WO1998008090A1

WO1998008090A1 - Anti-idiotypic antibodies to an epiglycanin

Info

Publication number: WO1998008090A1
Application number: PCT/US1996/013231
Authority: WO
Inventors: John F. Codington
Original assignee: Epigen, Inc.
Priority date: 1995-06-06
Filing date: 1996-08-16
Publication date: 1998-02-26

Abstract

Methods of assaying for the presence of Human Carcinoma Antigen (HCA). Also disclosed are methods of in vivo imaging of a tumor and methods for treating and/or preventing a carcinoma. Further disclosed are anti-idiotypic antibodies which bind to an antibody which binds HCA; anti-anti-idiotypic antibodies which bind to a member of the group consisting of epiglycanin, HCA and an anti-idiotypic antibody which binds to an antibody which binds to HCA; and kits for detecting human carcinoma antigen.

Description

ANTI-IDIOTYPIC ANTIBODIES TO AN EPIGLYCANIN

Description

Background of the Invention

Epiglycanin is a sialo ucin isolated from the mouse carcinoma cell line TA3-Ha and reported by Codington, et al . in 1972 (Biochemistry 11:2559-2564). Mild proteolysis of viable TA3-Ha ascitic cells with TPCK-trypsin, followed by fractionation of the released peptides by gel exclusion chromatography, gave a peak of glycopeptide material of high molecular weight. Epiglycanin is generally characterized as having a molecular weight of 500,000 dalton, about 80% of which is carbohydrate, largely beta- Gal (1-3) GalNAc side chains linked to serine or threonine residues in a polypeptide chain of about 1,300 amino acids. See, Van den Eijnden et al . , (1979) J. Biol. Chem.

254 : 12153-12159. Polyclonal and monoclonal antibodies to epiglycanin have been reported by Codington. United States Patent No. 4,837,171, which is incorporated herein by reference. The polyclonal antibodies were reported to react with a substance in peritoneal and pleural fluid in sera of patients with metastatic cancer. Codington et al . , (1984) J. Nat'l Cancer Inst. 73:1029-1038.

Summary of the Invention

The invention is based upon the discovery of improved methods for assaying for the presence of cancer cells or diagnosing, particularly carcinoma or metastatic cancer cells characterized by the presence of the human homolog of epiglycanin, termed Human Carcinoma Antigen (HCA) . The invention relates to a method of assaying for the presence of Human Carcinoma Antigen (HCA) in a sample comprising contacting the sample with a first antibody in the presence or absence of an antiidiotypic antibody which binds to an antibody which binds HCA (hereinafter "HCA antiidiotypic antibody" or "HCA anti-Id") , to produce a mixture. The first antibody is selected from the group consisting of (1) a monoclonal antibody which binds to HCA and (2) an anti- antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an HCA antiidiotypic antibody. When the HCA antiidiotypic antibody is absent, the first antibody is an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an HCA antiidiotypic antibody. The resulting mixture is assayed for binding of HCA to the first antibody. In one embodiment, the resulting mixture is assayed for binding of HCA to the first antibody by an immunoassay procedure, such as a competitive immunoassay or a sandwich immunoassay. In a second embodiment, the resulting mixture is assayed for binding of HCA to the first antibody by determining the level of binding of the first antibody to the antiidiotypic antibody and comparing the level determined with a control level. If the level of binding of HCA to the first antibody is less than the control level, binding of HCA has been determined. Binding of HCA reflects the presence of HCA in the sample, thereby supporting a diagnosis of cancer, such as the presence of metastatic cancer.

In a third embodiment, an HCA antiidiotypic antibody is immobilized on a solid surface and the first antibody is labeled with a detectable marker. In a fourth embodiment, the first antibody is immobilized on a solid surface and the antiidiotypic antibody is labeled with a detectable marker .

In a fifth embodiment, the method of assaying for the presence of HCA in a sample comprises contacting the sample with an HCA anti-antiidiotypic antibody, in the presence or absence of an HCA antiidiotypic antibody to produce a mixture. The resulting mixture is assayed for binding of HCA to the anti-antiidiotypic antibody. In a particular embodiment, the anti-antiidiotypic antibody is immobilized on a solid surface and the resulting mixture is assayed for binding of HCA to the anti-antiidiotypic antibody after contacting a labeled antibody which binds to HCA with the mixture by determining the level of binding of said second antibody to HCA.

