO : 2 ) : a BLASTP search of the Rabat database using S2C6 VL as the query yielded no hits with 100% identity and numerous hits with 89-91% identity to the query. The first 5 are shown:
KADBID 005591, mouse IG RAPPA LIGHT CHAIN VARIABLE
REGION (5-14. X ,
KADBID 005594, mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (10VA. X ,
KADBID 005593 mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (12-4...) , KADBID 005603, mouse IG KAPPA LIGHT CHAIN VARIABLE REGION (17s.... ) ,
KADBID 005588, mouse IG KAPPA LIGHT CHAIN VARIABLE REGION (TEPC... ) .
VL CDRl (SEQ ID NO: 3) : a BLASTP search of the Rabat database with VL CDRl as the query yielded no hits with 100% identity and numerous hits with 93% identity (15/16) . The first 5 are shown below: KADBID 005720, mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (BW24... ) ,
KADBID 005614, mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (PME7... ) ,
KADBID 005624, mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (C5-7... ) ,
KADBID 005621, mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (40-6... ) ,
KADBID 005640, mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (40-9... ) .
VL CDR2 (SEQ ID NO : 4 ) : a BLASTP search of the Rabat database with VL CDR2 as the query yielded no hits.
VL CDR3 (SEQ ID NO: 5) : a BLASTP search of the Rabat database with VL CDR3 as the query yielded 1 hit with 100% identity to the query:
KADBID 005681, mouse IG KAPPA LIGHT CHAIN VARIABLE
REGION (NC10... ) .
S2C6 VH (SEQ ID NO: 7) : a BLASTP search of the Rabat database using S2C6 VH as the query yielded no hits with 100% identity and numerous hits with 79-85% identity to the query. The first 5 of the hits are shown below:
KADBID 001498, mouse IG HEAVY CHAIN VARIABLE REGION (HDEX24) , KADBID 001494, mouse IG HEAVY CHAIN VARIABLE REGION (HDEX5) , KADBID 001529, mouse IG HEAVY CHAIN VARIABLE REGION (163.72 'CD ,
RADBID 001500, mouse IG HEAVY CHAIN VARIABLE REGION (HDEX37) , KADBID 001597, mouse IG HEAVY CHAIN VARIABLE REGION (BB128 'CD ,
VH CDRl (SEQ ID NO: 8) : a BLASTP search of the Rabat database with VH CDRl as the query yielded no hits 0
VH CDR2 (SEQ ID NO: 9) : a BLASTP search of the Rabat database with VH CDR as the query yielded no hits with 100% identity and 10 hits with 87-88% identity to the query. The first 5 are shown :
KADBID 001535, mouse IG HEAVY CHAIN VARIABLE REGION (H10"CL) ,
KADBID 001534, mouse IG HEAVY CHAIN VARIABLE REGION (Hβl'CL) ,
KADBID 001533, mouse IG HEAVY CHAIN VARIABLE REGION Q (H50-CL),
KADBID 019741, mouse IG HEAVY CHAIN VARIABLE REGION (Clone F'CL) ,
RADBID 001529, mouse IG HEAVY CHAIN VARIABLE REGION (163.72 'CD ,
VH CDR3 (SEQ ID NO: 10): BLASTP search of the Rabat database with VH CDR3 as the query yielded no hits.
7. EXAMPLE : BIOLOGIC ACTIVITY OF S2C6
0
7.1 MATERIALS AND METHODS
7.1.1 ANTI-CD40 ANTIBODY PREPARATION The S2C6 hybridoma was cultured at 37°C in complete IMDM (Gibco BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS) , 100 units/ml penicillin and 100 mg/ml 5 streptomycin. The culture was harvested by centπfugation and the supernatant was collected by filtration using a 0.2 micron filter. Subsequently the supernatant was loaded onto a GammaBind™ Sepharose column (Pierce) , washed with phosphate buffered saline (PBS), and eluted with 0.1 M glycine pH 2.5. Immediately upon elution, the antibody was neutralized with 1 M Tris pH 8.0, dialyzed into PBS, and filter sterilized. MAb preparations were analyzed by size exclusion chromatography. Only samples of greater than 99% monomeric protein were used for the studies described herein.
