WO1996040754A1 - Immunogenic peptides of prostate specific antigen - Google Patents
Immunogenic peptides of prostate specific antigen Download PDFInfo
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- WO1996040754A1 WO1996040754A1 PCT/US1996/009303 US9609303W WO9640754A1 WO 1996040754 A1 WO1996040754 A1 WO 1996040754A1 US 9609303 W US9609303 W US 9609303W WO 9640754 A1 WO9640754 A1 WO 9640754A1
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- amino acid
- psa
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates generally to the field of cancer diagnosis. More particularly, there is described an immunological method, and accompanying reagents, for the production of antibodies useful in the diagnosis of prostate cancer.
- Prostate cancer is prevalent in old age, with approximately one-half of all males over the age of 70 having been shown to develop prostatic cancer. This high incidence rate has led to the search for markers that may be useful in the detection of prostate malignancy. For example, serum acid phosphatase activity is elevated in patients having metastasized prostate carcinoma. Gutman et al . , J. Clin . Invest . 17:473 (1938) . Numerous studies of this enzyme and its relation to prostatic cancer have been made, but attempts to develop a specific test for prostatic acid phosphatase have met with only limited success. One problem is that there are difficulties associated with accounting for biological and immunological activity of unrelated phosphatases.
- Immunologic assays suffer additional limitations in terms of sensitivity.
- PSA Prostate specific antigen
- PSA serum concentrations correlate with the clinical stage of prostatic cancer in untreated patients and are increased in 86% of patients with benign prostatic hyperplasia.
- PSA also characterized independently as seminal plasma protein p30 (Sensabaugh and Blake, J. Urol . (UNITED STATES) Dec 1990, 144 (6) pl523-6) or as [ ⁇ ] -seminoprotein, (Hara and Kimura, J. Lab . Clin . Med. (UNITED STATES) May 1989, 113 (5) p541-8) , is a glycoprotein of 33-34 kDa.
- PSA is synthesized in, and secreted by, the epithelial cells of the prostate gland.
- Testing for PSA expression has become an important methods in diagnosing and managing prostate cancer.
- the test is widely used, along with digital rectal examination and diagnostic ultrasonography, to permit an early diagnosis of prostate cancer in high-risk men.
- After radical prostatectomy a fall in the serum PSA concentration to undetectable levels gives assurance of curative therapy, while measurable PSA concentrations which persist after surgery indicate the existence of extra-capsular disease and the need for additional therapy.
- Serial PSA monitoring of patients after curative therapy can identify patients who develop recurrent disease, in many cases long before clinical symptoms are evident.
- discontinuous epitopes are composed of sequences from throughout an antigen and rely on folding of the protein to bring the sequences into close proximity of one another. Clearly, oligopeptides are incapable of representing such epitopes. Moreover, even though continuous epitopes are structurally less complicated than discontinuous ones, there remains only a poor understanding of how the immune system recognizes and responds to these antigenic species.
- an object of the present invention to provide a panel of immunodominant peptides suitable for the generation of antibodies to PSA. It also is an object of the present invention to provide compositions including immunodominant PSA peptides suitable for the immunization of animals. It is yet another object of the present invention to provide methods for the generation of polyclonal and monoclonal antibodies reactive with PSA peptides and to PSA itself.
- a peptide of twelve to twenty-one contiguous amino acid residues from the sequence of PSA wherein said peptide is defined by a motif of about fifteen to twenty-one amino acid residues in length having two hydrophobic and one hydrophilic regions arranged in the following manner:
- each of said hydrophobic and hydrophilic regions are of about five amino acid residues in length.
- the motif further is defined by a proline residue (i) within said hydrophilic region, (ii) at the border of said hydrophilic region or (iii) at least five amino acid residues from one end of said peptide.
- the peptide further comprises a leucine, isoleucine or asparagine residue within one or both of said hydrophobic regions.
- the peptide further is characterized by an approximated 360° (-) cosine hydrophilicity pattern.
- the peptide corresponds to the twenty-one N- or C-terminal residues of the sequence of PSA.
- the amino acid sequence of the peptide is selected from the group consisting of LYTKWHYRKWIKDTIVANP, AVKVMDLPQEPALGTTCYA, IVGGWECEKHSQPWQVLVAS, CAQVHPQKVTKFML, YLMLLRLSEPAELTDDAVKV ,
- LLKNRFLRPGDDSSHDLMLLY and ILLGRHSLFHPEDTGQVFQVY.
- a preferred embodiment is a peptide having the sequence CAQVHPQKVTKFML.
- variants of peptides having the above amino acid sequence where the variant have one, two, three, four or five residues that are conservative variations of the selected sequence.
- antigenic compositions comprising any of the foregoing peptides and a carrier molecule and/or an adjuvant.