In another embodiment, the method of assaying for the presence of HCA in a sample comprises contacting the sample with a solid-phase immunoadsorbent containing immobilized HCA antiidiotypic antibody, and a labeled HCA anti- antiidiotypic antibody. The level of labeled HCA anti- antiidiotypic antibody bound to said solid-phase immunoadsorbent is determined and compared to a control level. If the level of binding of labeled anti- antiidiotypic antibody to the solid-phase immunoadsorbent is less than the control level, binding of HCA has been determined. In a further embodiment, the method of assaying for the presence of HCA in a sample comprises contacting the sample with (1) a solid-phase immunoadsorbent containing immobilized HCA anti-antiidiotypic antibody; and (2) a labeled HCA antiidiotypic antibody. The level of labeled antiidiotypic antibody bound to the solid-phase immunoadsorbent is determined and compared to a control level. If the level of binding of labeled antiidiotypic antibody to the solid-phase immunoadsorbent is less than the control level, binding of HCA has been determined. In a further embodiment, the resulting mixture is assayed for binding of labeled monoclonal antibody to the solid-phase immunoadsorbent by determining the level of labeled monoclonal antibody bound to the solid-phase immunoadsorbent and comparing the level of labeled monoclonal antibody bound to the solid-phase iinmunoadsorbent with a control level. If the level of labeled monoclonal antibody bound to the solid-phase immunoadsorbent is less than the control level, binding of HCA is determined. In another embodiment, the method of assaying for the presence of HCA in a sample comprises contacting the sample with (1) a solid-phase immunoadsorbent containing immobilized HCA anti-antiidiotypic antibody; and (2) a labeled monoclonal antibody which binds HCA. The level of labeled antibody bound to the solid-phase immunoadsorbent is determined and compared to a control level. If the level of binding of labeled antibody to the solid-phase immunoadsorbent is less than the control level, binding of HCA has been determined. In still another embodiment, the method of assaying for the presence of HCA in a sample comprises contacting the sample with (1) a solid-phase immunoadsorbent containing immobilized monoclonal antibody which binds HCA; and (2) a labeled HCA anti-antiidiotypic antibody. The level of labeled antibody bound to the solid-phase immunoadsorbent is determined and compared to a control level. If the level of binding of labeled antibody to the solid-phase immunoadsorbent is less than the control level, binding of HCA has been determined. As set forth herein in more detail, an inhibition immunoassay, in essence, measures the inhibition of binding of an antibody to an inhibitor by an antigen present in the sample. Generally, the antigen and the inhibitor are the same or possess the same binding specificity to the antibody. A control level in an inhibition assay can be determined by repeating the assay in the absence of sample and, thus, measures the binding of antibody and inhibitor in the absence of a competing antigen.

The invention further relates to an improvement in an assay for carcinoma which employs epiglycanin and an

SUBSTITUTE SHEET (RULE 25) antibody which binds epiglycanin, wherein the improvement comprises substituting for epiglycanin an antiidiotypic antibody which binds to an antibody which binds human carcinoma antigen. The invention also relates to a method of in vivo imaging (selectively labeling) human tumor tissue in a patient comprising administering to the patient an anti- antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA, coupled (bound) to detectable labels (imaging agents), such as radioisotopes.

The invention further relates to vaccines which induce or elicit an immune response against HCA. In one embodiment, the vaccine comprises (1) an antiidiotypic antibody which binds to an antibody which binds HCA, or a vector which comprises a nucleic acid which encodes the antibody and (2) a pharmaceutically acceptable carrier. Also described herein are pharmaceutical compositions which comprise antibodies which bind to HCA. In this embodiment, the composition comprises (1) an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA, and (2) a pharmaceutically acceptable carrier.

The invention also relates to methods for treating and/or preventing a carcinoma in a patient in need thereof comprising administering to the patient a vaccine or pharmaceutical composition as described herein. For example, the method for treating and/or preventing a carcinoma in a patient in need thereof comprises administering to the patient an antiidiotypic antibody which binds to an antibody which binds HCA, in a therapeutically effective amount. The method for treating and/or preventing a carcinoma comprises administering to the patient an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA, in a therapeutically effective amount. The invention relates to antiidiotypic antibodies which bind to an antibody which binds HCA.

The invention also relates to anti-antiidiotypic antibodies which bind to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA.

The invention further relates to kits for detecting human carcinoma antigen comprising:

(a) a first antibody selected from the group consisting of: i) a monoclonal antibody which binds to HCA; and ii) an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA,

(b) an inhibitor selected from the group consisting of: i) epiglycanin, a mutant or a fragment thereof which binds to said first antibody; ii) HCA, a mutant or fragment thereof which binds to said first antibody; and iii) an antiidiotypic antibody or a fragment thereof which binds to said first antibody; and wherein one of said first antibody and said inhibitor is labeled.

The assays and kits of the invention can be used to determine HCA in samples including urine, plasma, serum, peritoneal fluid or lymphatic fluid, or solid tissue biopsies, such as breast carcinoma. Brief Description of the Drawings

Figure 1 is a bar graph showing the results of immunizations of C57BL mice with a monoclonal antibody which binds Human Carcinoma Antigen (HCA) . The plate is coated with epiglycanin then incubated with mouse sera.