10
7.1.2 HUMAN TUMOR XENOGRAFT MODELS
Ninety female C.B.-17 SCID mice were obtained (Taconic Labs, Germantown, NY) at age 6 to 8 weeks and quarantined for 2 weeks. Control groups of mice were injected intravenously ις (i.v.) with a human B cell tumor line: Ramos (non-Hodgkins lymphoma) , HS Sultan (multiple myeloma) or IM-9 (multiple myeloma) cells (Ixl06-2xl06 cells) . The remaining mice were divided into two groups; half were treated with 200 μl of a 1:10 dilution of anti-asialo-GMl (Wako Chemicals, Richmond, VA) i.v., one day prior to the injection of tumor cells, to 0 remove host natural killer cells (Murphy et al . , 1992, Eur. J. Immunol. 22:241) . Mice in the two groups were injected i.v. with Ramos, HS Sultan or IM-9 cells (Ixl06-2xl0scells) . Mice in the test groups were then injected intraperitoneally (i.p.) with 1 mg/kg of S2C6 IgG prepared as described in 5 Section 7.1.2 starting on day 1 or day 5 post tumor implant, according to the following schedule and were monitored for partial paralysis or other signs of disease.
Xenograft Tumor Model Schedule 0
5
Figure imgf000052_0001
Figure imgf000053_0001
7.1.3 PERIPHERAL BLOOD B CELL ISOLATION Peripheral blood B cells were isolated by positive selection using immobilized antibodies against both CD19 and CD20. The final isolated cell population contained greater than 85% B cells as determined by flow cytometry. For storage, the cells were diluted to 4xl07 cells/ml m fetal bovine serum (FBS) containing 10% dimethyl sulfoxide and stored in a liquid nitrogen freezer.
7.1.4 B CELL PROLIFERATION ASSAY Human peripheral blood B cells were thawed and incubated m 96-well tissue culture plates at lxlO5 per well IMDM medium plus 10% FBS m the presence of 5 ng/ml recombmant human IL-4 (Biosource) and various dilutions of an antι-CD40 mAb: S2C6, G28-5 (Bristol-Myers Squibb) or M3 (Genzyme #80- 3702-04) . As a control, cells were incubated with IL-4 and an irrelevant control mAb, EXA2-1H8 (ant i - Pseudomonas exotoxm) . The plates were incubated at 37°C for 3 days and then pulsed for 16 h with 0.5 mCi 3H-thymιdme/well . Cells were harvested onto 96-well glass fiber filters using a Filtermate 196 Harvester™ (Packard Instruments) and combined with scintillation fluid. The extent of 3H-thymιdme incorporated into nascent DNA was measured by liquid scintillation counting using a Topcount LSC™ (Packard Instruments) .
A Jurkat cell line selected to express constitutive high levels of CD40L ( "Jurkat/CD40L" ) , was used as CD40L stimulator cells (Malik et al . , 1996, J. Immunol. 156:3952- 60) . To eliminate proliferation of the stimulator cells, they were treated with mitomycm C (50 mg/ml) PBS for 20 mm at 37°C followed by 3 washes m PBS prior to combining with B cells. B cells (lxl05/well) were combined with Jurkat/CD40L cells and assayed as above. B cells and IL-4 were initially combined with stimulator cells (2.5xl04/well) directly followed by addition of the antι-CD40 mAbs. Monoclonal antibodies were titrated with either a fixed concentration of stimulator cells or stimulator cells were titrated with a fixed concentration of mAb.
7.1.5 CD40/CD40L BINDING ASSAY
The Jurkat/CD40L cell line was used as a target cell line m these assays. Cells were adjusted to a density to 2xl07/ml at 50 μl per sample. Binding was performed m RPMI 1640 media (Gibco) + 10% FBS. To determine receptor saturation, Jurkat/CD40L cells were incubated with increasing concentrations of CD40-Ig (a soluble fusion protein of CD40 and human lmmunoglobulm) (Noelle et al . , 1992, Proc. Natl. Acad. Sci. USA 89:6550-6554), washed and incubated with fluorescem isothiocyanate conjugated to antI -human lmmunoglobulm ( "FITC-anti-human Ig"). The resultant binding was evaluated using a FacScan™ flow cytometer (Becton Dickinson) . Recombmant soluble CD40-Ig (25 μg/ml) was pre- mcubated for 1 h on ice with increasing concentrations of mAb S2C6. The antι-CD40 mAb G28-5; M3 ; and anti - Pseudomonas exotoxm, an lsotype control, were usedJ f sor comparison. The recombmant human soluble CD40 ligand (CD154-muCD8) , produced as a fusion protein with murme CD8 and labeled with FITC, was obtained from Research Diagnostics, Inc. (Flanders, NJ) .