- polyclonal antisera specific for a peptide of twelve to twenty-one contiguous amino acid residues from the sequence of PSA wherein said peptide is defined by a motif of about twelve to twenty-one amino acid residues in length having two hydrophobic and one hydrophilic regions arranged in the following manner:
- each of said hydrophobic and hydrophilic regions are of about five amino acid residues in length.
- the motif further is defined by a proline residue (i) within said hydrophilic region, (ii) at the border of said hydrophilic region or (iii) at least five amino acid residues from one end of said peptide.
- a preferred antisera is specific for CAQVHPQKVTKFML.
- polyclonal antisera generated by immunization of an animal with a peptide of twelve to twenty-one contiguous amino acid residues from the sequence of PSA, wherein said peptide is defined by a motif of about fifteen to twenty-one amino acid residues in length having two hydrophobic and one hydrophilic regions arranged in the following manner:
- each of said hydrophobic and hydrophilic regions are of about five amino acid residues in length.
- at least 80% of the antibodies of said antisera bind to the same determinant of said peptide.
- a preferred embodiment is a peptide with the sequence CAQVHPQKVTKFML.
- a method for diagnosing prostate cancer comprising the steps of (i) providing a sample; (ii) contacting said sample with antisera specific for a peptide as defined above; and (iii) detecting the binding said antibody to a polypeptide in said sample.
- the method is practiced in vivo .
- the method can be practiced in vi tro .
- the antisera is a polyclonal antisera.
- the method is an ELISA.
- FIG. 1 The amino acid sequence for PSA is shown. V. Detailed Description of the Invention
- PSA is a potential target for diagnosing prostate cancer
- the development of immunologic reagents for the identification of PSA is of considerable interest to both patients and clinicians.
- PSA bears a striking homology to certain kallikreins and, therefore, a significant chance for cross-reactivity with non-PSA antigens exists.
- fragments of PSA In order to generate PSA specific antibodies, it will be necessary to use fragments of PSA and, in all probability, relatively short peptides that represent sequences unique to PSA.
- an Immunologic Model for Identifying Linear Epitopes One embodiment of the present invention involves the identification of immunologic epitopes within PSA.
- the present inventors developed a method for the identification of immunodominant epitopes within polypeptides.
- the method is premised on the notion that linear epitopes are defined by two "descriptors.”
- the first descriptor is a continuous sequence of approximately twelve to twenty-one amino acids that forms a "relative" hydrophobic- hydrophilic-hydrophobic motif, with each of these three charged regions containing about five amino acids.
- “relative” it is meant that there need not be absolute hydrophobicity or hydrophilicity as defined by classic hydrophobicity or hydrophilicity determinations but, rather, that the regions be hydrophobic or hydrophilic with respect to each other and to the flanking sequences.
- the second descriptor is a proline residue at one of the hydrophilic-hydrophobic borders or within the hydrophilic region.
- a portion of an antigen contains both of these descriptors, it is expected to represent a particularly immunogenic portion of the molecule.
- N- and C-terminal peptides having the first, but not the second descriptor also are considered to be promising epitopes.
- a series of rules are applied as follows. First, potential epitopes are identified by locating every proline residue within the polypeptide. Second, roughly twenty-one amino acid "arrays" are defined for each proline, where the proline is placed at the 11th residue of the 21-mer. Third, all non-overlapping arrays are identified. Fourth, a relative hydrophilic or hydrophobic label is assigned to each residue within the unique arrays according to the method of Jainin, Nature 277: 491-492 (1979) . Hydrophobic-hydrophilic-hydrophobic patterns are identified where the proline falls within one of the relatively hydrophilic regions. Arrays thus identified have a high probability of representing immunogenic epitopes.
- the hydrophobic region may prove useful to further rank the epitopes identified according to the above-noted rules. For example, it is considered even more desirable that the hydrophobic region contain a leucine, isoleucine or lysine.
- Another positive indicator of immunodominance is an approximated 360° (-) cosine pattern resulting from a Kyte-Doolittle (1982) hydrophilicity plot.
- the specific definition of the negative cosine curve was as provided in the Microsoft Fortran Library, version 5.1 or PC's.
- the Kyte-Doolittle measurement scale assigns a hydropathy score to each natural amino acid based on side chain (i) interior- exterior distribution and (ii) water-vapor transfer free energy as determined by water-vapor partition coefficients. The values are average over a 5-7 residue window and a value assigned to each window.
- the cDNA for PSA predicts a 362 amino acid preproprotein. This form contains two cleaved sequences not present in the mature molecule. The first is a seventeen amino acid hydrophobic leader sequence and the second is a seven amino acid "pro" sequence. Thus, the mature molecule is 238 amino acids, with the first amino acid of the mature protein being the twenty-fifth predicted from the cDNA.
- PSA was divided into 14 peptides ranging in size from 16 to 21 amino acids for the initial analysis.
- the epitopes defined in PSA will be 12 to 25 amino acids in length.