Figure 2 is a bar graph showing the results of immunizations of C57BL and BALB/c mice with an antiidiotypic antibody which binds to an antibody which binds HCA. Figure 3 is a flow chart showing the route of formation of the antiidiotypic antibodies and anti- antiidiotypic antibodies and their binding reactions.

Figure 4 is a graph showing the results of a competition binding assay where rabbit anti-E5 antibody is the binder, E-5 antibody is an inhibitor and F-2 antibody is an inhibitor.

Figure 5 is a graph showing the results of a competition binding assay where E-5 antibody is the binder and rabbit anti-E5 antibody is the inhibiter.

Detailed Description of the Invention

As set forth above, the invention relates, in one aspect, to an improved assay for the presence of HCA in a sample comprising contacting the sample with an antibody (a first antibody) which binds to HCA and determining binding. The assay is preferably a radioimmunoassay or, more preferably, a competitive inhibition assay. Where the assay is a competitive inhibition assay, the assay further comprises contacting the sample and first antibody with an inhibitor. The inhibitor is a compound which inhibits binding of the HCA to the antibody. Examples of suitable inhibitors are HCA, epiglycanin and antiidiotypic antibodies which bind the first antibody, or binding fragments of any of the above. Preferably, the inhibitor employed is an antiidiotypic antibody.

SUBSTITUTE SHEET(RULE 25) Suitable antibodies for use in the invention can be raised against (1) epiglycanin, (2) HCA or (3) antiidiotypic antibodies raised against antibodies which bind HCA (anti-antiidiotypic antibodies) . Antibodies which can be used herein can be polyclonal, monoclonal, chimeric, humanized or resurfaced antibodies or antigen-binding fragments thereof. Preferred antibodies are monoclonal antibodies.

Antibodies to epiglycanin and methods for their production have been described in the art. For example, monoclonal antibodies to epiglycanin and methods for their production are described, for example, in U.S. Patent No. 4,837,171, issued to John F. Codington; U.S. Patent No. 5,545,532, issued to John F. Codington et al . ; and Haavik et al . , Glycobiology 2:217-224 (1992), the teachings of all of which are entirely incorporated herein by reference. Hybridomas producing anti-murine epiglycanin antibodies, AE-1, AE-3 and AE-4, have been deposited with the American Type Culture Collection (ATCC) , 12301 Parkland Drive, Rockville, MD 20852 USA. For example, the hybridoma HAE-1 (producing monoclonal antibody AE-1) was deposited at the ATCC under accession no. HB-9466. The hybridoma HAE-3 (producing monoclonal antibody AE-3) was deposited at the ATCC under accession no. HB-9467. The hybridoma HAE-4 (producing monoclonal antibody AE-4) was deposited at the ATCC under accession no. HB-9468. Similar antibodies can be prepared by known methods. Epiglycanin can be obtained, for example, as described in U.S. Patent No. 4,837,171, issued to John F. Codington, the teaching of which is entirely incorporated herein by reference.

Polyclonal antibodies to epiglycanin and methods for their production have also been described, for example, in Codington et al . , J . Nat . Cancer Instit . 73:1029-1038 (1984) . Antibodies to human carcinoma antigen and methods for their production are described, for example, in U.S. Patent No. 5,545,532, issued to John F. Codington et al .

Antibodies (e.g., the first antibodies) useful in determining the presence of HCA bind to the antigen, HCA. It has been found that certain antibodies to epiglycanin cross-react with HCA. As such, where an antibody for use on the present invention is produced employing epiglycanin, or an immunogenic fragment thereof, as the immunogen, it is important that the resulting antibodies are screened for their ability to cross-react and bind HCA. An example of such an antibody, AE-3 is described in United States Serial No. 08/192,840, filed February 7, 1994 now US Patent 5,545,532, incorporated herein by reference. As set forth above, the assay is preferably a competitive inhibition assay wherein an inhibitor is added to the assay mixture. A competitive inhibitor is one which competes with the antigen for binding to the antibody. Suitable inhibitors include HCA, epiglycanin, or antiidiotypic antibodies which bind the first antibody, as defined above. Other suitable inhibitors include antibody binding fragments of these compounds. In the case of HCA and epiglycanin, the antibody binding fragment is generally the fragment or region of the protein that comprises the epitope to which the antibody binds. It is apparent to the person of skill in the art that non-essential amino acids of the protein can be substituted (e.g., mutants), added (e.g., fusion proteins) or deleted (e.g., fragments) without substantially impacting upon the protein's ability to bind to the antibody. In the assay of the invention, it is preferred to employ anti-idiotypic antibodies, rather than HCA or epiglycanin. Anti-Idiotypic Antibodies

The present invention provides for anti-idiotypic (anti-Id, or a-Id) antibodies. An anti-Id antibody is an antibody which recognizes unique antigenic determinants (idiotypic determinants) generally associated with the antigen-binding region of another antibody. An anti-Id antibody can be prepared against a selected antibody by immunizing an animal (e.g. a mouse, rabbit, rat or goat) with the selected antibody. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and may produce antibodies to various antigenic determinants. Antibodies to the hypervariable (binding) region of the immunizing antibody, as in the present invention, may represent only a small fraction of the anti-antibody antibody-producing hybridomas.