Dilutions of soluble CD40-Ig and antι-CD40 mAbs were made at a 4-fold final concentration, pre-mcubated on ice for 1 h and then combined with Jurkat cells on ice for 1 h. Cells were washed and labeled with FITC-Goat anti-human F(ab')2,
(Jackson Labs, Fc-specific #109-096-098) . The extent of CD40 binding was determined by flow cytometry. 7 . 2 RESULTS
7.2.1 IN VITRO STUDIES: mAb S2C6 PROMOTES CD40/CD40L INTERACTION
To evaluate the effect of antι-CD40 mAbs on the binding of soluble CD40 to CD40L expressed on the surface of activated T cells, increasing concentrations of various CD40 mAbs were pre-mcubated with 25 μg/ml soluble CD40-Ig followed by incubation of the complexes with Jurkat/CD40L cells. CD40L expression on selected CD40L+ Jurkat T cells was initially verified by flow cytometry with FITC-labeled anti- CD40L (data not shown) . CD40 binding to CD40L on these target cells was then determined by flow cytometry of the Jurkat/CD40L cells using FITC-goat anti-human Ig to detect the bound CD40-Ig. Titration with CD40-Ig showed receptor saturation at approximately 25 μg/ml CD40-Ig. Using saturating concentrations of soluble CD40, S2C6 complexed with CD40 at ratios ranging from 0.25 to 2:1 (mass:mass) resulted m a dose-dependent increase in CD40 binding to CD40L (approximately 50%, 100%, 146% and 220% at concentrations of approximately 6 μg/ml, 13 μg/ml, 25 μg/ml, and 50 μg/ml, respectively) (Figure 4) . A similar titration with the inhibitory antibody M3 blocked CD40/CD40L binding in a dose dependent manner. mAb G28-5 showed no effect of CD40/CD40L binding at concentrations up to 25 μg/ml and was only slightly stimulatory at the highest concentration tested (50 μg/ml) , relative to control EXA2-1H8 Ig.
These data clearly indicate mAb S2C6 promotes CD40/CD40L interaction. Further, S2C6 differs from G28-5 and M3 in its ability to increase CD40/CD40L interaction.
In a reciprocal assay, the effect of antι-CD40 mAbs on the binding of soluble CD40L to membrane-bound CD40 expressed on the surface of B cells was evaluated. Titration with soluble CD40L showed Ramos B cell surface CD40 saturation at approximately 10 μg/ml. Increasing concentrations of various antι-CD40 mAbs were pre-mcubated with CD40 -expressing B cells followed by incubation of the cells with FITC-labeled soluble CD40L. The labeled CD40L binding to CD40 on target B cells was then determined by flow cytometry of the Ramos cells. Using saturating concentrations of soluble CD40L, mAb S2C6 complexed with CD40 -expressing cells resulted m a maximal increase m CD40L binding of approximately 51% to 68% at concentrations ranging from 0.04 to 2 μg/ml (Figure 5) . In contrast to the above results with soluble CD40, m which mAb G28-5 had little effect on CD40/CD40L interaction, G28-5 showed inhibition of soluble ligand binding to CD40 at all concentrations tested. A similar titration with the inhibitory mAb M3 also blocked CD40L/CD40 binding m a dose dependent manner.
These data indicate that S2C6 differs surprisingly from G28-5 and M3 m its ability to increase CD40L/CD40 interaction. Moreover, under these conditions, both mAb G28-5 and mAb M3 inhibit the interaction of soluble CD40L with CD40 at concentrations as low as 40 ng/ml.
7.2.2 IN VITRO STUDIES: mAb S2C6 INCREASES B CELL RESPONSE TO CD40/CD40L
The growth response of primary peripheral B cells to CD40L-expressing cells was measured the presence of an antι-CD40 mAb (S2C6, G28-5 or M3 ) . First, B cells were combined with increasing numbers of non-proliferatmg, Jurkat/CD40L cells m the presence or absence of a fixed level (30 ng/ml) of the various mAbs. B cell activation m response to treatment was then measured by 3H-thymιdme incorporation at 72 h post-stimulus. T cell titration the presence of mAb M3 resulted in B cell proliferation similar to that seen with control Ig (Figure 6) .
Although mAb G28-5 provided some B cell activation m the absence of ligand (Figure 7) , CD40L+ T cell titration m the presence of G28-5 only nominally increased B cell proliferation (1.3-fold) over the level seen with G28-5 alone. In contrast, B cell proliferation increased m the presence of S2C6 m a dose dependent manner with increasing numbers of T cell stimulator cells to 3 -fold above mAb-only stimulation with a B cell to T cell stimulator ratio of 4:1. These data demonstrate that unlike M3 and G28-5, S2C6 can surprisingly synergize with CD40L to promote B cell proliferation via CD40.