- Antibodies will bind a peptide epitope having an immunodominant epitope motif defined by an adjacent hydrophobic, hydrophilic, and hydrophobic region with a proline in the hydrophilic region.
- PSA peptides identified in this application may be modified for particular purposes according to methods well known to those of skill in the art.
- particular peptide residues may be derivatized or chemically modified in order to alter the immune response or to permit coupling of the peptide to other agents.
- the size and/or charge of the side chains also are relevant factors in determining which substitutions are conservative.
- Bioly functional equivalent protein or peptide is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity.
- Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted.
- Particular embodiments encompass variants that have one, two, three, four, five or more variations in the peptides sequence. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.
- arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
- the hydropathic index of amino acids also may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5) ; valine (+4.2) ; leucine (+3.8) ; phenylalanine (+2.8) ; cysteine/cystine (+2.5) ; methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7) ; serine (-0.8) ; tryptophan (-0.9) ; tyrosine (-1.3) ; proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5) ; aspartate (-3.5) ; asparagine (-3.5) ; lysine (-3.9) ; and arginine (-4.5) .
- peptidyl compounds suitable for use as immunogens may be formulated to mimic the key portions of the peptide structure.
- Such compounds which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents.
- the generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
- Another method for determining the "equivalence" of modified peptides involves a functional approach. For example, a given peptide can be used to generate monoclonal or polyclonal antibodies. These antibodies can then, in turn, be used to screen libraries of degenerate peptides that include thousand or hundreds of thousands of other peptides, thereby identifying structures that are, at least to a certain extent, immunologically equivalent. Of course, these structures may bear some primary sequence homology to the peptide used to generate the antibodies, but they also may be quite different.
- Lymphokines and Interferons IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ .
- Hormones and Growth Factors nerve growth factor, somatotropin, somatomedins, parathormone, FSH, LH, EGF, TSH, THS-releasing factor, HGH, GRHR, PDGF, IGF-I, IGF-II, TGF- ⁇ , GM-CSF, M-CSF, G-CSF, erythropoietin.
- Tumor Markers and Tumor Suppressors ⁇ -HCG, 4-N- acetylgalactosaminyltransferase, GM2, GD2, GD3, MAGE-1, MAGE-2, MAGE-3, MUC-1, MUC-2, MUC-3, MUC-4, MUC-18, ICAM-1, C-CAM, V-CAM, ELAM, NM23, EGFR, E- cadherin, N-CAM, CEA, DCC, PSA, Her2-neu, UTAA, melanoma antigen p75, K19, HKer 8, pMel 17, tyrosinase related proteins 1 and 2, p97, p53, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC and MCC.
- Oncogenes ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl .
- Clr Cls, C4, C2, Factor D, Factor B, properdin, C3, C5, C6, C7, C8, C9, Cllnh, Factor H, C4b-binding protein, DAF, membrane cofactor protein, anaphylatoxin inactivator S protein, HRF, MIRL, CRl, CR2, CR3, CR4, C3a/C4a receptor, C5a receptor.
- Viral Antigens HIV (gag, pol, gp41, gpl20, vif, tat, rev, nef , vpr, vpu, vpx) , HSV (ribonucleotide reductase, ⁇ -TIF, ICP4, ICP8, ICP35, LAT-related proteins, gB, gC, gD, gE, gH, gl, gj) , influenza
- HIV gag, pol, gp41, gpl20, vif, tat, rev, nef , vpr, vpu, vpx
- HSV ribonucleotide reductase, ⁇ -TIF, ICP4, ICP8, ICP35, LAT-related proteins, gB, gC, gD, gE, gH, gl, gj
- EBNA Epstein-Barr Virus
- hydrophilic region as a defining characteristic of epitope motifs in proteins has been accepted for some time.
- Such an epitope's hydrophilic amino acids are generally thought to be important in that they situate the epitope on the external portion of the antigen.
- the present invention contemplates a more complex site having hydrophobic-hydrophilic-hydrophobic structure including at least one proline.
- hydrophilic interactions probably provide some structural features and ensure localization of the epitope on the external portion of the antigen.
- the flanking hydrophobic interactions at both ends of the hydrophilic region may help dock the epitope in the antibody binding site.
- the bend in the amino acid sequence at proline may provide a rudimentary, but easily recognizable three- dimensional structure that facilitates the transition from hydrophilic amino acids regions in the complete epitope of a protein.
- I most likely I, is important in anchoring the N-terminus of the epitope to an antibody binding site.
- the middle sections in both of these examples are relatively hydrophilic, do not bind antibody by themselves, and a proline is located at the border within the relatively hydrophilic region of the peptide in both, although the sequence order is from the C-terminal to N-terminal in the lower example. Hydrogen bonding utilizing the middle hydrophilic amino acids may play a significant role in determining epitope specificity, even though not contributing greatly to the overall strength of the antibody-epitope bond.