In one embodiment, anti-epiglycanin antibodies, anti- HCA antibodies, including antibodies which crossreact, or antigen binding fragments thereof, are used as immunogens to raise anti-Id antibodies to epiglycanin (e.g., idiotypic antibodies that will bind to epiglycanin-binding antibodies) .

The antiidiotypic antibodies further bind to an antibody which binds HCA and epiglycanin. In a particular embodiment, the antiidiotypic antibody is the F-2 antibody or the E-5 antibody. The F-2 antibody is also referred to herein as the C8F-2 of C8F2 antibody. An antiidiotypic antibody which binds to an antibody which binds HCA is also referred to herein as an Ab-2 antibody. Thus, the F-2 antibody and the E-5 antibody are examples of Ab-2 antibodies as defined herein.

The work herein demonstrates that the F-2 antibody binds only to the hypervariable region of the AE-3 antibody. The F-2 antibody therefore has the specificity of the epitope in epiglycanin which binds to the AE-3 antibody. The F-2 antibody should, therefore, be highly specific and should be able to mimic epiglycanin in immunological reactions.

As a result of Applicant's discovery, improved methods for assaying for the presence of cancer cells or diagnosing, particularly carcinoma or metastatic cancer cells characterized by the presence of the human homolog of epiglycanin, termed HCA, are provided. HCA and the method of producing HCA is described in U.S. Patent No. 5,545,532, issued to Codington et al . ; in U.S. Application No. 08/484,061, filed June 6, 1995; and in U.S. Application No. 08/468,276, filed June 6, 1995, the teachings of all of which are entirely incorporated herein by reference.

In a particular embodiment, monoclonal antibody AE-3 is used to raise anti-Id antibodies. The anti-Id antibodies that are produced bind to the hypervariable region of the immunizing antibody. These antiidiotypic antibodies can competitively inhibit the binding of HCA and AE3. As such, it is useful in the present invention.

In fact, the invention, at least in part, relates to the discovery that the accuracy of the assay significantly improves upon the use of antiidiotypic antibodies, rather than antigen, as the competitive inhibitor, resulting in a more dramatic or steeper slope in the standard curve (control) . Furthermore, the antiidiotypic antibody can be easier to produce, can have a longer shelf life and can be generally more stable.

In a particular example, the F-2 antibody has been shown to substitute for epiglycanin in every respect in the "Cod Test", a competitive binding assay described in Example 9. In addition, when the F-2 antibody is used in the Cod Test, for coating the wells of an immunoplate, the standard curve, using epiglycanin as a standard, is essentially identical to that obtained if epiglycanin is used for coating the plate. However, when used as a standard in the test, F-2 gives a curve of a steeper slope than that given by epiglycanin. Thus, the F-2 antibody exhibits a greater potential for diagnostics.

These results demonstrate a significant advantage in methods for assaying for the presence of cancer cells or diagnosing, particularly carcinoma or metastatic cancer cells characterized by the presence of HCA, where antiidiotypic antibodies which bind to an antibody which binds HCA are used. Antiidiotypic antibodies which bind to an antibody which binds HCA, also referred to herein as Ab-2 antibodies, provide greater stability, consistency, specificity and sensitivity to the methods. For example, using the F-2 antibody in the Cod Test provides greater stability, consistency, specificity and sensitivity in the test. A monoclonal antibody is essentially immortal, and with a good supply of frozen vials of the hybridoma in stock, the hybridoma and antibody itself should be reproducible at any time. Furthermore, the purified antibody itself can be frozen and can be removed from the freezer whenever it is needed. In contrast, epiglycanin is subject to a phenomenon known as microheterogenity where variations in the carbohydrate structures can occur.

Anti-Anti-Idiotypic Antibodies

The invention further provides for anti-anti-idiotypic (anti-anti-Id) antibodies. An anti-anti-Id antibody can be prepared against a selected anti-Id antibody by immunizing an animal (e.g., a mouse, rabbit, rat or goat) with an antild antibody. The anti-anti-Id antibody can be similar or identical in antigenic specificity to the original immunizing antibody (e.g., the antibody used to raise the anti-Id antibody) . HCA anti-anti-idiotypic antibodies are also referred to herein as "Ab-3" antibodies.