In a second assay of this type, B cells were either 5 titrated with an antι-CD40 mAb or combined with non- proliferat g CD40L* T stimulator cells at a fixed ratio of 4:1 (B:T) and titrated with an antι-CD40 mAb (Figure 7) .
These results demonstrate that, under these conditions, activation of primary human peripheral blood B cells JO increased 2-fold at 10 μg/ml of mAb G28-5 and ligand, as compared to G28-5 alone. To a surprising degree, S2C6 was significantly more active and m the presence of ligand increased B cell proliferation m a dose dependent manner to 16.2-fold at 10 μg/ml (the highest level tested) as compared to S2C6 alone.
Taken together, these data indicate that S2C6 complexed to CD40 increases CD40L binding. Although S2C6 by itself will stimulate B cell proliferation m a manner similar to G28-5, S2C6 is distinguished from G28-5 by its ability to increase CD40L binding and the subsequent magnitude of the CD40L-medιated activation signal.
8. EXAMPLE: MONOCLONAL ANTIBODY S2C6 INHIBITS TUMOR GROWTH
To evaluate the anti -tumor activity of native mAb S2C6,
25 female C.B.-17 SCID mice were divided into two groups (20 mice/group) . Half of the mice of each group were treated with anti-asialo-GMl to blunt host natural killer cell activity
(Murphy et al . , 1992, Eur . J. Immunol. 22:241) . The following day, mice were injected i.v. with Ramos, HS Sultan
TJ-, or IM-9 cells (1x10s cells) . Mice were then injected i.p. with 1 mg/kg of mAb S2C6 IgG, as described m Materials and
Methods m Section 7 supra and monitored for partial paralysis or other signs of disease onset.
Monoclonal antibody S2C6 treatment of animals harboring
Ramos human B cell lymphoma (Figure 8A) , HS Sultan multiple myeloma (Figure 8B) , or IM-9 multiple myeloma (Figure 8C) , resulted m significant reduction in tumor mass and subsequent tumor-related morbidity and mortality. In parallel studies, efficacy was sustained m the presence of anti-asialo-GMl , suggesting that the increased survival the presence of mAb S2C6 was not due to nonspecific NR activity. The IM-9 cell line is an aggressive tumor model that, like multiple myeloma, secretes human Ig as a surrogate marker of disease.
Treatment of IM-9 diseased mice with mAb S2C6 significantly increased animal survival . These studies clearly demonstrate that S2C6 has potent anti -tumor activity against engrafted human tumors m mice.
9. EXAMPLE: A SINGLE-CHAIN ANTI-CD40 IMMUNOTOXIN FUSION PROTEIN BINDS CD40-Iq
BD1-S2C6 sFv (single-chain antι-CD40 immunotoxm, a fusion protein consisting of the ammo acid sequence of bryod 1 (BD1) (Francisco et al . , 1997, J. Biol. Chem.
272 (39) :24165-24169) fused to the variable regions of monoclonal antibody S2C6) was expressed in E. coll as inclusion bodies, denatured and refolded. Briefly, total RNA was isolated from S2C6 hybridoma cells using TRIZOL reagent (Life Technologies) following the manufacturer's recommendations. First strand cDNA synthesis of the light chain and heavy chain variable regions was performed essentially as described by Gilliland et al . (Tissue Antigens, 47:1-20(1996)) using primers which are complementary to sequences approximately 100 bases downstream of the J-C junctions. The first strands were then poly-G tailed and amplified by PCR using a poly-C anchor primer, which is complementary to the poly-G tail, and a primer nested approximately 50 bases mside the one used for first strand synthesis. The PCR primers were designed to generate unique restriction sites at the 5 ' and 3 ' ends of the PCR products. The two PCR products, containing the sequences coding for the light chain and heavy chain variable regions, were digested with EcoRI and HINDIII and ligated into pUC19 which had been digested with the same enzymes. The resulting plasmids, pSG5 and pSGlO, contain the DNA coding for S2C6 VL and S2C6 VH, respectively. The DNA of both plasmids was sequenced and verified to match the ammo-terminal acid sequence of the parental monoclonal antibody. The VH and VL fragments of S2C6 were "sewn" together (overlap extension PCR) as described by Gilliland et al . m the VH-VL orientation and ligated into a cloning vector. Subsequently the sFv fragment of BD1-G28-5 sFv (Francisco et al . , 1997, J. Biol . Chem . 272:24165-24169) was removed from pSE151 by restriction digestion and S2C6 sFv was ligated m its place. The resulting plasmid, pSG40, contains the gene coding for BD1-S2C6 sFv under the control of the mducible T7 promoter .