- the motif was used to successfully locate immunodominant and neutralizing antibody epitopes for the following:
- the immunodominant motif identifies regions in various proteins that elicit high titer antibody responses because it defines loop or pseudo-loop structures in proteins known to elicit high titer functional antibodies in proteins such as HIV-1 gpl20.
- oligopeptides identified above will be used in the form of antigen compositions for the immunization of animals. Even though some oligopeptides may be immunogenic in and of themselves, it often will be desirable to conjugate oligopeptides to higher molecular weight carrier molecules.
- BSA bovine serum albumin
- KLH keyhole limpet hemocyanin
- Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as carriers. Synthetic carriers also are used and are readily available.
- Means for conjugating a polypeptide to a carrier protein are also well known in the art and include glutaraldehyde, .n-maleimidobencoyl-N- hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
- PSA immunogens can be used to produce monoclonal antibodies according to the present invention.
- peptides may be chemically- synthesized, based on the identified PSA sequences.
- these immunogens can be synthesized chemically, either as a single molecule or separately followed by conjugation.
- a host cell e . g. , a bacterial, insect, yeast or mammalian cell
- Transformation of host cells may be either transient or stable.
- Stable transformation may be achieved by either integration into the host genome or by episomal maintenance of an expression vector.
- Bacterial hosts are desired where post-translational modifications of the protein to be produced are minimal. Where post- translational modifications are significant, it may be necessary to use eukaryotic hosts such as insect, yeast, mammalian or even human cells.
- adjuvants that enhance the immune response to PSA peptides.
- Such adjuvants include all acceptable immunostimulatory compounds such as cytokines, toxins or synthetic compositions. Examples of these are IL-1, IL-2, BCG, aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thur-MDP) , N-acetyl-nor-muramyl- L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP) , N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine- 2- (1' -2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) - ethylamine (CGP) 1983A, referred to as MTP-PE) , lipid A, MPL and RIBI
- Liposomes also can be used as adjuvants.
- BRM biologic response modifiers
- Such BRM's include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA) ; Indomethacin (IND; 150 mg/d) (Lederle, NJ) ; or low-dose Cyclophosphamide (CYP; 75, 150 or 300 mg/m 2 ) (Johnson/Mead, NJ) .
- Antigen compositions of the present invention include PSA antigens advantageously administered in the form of injectable, pharmaceutical compositions.
- a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
- the composition may contain about 10 mg of human serum albumin and from about 20 to 200 micrograms of the labeled monoclonal antibody or fragment thereof per milliliter of phosphate buffer containing NaCl.
- Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc .
- Intravenous vehicles include fluid and nutrient replenishers.
- Preservatives include antimicrobial, anti ⁇ oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art.
- one of the uses for PSA peptides according to the present invention is to generate antibodies.
- Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies, and parts thereof, either alone or conjugated with other moieties.
- Antibody parts include Fab and F(ab) 2 fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals or in vi tro using recombinant DNA techniques.
- polyclonal antisera is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal.
- a wide range of animal species can be used for the production of antisera.
- the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig, a goat, a sheep or a chicken. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
- the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization.
- a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal) .
- the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved.
- the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs (below) .
- polyclonal antisera is derived from a variety of different "clones,” i.e., B- cells of different lineage.
- Monoclonal antibodies by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.
- peptides When peptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed. Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well assume multiple (and probably non- native) conformations. As a result, even short peptides can produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule.
- Polyclonal antisera according to present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher.
- the present invention provides for polyclonal antisera where the clonality, i.e., the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality - 90%, 95% or greater - is contemplated.
- Anti-PSA antisera or monoclonal antibodies can be identified in a fairly straightforward manner using any one of variety of immunological screening assays in which antibody competition can be assessed.
- test antibodies can be used in simple competition assays. A known antibody preparation and the test antibody are premixed, incubated with the antigen composition and compete for binding to the PSA peptide.
- Antigen composition means any composition that contains some version of PSA, whole or fragment, in an accessible form. Antigen-coated wells of an ELISA plate are particularly preferred.
- Antibody competition assays are particularly suitable for use in an ELISA or RIA microtiter well format, but any immunoassay may be used. Most preferably, the assay will be capable of generating quantitative results. An antibody that binds to the antigen composition will be able to effectively compete for binding of the known antibody and thus will significantly reduce binding of the latter. The reactivity of the known antibodies in the absence of any test antibody is the control. A significant reduction in reactivity in the presence of a test antibody is indicative of a test antibody that binds to the PSA peptide, i.e., one that "cross-reacts" with the known antibody.
- MAbs may be readily prepared through use of well- known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
- this technique involves immunizing a suitable animal with a selected immunogen composition, e . g. , purified or partially purified protein, synthetic protein or fragments thereof.
- the immunizing composition is administered in a manner effective to stimulate antibody producing cells .
- Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is possible. The use of rats may provide certain advantages, but mice are preferred, with the
- BALB/c mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions.