For example, a suitable anti-antiidiotypic antibody for use in the claimed invention can be raised against the antiidiotypic antibody, F-2, specifically, the hypervariable region or antigen-binding region.

Antibodies and Methods

The term "antibody" is meant to include polyclonal antibodies and monoclonal antibodies (mAbs) . Monoclonal antibodies can include chimeric antibodies, resurfaced antibodies, anti-idiotypic (anti-Id) antibodies, anti-antiidiotypic (anti-anti-Id) antibodies, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or reco binant techniques.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody contains a substantially homogeneous population of antibodies specific to an epitope present in an antigen. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al . , eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1987, 1992); and Harlow and Lane ANTIBODIES : A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988); Colligan et al . , eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), the contents of which references are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG (including IgGl, IgG2, lgG3 and lgG4) , IgM, IgE, IgA, GILD and any subclass thereof. It is preferred that the anti-antiidiotypic antibody be an IgG, such as an IgGl. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo . Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production. Chimeric antibodies are immunoglobulin molecules characterized by two are more segments or portions derived from different animal species. Generally, the variable region of the chimeric antibody is derived from a non-human mammalian antibody, such as a murine mAb, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used.

As used herein, the term "chimeric antibody" includes monovalent, divalent or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer (HL) ) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric antibody is a tetramer (H2L2) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody can also be produced, for example, by employing a CH region that aggregates (e.g., from an IgM H chain, or μ chain) .

Chimeric antibodies and methods for their production have been described in the art (Morrison et al . , Pro . Natl . Acad . Sci . USA 81:6851-6855 (1984); Boulianne et al . , Nature 312:643-646 (1984); Neuberger et al . , Nature 314 : 268-270 (1985); Taniguchi et al . , European Patent Application 171496 (published February 19, 1985); Morrison et al . , European Patent Application 173494 (published March 5, 1986); Neuberger et al . , PCT Application WO 86/01533, (published March 13, 1986); Kudo et al . , European Patent Application 184187 (published June 11, 1986); Morrison et al., European Patent Application 173494 (published March 5, 1986); Sahagan et al . , J . Immunol . 137:1066-1074 (1986); Robinson et al . , International Publication No. PCT/US86/02269 (published 7 May 1987); Liu et al . , Proc. Natl . Acad . Sci . USA 84:3439-3443 (1987); Sun et al . , Proc . Natl . Acad . Sci . USA 84:214-218 (1987); Better et al . , Science 240:1041-1043 (1988); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor

Laboratory, New York, 1988) . These references are entirely incorporated herein by reference.

Methods for humanizing and resurfacing antibody are described in the art. See, for example, Winter (U.S. Patent No. 5,225,539 and EP 239,400 Bl) , Padlan et al . (EP 519,596 Al) and Pedersen et al . (EP 592,106 Al) . These references are incorporated herein by reference. Humanizing and/or resurfacing an antibody can further reduce the immunogenicity of the antibody. Reducing the immunogenicity of an antibody is especially advantageous for in vivo applications, such as for use in human therapies.

Methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow, et al . , Antibodies : A Laboratory Manual , Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY, 1988) , Colligan et al . , eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth . Enzymol . 92:589-601 (1983), which references are entirely incorporated herein by reference. Preferred antibodies possess good to high affinity to the antigen and inhibitor.

As used herein, the term "antigen binding region" and "antigen binding fragment" refer to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Generally, the antigen binding region will be of murine origin. In other embodiments, the antigen binding region can be derived from other animal species, such as sheep, rabbit, rat or hamster. Preferred sources for the DNA encoding such a non-human antibody include cell lines which produce antibody, preferably hybrid cell lines commonly known as hybridomas.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen can have one or more than one epitope.

The term "epitope" is meant to refer to that portion of the antigen capable of being recognized by and bound by an antibody at one or more of the antibody's antigen binding region. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics . "Fragments" or antigen binding fragments of the antibodies include, for example, Fab, Fab', F(ab')- and Fv. These fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody (Wahl et al . , J. Nucl . Med . 24:316-325 (1983)). These fragments are produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

Antibody Production Using Hybridomas

The cell fusions are accomplished by standard procedures well known to those skilled in the field of immunology. Fusion partner cell lines and methods for fusing and selecting hybridomas and screening for mAbs are well known in the art. See, e.g, Ausubel infra, Harlow infra, and Colligan infra, the contents of which references are incorporated entirely herein by reference.