For expression, pSG40 was transformed into competent E. coli strain BL21 (DE3 ) pLysS cells and the cells were grown m
T-broth at 37C°. When the culture reached OD600 = 1.0 the cells were induced with 1 mM isopropyl-β-D- thiogalactopyranoside (IPTG) for 3 h. Subsequently, the cells were harvested by centrifugation, lysed by sonication, and the BD1-S2C6 sFv fusion was isolated as insoluble inclusion bodies by centrifugation, which were denatured and refolded as follows: Inclusion bodies were solubilized m 7M guanidme at 5 mg/ml, refolded by rapid dilution (1:100) into
PBS containing 0.3M L-arginme and 2 mM DTT, and dialyzed against 20 mM sodium phosphate buffer, pH 7.4, for subsequent purification.
The refolded protein was isolated using Blue Sepharose followed by affinity chromatography over immobilized CD40-Ig. The purified protein was then tested for binding to immobilized CD40-Ig in ELISA. Microtiter plates were coated with CD40-Ig at 0.5 μg/ml followed by the addition of dilutions of purified BD1-S2C6 sFv in PBS (pH 7.4) with 1% bovme serum albumin and 0.05% Tween-20 m the presence of 25 μg/ml S2C6 mAb (A) , 25 μg/ml control antibody BR96 (•) , or no excess antibody (■) . Binding of BD1-S2C6 sFv to the immobilized receptor was detected by the addition of BD1- specific rabbit antiseru (Seattle Genetics, Inc., Bothell, Washington) followed by the addition of horseradish peroxidase conjugated goat ant1-rabbit Ig.
The binding of BD1-S2C6 sFv to CD40-Ig was completely inhibited by the addition of excess S2C6 mAb but not by the 5 addition of the control mAb (Figure 9) .
10. EXAMPLE: ADMINISTRATION OF RECOMBINANT S2C6 IN THE TREATMENT OF CD40- POSITIVE MALIGNANCIES
Patients with CD40-posιtιve malignancies such as Non- 10 Hodgkin's Lymphoma, Multiple Myeloma, and colon or other carcinomas are injected with recombmant humanized S2C6-antι- CD40 monoclonal antibody (with murine CDRs and human framework regions) or a recombmant chimeric antibody comprising the variable region of S2C6 and the constant 15 region of a human antibody. The recombmant antibody is prepared m vi tro . Treatment can commence at any time during the course of the disease, m the presence or absence of concomitant chemotherapy.
The treatment regimen includes weekly injections of the 20 agent diluted in saline or other physiologically compatible solution.
The dosage used for recombmant S2C6 is m the range of 0.1 mg/m2 (of body surface area of the patient) to 1000 mg/m2 with the preferred dosage being 100-500 mg/m2. 25 The route of injection is intravenous through either a peripheral IV access line or a central IV access line. The agent is administered as an infusion and not an IV push.
The effect of therapy with recombmant S2C6 is monitored by measuring: a) total lymphocyte and T and B lymphocyte .„ counts m the peripheral blood; b) activity of T lymphocytes (helper T4 lymphocytes and cytolytic T8 lymphocytes) in vi tro; and/or c) changes m the morphology of tumors using techniques such as a computed tomographic (CT) scan, magnetic resonance imaging (MRI) scan, x-radiographic imaging, bone scan imaging and tumor biopsy sampling including bone marrow aspiration (BMA) . Depending on the results obtained, the therapeutic regimen is developed to optimally treat CD40-posιtιve malignancies with minimal impact on the competency of the immune system with the ultimate goal of achieving tumor regression and complete eradication of tumor cells.
11. DEPOSIT OF MICROORGANISM Hybridoma S2C6, secreting native monoclonal antibody S2C6, was deposited on May 25, 1999, with the American Type Culture Collection (ATCC) , 10801 University Boulevard,
Manassass, Virginia 20110-2209, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures, and assigned accession number PTA-110.
12. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures . Such modifications are intended to fall within the scope of the appended claims.
Various references, including patent applications, patents, and scientific publications, are cited herein, the disclosures of which are incorporated herein by reference in their entireties.
MICROORGANISMS
Optional Sheet in connection with the microorganism referred to on pages 60 , lines 7-14 of the description '
A. IDENTIFICATION OF DEPOSIT ■
Further deposits are identified on an additional sheet
Name of depositary institution ' American Type Culture Collection
Address of depositary institution (including postal code and country)
10801 University Blvd. Manassas, VA 201 10-2209 US
Date of deposit ' May 25, 1999 Accession Number • PTA-1 10
B. ADDITIONAL INDICATIONS ' (leave blank if not applicable) This information is continued on a separate attached sheet
C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' on*
D. SEPARATE FURNISHING OF INDICATIONS ' (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau later ' (Specify the general nature of the indications e.g., "Accession Number of Deposit")
E. M T Tbhis sheet was received with the International application when filed (to be checked by ±e receiving Office)
(AuthonzedOfficer) ■/-*) 016 ?"