- somatic cells with the potential for producing antibodies are selected for use in the MAb generating protocol .
- B cells B lymphocytes
- These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
- a panel of animals will have been immunized and the spleen of animal with the highest antibody titer removed.
- Spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
- a spleen from an immunized mouse contains approximately 5 X 10 7 to 2 X 10 8 lymphocytes.
- the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
- Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells, called "hybridomas.
- any one of a number of myeloma cells may be used and these are known to those of skill in the art.
- the immunized animal is a mouse
- rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
- NS-1 myeloma cell line also termed P3-NS-l-Ag4-l
- P3-NS-l-Ag4-l NS-1 myeloma cell line
- Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
- Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes .
- Fusion methods using Sendai virus have been described by Kohler and Milstein Nature 256:495-497 (1975) and Eur. J. Immunol . 6:511-519 (1976) , and those using polyethylene glycol (PEG) , such as 37% (v/v) PEG, by Gefter et al . Somatic Cell Genet . 3:231-236 (1977) .
- PEG polyethylene glycol
- Somatic Cell Genet . 3:231-236 (1977) The use of electrically induced fusion methods is also appropriate.
- Fusion procedures usually produce viable hybrids at low frequencies, about 1 X 10 "6 to 1 X 10 "8 . This does not pose a problem, however, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culture in a selective medium.
- the selective medium generally is one that contains an agent that blocks the e novo synthesis of nucleotides in the tissue culture media.
- Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
- the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium) .
- HAT medium a source of nucleotides
- azaserine the media is supplemented with hypoxanthine.
- the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
- the myeloma cells are defective in key enzymes of the salvage pathway, e . g. , hypoxanthine phosphoribosyl transferase (HPRT) , and they cannot survive.
- the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells .
- This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by single-clone dilution in microtiter plates, followed by testing the individual clonal supematants (after about two to three weeks) for the desired reactivity.
- the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
- the selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
- the cell lines may be exploited for MAb production in two basic ways.
- a sample of the hybridoma can be injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
- the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
- the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
- the individual cell lines could also be cultured in vi tro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations .
- MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
- Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art.
- Monoclonal antibodies according to the present invention also may be monoclonal heteroconjugates, i.e., hybrids of two or more antibody molecules.
- monoclonal antibodies according to the invention are chimeric monoclonal antibodies.
- the chimeric monoclonal antibody is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse anti-PSA producing cell and the constant-region exons from a human antibody gene.
- the antibody encoded by such a recombinant gene is a mouse- human chimera. Its antibody specificity is determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.
- monoclonal antibodies according to the present invention is a "humanized” monoclonal antibody, produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V- chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the framework regions of their murine counterparts.
- CDRs mouse complementary determining regions
- Humanized monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods.
- the monoclonal antibodies and fragments thereof according to this invention can be multiplied according to in vi tro and in vivo methods well-known in the art.
- Multiplication in vi tro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e . g. , feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
- suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium
- a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e . g.
- feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
- vi tro production provides relatively pure antibody preparations and allows scale-up
- Techniques for large scale hybridoma cultivation under tissue culture conditions include homogenous suspension culture, e . g. , in an airlift reactor or in a continuous stirrer reactor or immobilized or entrapped cell culture.
- Large amounts of the monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo .
- Cell clones are injected into mammals which are histocompatible with the parent cells, e . g. , syngeneic mice, to cause growth of antibody- producing tumors.
- the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.
- fragments of the monoclonal antibody of the invention can be obtained from monoclonal antibodies produced as described above, by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction.
- monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art.
- the monoclonal conjugates of the present invention are prepared by methods known in the art, e . g. , by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
- a coupling agent such as glutaraldehyde or periodate.
- Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as 3 H, 125 I, 131 I 32 P, 35 S; 14 C# 51 Cr# 36 C1/ 57 C ⁇ / 58 C ⁇ / 59 ⁇ 75 Se#
- Radioactively labeled monoclonal antibodies of the present invention are produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium-"m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e . g. , by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
- a reducing agent such as SNC1 2
- a buffer solution such as sodium-potassium phthalate solution
- the anti-PSA antibodies will have significant utility in assays for the detection of the PSA antigen, as needed in diagnosis and prognostic monitoring.
- Immunoassays encompassed by the present invention include, but are not limited to those described in U.S.
- assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vi tro and in vivo .
- Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.
- the anti-PSA antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the PSA antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound PSA antigen may be detected. Detection is generally achieved by the addition of another anti-PSA antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti-PSA antibody, followed by the addition of a third antibody that has binding affinity for the second anti-PSA antibody, with the third antibody being linked to a detectable label.
- the samples suspected of containing the PSA antigen are immobilized onto the well surface and then contacted with the anti-PSA antibodies of the invention. After binding and appropriate washing, the bound immune complexes are detected. Where the initial anti-PSA antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-PSA antibody, with the second antibody being linked to a detectable label.