Immunoassays

As set forth above, the invention relates to immunoassays for detecting the presence of HCA in a sample. Immunoassays useful in the present invention is any assay that can detect the binding (or absence of binding) of an antigen to an antibody. Examples of suitable immunoassays include sandwich assays, radioimmunoassays and, preferably, competitive inhibition assays. In the present section, the use of the term "antibody" is intended to include any antibody or fragment thereof which binds HCA, including, for example, anti-antiidiotypic antibodies. The use of the term "antigen" or "inhibitor" in the context of a reagent in the assay is intended to include HCA, epiglycanin and antiidiotypic antibodies or antibody-binding fragments thereof. In a radioimmunoassay (RIA) , the amount of antigen present in a sample is measured indirectly employing a limited amount of antibody to compete for labeled antigen. In an IRMA (immunoradiometric assay) , antigen is assayed directly by reacting the antigen with excess labeled antibody.

In one class of IRMA assays, the unknown antigen is insolubilized and reacted with labeled antibody. When the antigen is insolubilized by reaction with solid-phase antibody, the assay is termed a "two-site IRMA", "junction test", or "sandwich assay". Sandwich assays are further classified according to their methodology as forward, reverse or simultaneous sandwich assays.

In a forward sandwich immunoassay, a sample containing the antigen can be first incubated with a solid-phase immunoadsorbent containing immobilized antibody. Incubation is continued for a sufficient period of time to allow antigen in the sample to bind to immobilized antibody on the solid-phase immunoadsorbent. The solid-phase immunoadsorbent can then be separated from the incubation mixture and washed to remove excess antigen and other substances which also may be present in the sample. The solid-phase immunoadsorbent containing antigen (if any) bound to immobilized antibody can be subsequently incubated with labeled antibody capable of binding to the antigen. After the second incubation, another wash is performed to remove unbound labeled antibody from the solid-phase immunoadsorbent thereby removing non-specifically bound labeled antibody. Labeled antibody bound to the solid- phase immunoadsorbent is then detected and the amount of labeled antibody detected can serve as a direct measure of the amount of antigen present in the sample. Such forward sandwich assays are described in the patent literature, and in particular, in U.S. Patent Nos. 3,867,517 and 4,012,294, issued to Chung-Mei Ling, which ar incorporated herein by reference.

In a reverse sandwich assay, a sample can be incubated with labeled antibody after which the solid-phase immunoadsorbent containing immobilized antibody is added and incubated. A washing step can be performed after the second incubation period. A reverse sandwich assay has been described in the patent literature in U.S. Patent No. 4,098,876, issued to Roger N. Piasio et al .

In a simultaneous sandwich assay, a sample can be incubated simultaneously in one step with both an immunoadsorbent containing immobilized antibody for the antigen and labeled antibody for the antigen. Thereafter, labeled antibody bound to the immunoadsorbent can be detected as an indication of the amount of antigen present in the sample. A simultaneous sandwich assay has been described in the patent literature in U.S. Patent

No. 4,837,167, issued to Hubert J.P. Schoe aker et al .

Many solid-phase immunoadsorbents can be employed. Well-known immunoadsorbents include beads formed from glass polystyrene, polypropylene, dextran, and other materials. Preferably, the solid support is a plate, stick, tube or well formed from or coated with such materials; etc. The antibody can be either covalently or physically bound to the solid-phase immunoadsorbent by techniques such as covalent bonding via an amide or ester linkage or adsorption. Many other suitable solid-phase immunoadsorbents and methods for immobilizing antibodies thereon are known in the art.

The preferred embodiment of the claimed invention is the competitive inhibition immunoassay, which has been used to determine the presence of an antigen in a sample by measuring the inhibition of formation of a competitive inhibitor-antibody complex, one of which is typically bound and the other of which is typically labeled, by free antigen in the sample. In addition, a typical quantitative immunoassay kit can include a standardized sample of pure inhibitor, such as an antigen or antiidiotypic antibody, so that a reference solution can be run together with the sample to minimize sampling errors and to assure precision. Competitive immunoassays (e.g., radioimmunoassay (RIA) , enzyme-linked immunoadsorbant assay (ELISA) ) are used to detect and quantitate the presence of antigen in a sample by determining the extent of inhibition by the antigen of a competitive inhibitor/antibody reaction. Typically, either the inhibitor or the antibody is bound to a solid support (as described above) , while the other component of the pair is labeled in some fashion to render it detectable. Such labels are well known in the art and include, e.g., radioisotopes, enzymes (such as horse radish peroxidase) , dyes (chromophores) , and fluorescent markers. Biotin/avidin labeling systems can be used. Coupled assays can also be used for detecting labels.