D The date of receipt (from the applicant) by the International Bureau *
(Authorized Officer) Form PCT/RO/134 (January 1981 )

Claims

WHAT IS CLAIMED IS:
1. A molecule comprising SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (a)
5 immunospecifically binds CD40, and (b) comprises one or more substitutions or insertions primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110.
10
2. A molecule comprising SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (a) immunospecifically binds CD40, and (b) is not monoclonal
. _. antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and does not result from cleavage of S2C6 with papam or pepsin.
3. The molecule of claim 1 or 2 comprising the ammo acid sequence of SEQ ID NO: 2 or the ammo acid sequence of SEQ ID NO: 7 or the ammo acid sequences of both SEQ ID NO : 2 and NO: 7.
4. The molecule of claim 1 or 2 which is an antibody.
25
5. The molecule of claim 1 or 2 which is a fusion protein comprising the ammo acid sequence of a second molecule that is not an antibody.
6. The molecule of claim 5 that comprises an ammo
30 acid sequence of bryodm (BDl) fused to SEQ ID NO : 7 fused to
SEQ ID NO:2.
7. The molecule of claim 1 or 2 which is an antibody comprising a variable domain of monoclonal antibody S2C6 as JJ secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and a human constant region.
8. The molecule of any one of claims 1-4 which is purified.
9. A purified protein comprising an am o acid sequence that has at least 95% identity to SEQ ID NO : 2 or SEQ ID NO : 7 , which protein (a) immunospecifically binds CD40; and (b) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110.
<- 10. A purified protein, which protein (a) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (b) increases the binding of CD40 ligand to CD40 by at least 45%, and (c) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110.
11. A purified protein, which protein (a) competes for 5 binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (b) increases the binding of CD40 ligand to
CD40 by at least 45%, and (c) is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and does not result from cleavage of S2C6 with papam or pepsin.
12. An isolated nucleic acid comprising SEQ ID NO : 1 , SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
13. An isolated nucleic acid comprising a nucleotide sequence encoding a protein comprising SEQ ID NO : 3 , SEQ ID NO: 4, SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10.
14. The isolated nucleic acid of claim 13 comprising a nucleotide sequence encoding a protein comprising (a) a heavy chain variable domain of monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and (b) a human constant region.
15. An isolated nucleic acid comprising a nucleotide sequence encoding a protein comprising an ammo acid sequence that has at least 95% identity to SEQ ID NO : 2 or SEQ ID NO : 7.
16. An isolated nucleic acid comprising a nucleotide sequence encoding a protein, which protein competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and which protein increases the binding of
CD40 ligand to CD40 by at least 45%.
17. An isolated nucleic acid comprising a nucleotide sequence encoding a fusion protein, said fusion protein comprising an ammo acid sequence of bryodm 1 (BDl) fused to SEQ ID NO: 7 fused to SEQ ID NO : 2.
18. An isolated nucleic acid which hybridizes to the reverse complement of a DNA consisting of a coding DNA sequence encoding a protein consisting of an ammo acid sequence selected from the group consisting of SEQ ID NO : 2 and SEQ ID NO : 7 , under highly stringent conditions, which isolated nucleic acid encodes a protein that immunospecifically binds CD40.
19. A recombmant cell containing a recombmant nucleic acid vector comprising a nucleotide sequence encoding a protein, which protein competes for binding to CD40 with
-6^ monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA- 110, and which protein increases the binding of CD40 ligand to CD40 by at least 45%.
20. A recombmant cell containing a recombmant nucleic acid vector comprising SEQ ID NO:l, SEQ ID NO: 6, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
21. A method of producing a protein comprising:
(a) growing a cell containing a recombmant nucleotide sequence encoding a protein, which protein competes for binding to CD40 with monoclonal antibody S2C6 as deposited with the ATCC and assigned accession number PTA-110, and which protein increases the binding of CD40 ligand to CD40 by at least 45%, such that the protein is expressed by the cell; and
(b) recovering the expressed protein.
22. A method of producing a protein comprising:
(a) growing a cell containing a recombmant nucleotide sequence encoding a protein comprising SEQ ID NO : 2 , SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , or SEQ ID NO: 10, such that a protein encoded by said nucleotide sequence is expressed by the cell; and
(b) recovering the expressed protein.