- Competition ELISAs are also possible in which test samples compete for binding with known amounts of labeled PSA antigens or antibodies.
- the amount of reactive species in the unknown sample is determined by mixing the sample with the known labelled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
- ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described as below.
- a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material . Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA) , casein and solutions of milk powder.
- BSA bovine serum albumin
- the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
- a secondary or tertiary detection means rather than a direct procedure.
- the immobilizing surface is contacted with the clinical or biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
- Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) /Tween. These added agents also tend to assist in the reduction of nonspecific background.
- BSA bovine gamma globulin
- PBS phosphate buffered saline
- the suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C, or may be overnight at about 4°C or so.
- the contacted surface is washed so as to remove non-complexed material . Washing often includes washing with a solution of PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
- the second or third antibody will have an associated label to allow detection.
- this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
- a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation, e . g. , incubation for 2 hours at room temperature in a PBS-containing solution such as PBS- Tween. After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.
- a chromogenic substrate such as urea and bromocresol purple or 2,2' -azino-di- (3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H 2 0 2 , in the case of peroxidase as the enzyme label .
- Quantification is then achieved by measuring the degree of color generation, e . g. , using a visible spectra spectrophotometer.
- the label may be a chemiluminescent one.
- the use of such labels is described in U.S. Patent Nos. 5,310,687, 5,238,808 and 5,221,605, all of which are incorporated by reference in their entirety.
- a typical application of immunoprecipitation involves the use of Staphylo •coccus aureus bacteria that have protein A expressed on the peptidoglycan cell wall.
- an antibody that recognizes the antigen of interest is quickly precipitated by the binding of the antibody to the protein A on the cells which are then easily precipitated by centrifugation (Kessler, S. W. Methods in Enzy ology, 73:442, 1981) .
- protein A-conjugated beads can be used in place of the S. aureus bacteria.
- Assays for PSA polypeptides also can determine normal/abnormal tissue distribution for diagnostic purposes.
- Methods for in vi tro and in si tu analysis are well known and involve assessing binding of PSA-specific antibodies to tissues, cells or cell extracts. These are conventional techniques well within the grasp of those skilled in the art.
- the antibodies of the present invention may be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared from study by immunohistochemistry (IHC) . Each tissue block may consist of 50 mg of residual "pulverized" prostate tumor. The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, e . g.
- frozen-sections may be prepared by rehydrating 50 ng of frozen pulverized prostate tumor at room temperature in PBS in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT) ; inverting the capsule and pelleting again by centrifugation; snap- freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections containing an average of about 500 remarkably intact tumor cells.
- OCT viscous embedding medium
- Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
- the invention also relates to an in vivo method of imaging a prostate-derived tumor. Specifically, this method involves administering to a subject an imaging- effective amount of a detectably-labeled anti-PSA monoclonal antibody or fragment thereof and a pharmaceutically-effective carrier and detecting the binding of the labeled monoclonal antibody to the diseased tissue.
- in vivo imaging refers to any method which permits the detection of a labeled monoclonal antibody of the present invention or fragment thereof that specifically binds to a target tissue located in the subject's body.
- a "subject” is a mammal, preferably a human.
- An “imaging effective amount” means that the amount of the detectably-labeled monoclonal antibody, or fragment thereof, administered is sufficient to enable detection of binding of the monoclonal antibody or fragment thereof to the diseased tissue.
- in vivo imaging relies on radionuclides.
- a factor to be considered in selecting a radionuclide for in vivo diagnosis is that the half-life must be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host, as well as background, is minimized.
- a radionuclide used for in vivo imaging will lack a particulate emission, but produce a large number of photons in a 140-2000 keV range, which may be readily detected by conventional gamma cameras.
- a radionuclide may be bound to an antibody either directly or indirectly by using an intermediary functional group.
- Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA) .
- DTPA diethylenetriaminepentaacetic acid
- EDTA ethylene diaminetetracetic acid
- metallic ions suitable for use in this invention are 99m Tc, 123 I, 131 I ⁇ :L1 In, 131 I, 97 Ru, 67 Cu, 67 Ga, 125 I, 68 Ga, 72 As, 89 Zr, and
- the monoclonal antibody or fragment thereof may be labeled by any of several techniques known to the art .
- the methods of the present invention also may use paramagnetic isotopes for purposes of in vivo detection.
- Elements particularly useful in Magnetic Resonance Imaging (“MRI") include 157 Gd, 55 Mn, 162 Dy, 52 Cr, and 56 Fe.