The label may be directly linked to the component (the inhibitor or antibody) or may be bound to it indirectly, e.g. , by attaching the label to another molecule capable of recognizing a component of the antigen/antibody pair. For example, an antibody can be indirectly labeled by attaching an enzyme, fluorescent marker or radioisotope to an isotype-specific antibody which recognizes the non-variable region of the antigen-specific antibody. Alternatively, the label can be attached to an antibody which recognizes an available epitope af the antigen after it has been bound to the specific antibody. Many other variants of this broad concept are possible and known in the art. In one preferred embodiment, the label is a dye (such as, nitrophenyl) attached to the unbound component or reagent (unbound inhibitor or antibody) via a phosphate linker. After incubation of the labeled component with the immobilized binding partner, the presence of binding can then be determined by subjecting the solid support to a phosphatase enzyme, causing hydrolysis of the dye. The presence (and amount) of the dye can then be measured by absorbance, indicating the amount of binding of the two components . In each assay, the sample, antibody and, optionally, the inhibitor is incubated under conditions and for a period of time sufficient to allow antigen to bind to both the immobilized antibody and the labeled antibody. In general, it is usually desirable to provide incubation conditions sufficient to bind as much antigen or inhibitor as possible because this maximizes the binding of labeled antibody to the solid phase thereby increasing the signal. Suitable temperatures are generally below the temperature at which denaturation can occur. The examples herein exemplify a temperature at 4°C. The pH can suitably be

SUBSTITUTE 5HEET (RULE 26) selected between about 5 and 8, preferably physiologic temperature and can be controlled by the addition of a buffer. Reactants are preferably added at a concentration and molar ratio to maximize rate of reaction.

Methods of Imaging

The antibodies and anti-antiidiotypic antibodies described herein can further be used to specifically image tumors, in vivo . For example, the antibodies and anti- anti-Id can be covalently bound to a label, such as radioisotopes suitable for in vivo imaging, such as ^mIn or ^99mTc or ¹³¹I. Those skilled in the art will understand that there are numerous well known techniques for generating the radioisotopes and binding them to antibodies. See, for example, Bartorelli, US 4,732,862, hereby incorporated herein by reference. Kits can be obtained, e. f . from various commercial sources, such as Amersham, Mediphysics, Ξyncor or Mallinckrodt .

The imaging agent can be administered by various known techniques including local injection or i.v. infusion. Readings can be taken by, for example, a body scanning scintigram using a gamma camera interfaced with a computer.

Vaccines and Methods of Therapy

As set forth above, the invention relates, in one aspect, to vaccines comprising antiidiotypic antibodies, HCA or epiglycanin, or antigenic fragments thereof and their use in the immunization of patients in need thereof. The principal of this aspect of the invention is to induce an immune response against the antiidiotypic antibodies or antigen, thereby inducing an immune response against HCA. The antiidiotypic antibodies, HCA, epiglycanin or antigenic fragments thereof (hereinafter "antigen") are administered such that an immune response is elicited or induced. As such, the vaccines comprise the antigen in a pharmaceutically acceptable carrier and in a size and manner to elicit an immune response. This can be accomplished by administering the antigen in association with particles of appropriate size, as described in

Kovacsovics-Bankowski, M. and Rock, K.L., Eur . J . Immunol . , 24:2421 (1994) and Rock, K.L. et al . , J . Immunol . , 150:438 (1993) , which are incorporated herein by reference in their entireties. In these antigen presenting cells particle associated proteins are interionized in phagolysosomes, gain access to the cytosol, and then share a common pathway with endogenously synthesized proteins for class I MHC presentation.

A preferred method of administering the antigen is in a vector encoding the antigen which, upon administration, results in the expression of the antigen. The vector can be, for example, a recombinant host cell or virus which comprises a recombinant nucleic acid molecule which encodes the antigen operably linked to a promoter. In this embodiment, the recombinant host cell expresses and, optionally, secretes the antigen. The recombinant host cell can be any suitable cell, such as a bacteria, yeast, fungus, plant or animal cell. Where the host cell is an animal cell containing, in its genome, a nucleic acid which encodes the antigen (or an allelic variant or homolog of the antigen) , the host cell can be transformed or transfected with a nucleic acid which increases expression of the nucleic acid encoding the antigen, thereby causing over-expression of the antigen upon administration. Where the vector is a viral vector containing a nucleic acid encoding the antigen, the vector, upon infection, results in the expression of the antigen. The delivery of proteins by this method are generally described in, for example, Anderson et al . , United States Patent No. 5,399,346, which is incorporated herein by reference. Examples of suitable viral vectors include retroviruses, vaccinia viruses, adenoviruses, polioviruses and herpesviruses, particularly attenuated viruses or other viruses characterized by altered genomes to decrease infectivity, as is known in the art.