23. A pharmaceutical composition comprising: (a) a molecule comprising SEQ ID NO : 2 , SEQ ID
NO: 3, SEQ ID NO : 4 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (l) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (in) comprises one or more substitutions or insertions in primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, in an amount effective for the treatment or prevention of cancer; and (b) a pharmaceutically acceptable carrier.
24. A pharmaceutical composition comprising:
(a) a purified protein, which protein (I) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (in) comprises one or more substitutions or insertions in primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, in an amount effective for the treatment or prevention of cancer; and
(b) a pharmaceutically acceptable carrier.
25. A pharmaceutical composition comprising: (a) a purified protein, which protein (l) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (m) is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and does not result from cleavage of S2C6 with papam
-6tø or pepsin, m an amount effective for the treatment or prevention of cancer; and (b) a pharmaceutically acceptable carrier.
26. A pharmaceutical composition comprising:
(a) a molecule comprising SEQ ID NO : 2 , SEQ ID NO: 3, SEQ ID NO : 4 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (I) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (m) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, m an amount effective for activating or augmenting an immune response; and
(b) a pharmaceutically acceptable carrier.
27. A pharmaceutical composition comprising:
(a) a purified protein, which protein (I) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (in) comprises one or more substitutions or insertions in primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, in an amount effective for activating or augmenting an immune response; and
(b) a pharmaceutically acceptable carrier.
28. A pharmaceutical composition comprising:
- en (a) a purified protein, which protein d) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (in) is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and does not result from cleavage of S2C6 with papam or pepsin, m an amount effective for activating or augmenting an immune response; and
(b) a pharmaceutically acceptable carrier.
29. The pharmaceutical composition of any one of claims 23-28 further comprising CD40 ligand.
30. A method for the treatment or prevention of cancer in a subject comprising: administering to the subject an amount of a molecule comprising SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ ID NO:4, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO: 10, which molecule (I) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (in) comprises one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, which amount is effective for the treatment or prevention of cancer.
31 A method for the treatment or prevention of cancer in a subject comprising: administering to the subject an amount of a purified protein, which protein (l) competes for
^6 binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (11) increases the binding of CD40 ligand to CD40 by at least 45%, and (in) comprises one or more substitutions or insertions primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110,
10 which amount is effective for the treatment or prevention of cancer.
32. A method for the treatment or prevention of cancer m a subject comprising:
1 _ administering to the subject an amount of a purified protein, which protein (I) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (in) is not monoclonal antibody
S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and does not result from cleavage of S2C6 with papam or pepsin, which amount is effective for the 25 treatment or prevention of cancer.
33. A method for activating or augmenting the immune response of a subject comprising: administering to the subject an amount of a
30 molecule comprising SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ
ID NO: 4, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, or SEQ ID NO:10, which molecule (l) immunospecifically binds CD40, (n) increases the binding of CD40 ligand to CD40 by at least 45%, and (m) comprises JJ one or more substitutions or insertions m primary ammo acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, which amount is such that the immune response of the subject is activated or 5 augmented.
34. A method for activating or augmenting the immune response of a subject comprising: administering to the subject an amount of a i purified protein, which protein (i) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (ii) increases the binding of CD40 ligand to CD40 by at
..- least 45%, and (iii) comprises one or more substitutions or insertions in primary amino acid sequence relative to native monoclonal antibody
S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, which amount is such that the immune response of 20 the subject is activated or augmented.
35. A method for activating or augmenting the immune response of a subject comprising: administering to the subject an amount of a
25 purified protein, which protein (i) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (ii) increases the binding of CD40 ligand to CD40 by at
30 least 45%, and (iii) is not monoclonal antibody
S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, and does not result from cleavage of S2C6 with papain or pepsin, which amount is such that the immune -5-*) response of the subject is activated or augmented.
36. A method for the treatment or prevention of an immune disorder m a subject comprising: administering to the subject an amount of a molecule comprising SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ
5 ID NO:4, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , or
SEQ ID NO: 10, which molecule (i) immunospecifically binds CD40, (ii) increases the binding of CD40 ligand to CD40 by at least 45%, and (iii) comprises one or more substitutions or insertions in primary in amino acid sequence relative to native monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, which amount is effective for the treatment or prevention of an immune disorder.