- Administration of the labeled antibody may be local or systemic and accomplished intravenously, intraarterially, via the spinal fluid or the like. Administration may also be intradermal or intracavitary, depending upon the body site under examination. After a sufficient time has lapsed for the monoclonal antibody or fragment thereof to bind with the diseased tissue, for example 30 min to 48 h, the area of the subject under investigation is examined by routine imaging techniques such as MRI, SPECT, planar scintillation imaging and emerging imaging techniques, as well. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan. The distribution of the bound radioactive isotope and its increase or decrease with time is then monitored and recorded. By comparing the results with data obtained from studies of clinically normal individuals, the presence and extent of the diseased tissue can be determined.
- an ELISA method is utilized.
- the peptide segment representing the immunodominant motif epitope is plated in an ELISA microtiter plate well as are other immunosubdominant epitope peptides in separate wells.
- Antiserum made to an immunodominant epitope will test positive only in the ELISA well in which that epitope peptide is plated and will test negative in wells that contain an epitope peptide segment to which no antibodies are present in the sample. The extent of the homogeneity of sample antibodies for the immunodominant epitope peptide in question can thus be quantitated.
- kits may comprise a receptacle being compartmentalized to receive one or more containers such as vials, tubes and the like, such containers holding separate elements of the invention.
- one container may contain a first antibody bound to an insoluble or partly soluble carrier.
- a second container may contain a soluble, detectably-labeled second antibody, in lyophilized form or in solution.
- the receptacle may also contain a third container holding a detectably labeled third antibody in lyophilized form or in solution.
- a kit of this nature can be used in a diagnostic assay according to the invention.
- the kit may comprise PSA oligopeptides, free or bound to carrier molecules.
- a second container may contain carrier or adjuvant.
- a kit of this nature can be used in the production of PSA- specific antibodies.
- the amino acid sequence of PSA was divided into fourteen peptide segments. Thirteen of the peptides were 19-mers to 21-mers and one peptide was a 16-mer. Seven of the selected peptides exhibited an antibody immunodominant epitope motif consisting of two descriptors: (1) a proline, usually centrally located, in or at the border of the middle hydrophilic region of sequential amino acids when present, and (2) adjacent hydrophobic-hydrophilic-hydrophobic regions in a linear sequence of from 12-21 amino acids as determined by Kyte- Doolittle hydrophilicity plots. Seven of the peptides displayed both of the descriptors while five of the peptides displayed only the proline descriptors. Two of the peptides lacked both the proline and the hydrophobic- hydrophilic-hydrophobic region descriptors.
- Sheep antiserum was raised against the PSA immunodominant linear epitope spanning PSA amino acids 163-176. This epitope was selected since it had both descriptors and also since its sequence was relatively non-homologous with another prostatic kallikrein, known as human glandular kallikrein (HgK) .
- HgK human glandular kallikrein
- PSA peptide 163- 176 was plated in duplicate ELISA microtiter plate well rows as were other potential PSA epitope peptides in their respective duplicate ELISA plate rows.
- the PSA antiserum was serially diluted and added to the entire set of duplicate ELISA well rows.
- the antiserum only tested significantly positive in the ELISA well rows that contained the immunogen epitope peptide comprised of PSA linear amino acid sequence 163-
- the sheep polyclonal sera described in Example 3 further was evaluated in terms of specificity for PSA, as opposed to the related kallikrein HgK.
- Antisera raised in sheep to various PSA peptides are used as a source of antibody.
- This antiserum is affinity purified on a PSA peptide column as follows.
- the antigen is coupled to Sepharose CL-6B beads (Pharmacia) at approximately 0.5 mg total protein per mg of gel following activation with carbonyldiimidazole in dioxan (0.4 g per ml of gel) . Coupling is carried out in 0.5M Na 2 C0 3 , 0.5M NaCl (pH9.5) at 0°C over two nights.
- the gel is washed alternatively in O.IM NaHC0 3 , O.IM NaCl (pH 8.0) and O.IM CH 3 .COONa.3H 2 0, O.IM glycine HCl (pH 4.5) and finally washed with ice-cold 0.1M glycine HCl (pH 2.5) .
- the gel is then neutralized and stored in 0.01M Tris, 0.15M NaCl (pH 8.0) .
- the antibodies against the PSA peptide are bound to the antigen column in Tris-saline buffer (pH 8.0) and specifically eluted with 0.1 M glycine-HCl buffer (pH 2.5) . Following elution, the eluant is adjusted to pH 8.0 using 2 M Tris. Antibody is concentrated on a YM30 filter and dialyzed into water. Precipitating fats, etc., are removed by centrifugation. The dialyzed antibody solution is then adjusted to 0.1 M NaHC0 3 , 0.5 M NaCl (pH 8.3) and incubated overnight at 4°C with CNBr- activated Sepharose. Unreacted groups are blocked with 1.0 M ethanolamine pH 8.0 overnight at 4°C. The
- Sepharose beads are then washed with 0.1 M NaHC0 3 , 0.5 M NaCl (pH 8.3) buffer followed by 0.1 M sodium acetate buffer and stored in Tris-saline buffer pH 8.0 containing 0.001% sodium azide as a preservative.
- the columns are washed with ice-cold 0.1 M glycine-HCl buffer (pH 2.5) immediately prior to use to remove any weakly bound antibody.
- Example 6 Estimation of Antibody Concentrations
- Antibody concentrations are estimated using a direct, non-competitive ELISA. Positive control PSA peptides are diluted in carbonate/bicarbonate buffer pH 9.6 to a final concentration of 1 ⁇ g/ml. Aliquots of 200 ⁇ l are added to each well of a microtiter plate. Following overnight incubation at 4°C, plates are washed with PBS containing 0.05% (w/v) Tween 20 and blocked with gelatin. The same buffer is used for subsequent coupling and washing steps. Primary antibody is diluted in PBS- Tween 20 and allowed to react for 1 h at 37°C.
- reaction with the primary antiserum is followed by a 45 min reaction with a 1:1200 dilution of biotinylated donkey anti-rabbit IgG, then 45 min with 1: 1200 peroxidase coupled streptavidin.
- Peroxidase activity is measured with 1 mg/ml 5- aminosalicylic acid and 1.7 mM hydrogen peroxide in 20 mM phosphate buffer pH 6.7. Absorbance values are measured using a Titretek Multiscan Plus Mk II ELISA reader interfaced with an IBM compatible personal computer. Data are processed using the Kinetic Linked Immunosorbent Assay (KELA) to yield a kinetic estimate of the peroxidase activity.
- KELA Kinetic Linked Immunosorbent Assay
- a preferred embodiment of the present invention is a monoclonal antibody that recognizes the peptide antigens disclosed herein.
- Means for preparing and characterizing antibodies are well known in the art (See, e . g. ,
- monoclonal antibodies To obtain monoclonal antibodies, one would initially immunize an experimental animal, often preferably a mouse, with an antigenic PSA peptide composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired PSA peptide.
- cell lines such as human or mouse myeloma strains
- spleen cells are removed and fused, using a standard fusion protocol (see, e . g. , The Cold Spring Harbor Manual for Hybridoma Development, incorporated herein by reference) with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against the peptide.
- Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods.
- Hybridoma clones can then be cultured in liquid media and the culture supematants purified to provide the PSA peptide-specific monoclonal antibodies.
- Example 8 The Immunodominant Epitope Motif Specifies Protein Loop Structures in Gelonin
- a protein loop structure by definition is comprised of a linear sequence of amino acids that originates beneath or at the surface of a protein, loops out into the aqueous environment, and then returns to and may travel beneath the outer surface of the protein. Carlacci and Englander, BIOPOLYMERS 33:1271-1286 (1993); Bruccoleri et al . , Nature 335:564-568 (1988); Fine et al .
- loop formation is facilitated by the two characteristics that define the immunodominant motif presented here: (1) a hydrophobic- hydrophilic-hydrophobic region in a linear sequence of from twelve to twenty amino acids and (2) a proline, usually centrally located in the sequence within or at the border of the middle hydrophilic region. Since the termini defined in the immunodominant motif are hydrophobic, the proposed starting and ending points of the protein loop are comprised of these hydrophobic amino acids and can interact hydrophobically with buried amino acids near the protein surface which most likely are also hydrophobic.
- the middle hydrophilic region of the immunodominant motif is the proposed location of the loop amino acids which are extended out from the surface of protein proper. These extended, hydrophilic amino acids are biochemically suited to interact with the aqueous environment surrounding proteins. The bend in the amino acid chain at the motif's centrally located proline aids in returning the loop to the protein's surface.
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AU60956/96A AU6095696A (en) | 1995-06-07 | 1996-06-06 | Immunogenic peptides of prostate specific antigen |
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WO1997029199A2 (en) * | 1996-02-06 | 1997-08-14 | Abbott Laboratories | Prostate specific antigen peptides and uses thereof |
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US6780598B1 (en) * | 1997-06-13 | 2004-08-24 | William J. Kokolus | Method of identifying and locating immunobiologically-active linear peptides |
EP1224466B1 (en) * | 1999-10-07 | 2007-01-03 | Ciphergen Biosystems, Inc. | Prostate cancer marker proteins |
EP1964573A2 (en) | 1999-10-22 | 2008-09-03 | Aventis Pasteur Limited | Method of inducing and/or enhancing an immune response to tumor antigens |
US8017590B1 (en) | 1999-10-22 | 2011-09-13 | Sanofi Pasteur Limited | Method of inducing and/or enhancing an immune response to tumor antigens |
Also Published As
Publication number | Publication date |
---|---|
EP0832105A4 (en) | 2002-02-06 |
US6326471B1 (en) | 2001-12-04 |
US5807978A (en) | 1998-09-15 |
AU6095696A (en) | 1996-12-30 |
EP0832105A1 (en) | 1998-04-01 |
CA2224149A1 (en) | 1996-12-19 |
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