15
37. A method for the treatment or prevention of an immune disorder in a subject comprising: administering to the subject an amount of a purified protein, which protein (i) competes for binding to CD40 with monoclonal antibody S2C6 as 0 a secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, (ii) increases the binding of CD40 ligand to CD40 by at least 45%, and (iii) comprises one or more substitutions or insertions in primary amino acid 5 sequence relative to native monoclonal antibody
S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA-110, which amount is effective for the treatment or prevention of an immune disorder. 0
38. A method for the treatment or prevention of an immune disorder in a subject comprising: administering to the subject an amount of a purified protein, which protein (i) competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC
-7/ and assigned accession number PTA-110, (ii) increases the binding of CD40 ligand to CD40 by at least 45%, and (iii) is not monoclonal antibody S2C6 as secreted by the hybridoma deposited with 5 the ATCC and assigned accession number PTA-110, and does not result from cleavage of S2C6 with papain or pepsin, which amount is effective for the treatment or prevention of an immune disorder.
iQ 39. The method of any one of claims 30-38 further comprising administering CD40 ligand to the subject.
40. The method of any one of claims 30-38 in which the subject is a human.
15
41. The antibody of claim 4 which is not isotype IgGl .
42. A transgenic non-human animal, plant, or an isolated cell containing one or more transgenes encoding a protein, which protein competes for binding to CD40 with monoclonal antibody S2C6 as secreted by the hybridoma deposited with the ATCC and assigned accession number PTA- 110, and which protein increases the binding of CD40 ligand to CD40 by at least 45%.
25
43. A pharmaceutical composition comprising in an amount effective for the treatment or prevention of cancer or an immune disorder, or for activating or augmenting an immune response: (a) a molecule that immunospecifically binds CD40, which molecule increases the binding of CD40 ligand to CD40;
30 (b) CD40 ligand; and (c) a pharmaceutically acceptable carrier .
4 . A method for the treatment or prevention of cancer or an immune disorder in a subject comprising administering
JJ to the subject, in an amount effective for said treatment or prevention: (a) a molecule that immunospecifically binds CD40, which molecule increases the binding of CD40 ligand to CD40; and (b) CD40 ligand.
45. The method of claim 44, wherein the method is for the treatment of cancer.
46. The method of any one of claims 30-32 and 45, wherein the cancer is a solid tumor.
47. The method of any one of claims 36-38, wherein the immune disorder is rheumatoid arthritis.
13
PCT/US2000/015749 1999-06-08 2000-06-08 Recombinant anti-cd40 antibody and uses thereof WO2000075348A1 (en)

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AU54731/00A AU784313B2 (en) 1999-06-08 2000-06-08 Recombinant anti-CD40 antibody and uses thereof
CA2376485A CA2376485C (en) 1999-06-08 2000-06-08 Recombinant anti-cd40 antibody and uses thereof
JP2001501630A JP2003519470A (en) 1999-06-08 2000-06-08 Recombinant anti-CD40 antibody and use thereof
AT00939679T ATE519501T1 (en) 1999-06-08 2000-06-08 RECOMBINANT ANTI-CD40 ANTIBODIES AND APPLICATIONS THEREOF
HU0301789A HUP0301789A2 (en) 1999-06-08 2000-06-08 Recombinant anti-cd40 antibody and uses thereof
IL14695000A IL146950A0 (en) 1999-06-08 2000-06-08 Recombinant anti-cd40 antibody and uses thereof
MXPA01012614A MXPA01012614A (en) 1999-06-08 2000-06-08 Recombinant anti-cd40 antibody and uses thereof.
EP00939679A EP1190074B1 (en) 1999-06-08 2000-06-08 Recombinant anti-cd40 antibody and uses thereof
BR0011394-8A BR0011394A (en) 1999-06-08 2000-06-08 Molecule, purified protein, isolated nucleic acid, recombinant cell, methods for producing a protein, for treating or preventing cancer and an immune disorder, and for activating or enhancing the immune response in an individual, non-human animal, plant or transgenic isolated cell, pharmaceutical composition and antibody
NZ516250A NZ516250A (en) 1999-06-08 2000-06-08 Recombinant anti-CD40 antibody and uses thereof
IL146950A IL146950A (en) 1999-06-08 2001-12-06 Recombinant anti-cd40 antibody and pharmaceutical compositions comprising it
NO20016007A NO20016007L (en) 1999-06-08 2001-12-07 Recombinant anti-CD40 antibody and uses thereof
HK03101596.3A HK1049500A1 (en) 1999-06-08 2003-03-04 Recombinant anti-cd40 antibody and uses thereof

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US09/328,296 US6946129B1 (en) 1999-06-08 1999-06-08 Recombinant anti-CD40 antibody and uses thereof

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