In a particularly preferred embodiment, the vector is a recombinant bacterium encoding the antigen. Particularly preferred bacteria are bacterial vaccines, such as is described in Cirillo et al . , Clinical Infect . Diseases, 20:1801-9 (1995), which is incorporated herein by reference. An example of a pharmaceutically acceptable bacterial vaccine is MycoJacterium bovis Bacillus Calmette- Guerin (BCG) . Transfection of foreign genes, including human genes, into BCG has been reported. See, for example, Aldovini a. and Young, R.A. , Nature, 351:479-482 (1991), Stover, C.K. et al . , J . Exp . Med . , 178:197-209 (1993), Lagranderie M. , et al . , Vaccine, 11:1283-1290 (1993), Connell, N.D., et al . , PNAS , 90:11473-11477 (1993) and Stover, C.K., et al . , Nature , 351:456-460 (1991), which are incorporated herein by reference.

The vaccines of the present invention can be administered locally or systemically, including orally (e.g., capsule, tablet or liquid formulation), parenterally (e.g., intra- uscularly, intravenously, subcutaneously) , topically, nasally, intravaginally or rectally, or via slow releasing microcarriers in dosage formulations containing a physiologically acceptable vehicle and optional adjuvants and preservatives. Suitable physiologically acceptable vehicles include saline sterile water, Ringer's solutions, and isotonic sodium chloride solutions. Specifically,

Sodium Chloride Injection USP (0.9%), Ringer's Injection USP, Lactated Ringer's Injection USP, Sodium Lactate Injection USP, Dextrose Injection USP (5% or 10%) , Bacteriostatic Water for Injection USP and Sterile Water for Injection USP can be used, for example. The specific dosage level of active ingredient will depend upon a number of factors, including biological activity of the particular preparation, age, body weight, sex, general health and the clinical stage of the disease. Alternatively or additionally, the invention relates to the pharmaceutical compositions and methods of treating or preventing a carcinoma in a patient in need thereof comprising administering to said patient an antibody or an anti-antiidiotypic antibody or an antigen binding fragment thereof which binds to a member of the group consisting of: epiglycanin, Human Carcinoma Antigen (HCA) and an antiidiotypic antibody which binds to an antibody which binds to HCA, in a therapeutically effective amount.

The antibody can be administered in a pharmaceutically acceptable carrier or in a vector encoding the antibody, as discussed above. That is, the antibody can be administered locally or systemically , including orally (e.g., capsule, tablet or liquid formulation), parenterally (e.g., intramuscularly, intravenously, subcutaneously) , topically, nasally, intravaginally or rectally, or via slow releasing microcarriers in dosage formulations containing a physiologically acceptable vehicle and optional adjuvants and preservatives. Suitable physiologically acceptable vehicles include saline sterile water, Ringer's solutions, and isotonic sodium chloride solutions. Specifically, Sodium Chloride Injection USP (0.9%), Ringer's Injection USP, Lactated Ringer's Injection USP, Sodium Lactate

Injection USP, Dextrose Injection USP (5% or 10%), Bacteriostatic Water for Injection USP and Sterile Water for Injection USP can be used, for example. The specific dosage level of active ingredient will depend upon a number of factors, including biological activity of the particular preparation, age, body weight, sex, general health and the clinical stage of the disease.

Where the compound being administered is an antibody, particularly antiidiotypic and anti-antiidiotypic antibodies, the antibody can be a chimeric antibody, a humanized antibody and a resurfaced antibody or fragment thereof .

Equivalents

Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

EXAMPLES

Example 1 Epiglycanin Monoclonal Antibodies

Anti-epiglycanin monoclonal antibodies were prepared and purified as described previously (U.S. Patent No.

4,837,171, issued to John F. Codington; Haavik et al . ,

Glycobiology 2:217-224 (1992); and U.S. Patent No. 5,545,532, issued to Codington et al . , the teachings of all of which are incorporated herein by reference) .

Example 2 Preparation of the Antibody-Keyhole Limpet Hemocvanin Complexes The immunogens used to raise antiidiotypic antibodies which bind to an antibody which binds HCA and anti- antiidiotypic antibodies which bind to a member of the group consisting of (1) epiglycanin, (2) HCA and (3) an antiidiotypic antibody which binds to an antibody which binds to HCA, were complexes consisting of the immunizing antibody and Keyhole Limpet Hemocyanin (KLH) . The solutions injected were made of equal volumes of the complex in phosphate-buffered saline (PBS) and an adjuvant. The adjuvant is a monophosphoryl lipidA + trehalose dicorynomycolate (MLP + TDM) emulsion, R-700 (Ribi ImmunoChem Research, Inc., Hamilton, MT) . Two complexes were prepared: the AE-3-KLH complex used for the induction of the antiidiotypic antibodies; and the C8F2-KLH complex used for the induction of the anti- antiidiotypic antibodies. The preparation of each complex was similar and followed the procedure supplied by the manufacturer of the chemicals used, Pierce Chemical Co. The first step involved the preparation of a sulfhydryl (-SH) -containing antibody. This was done by the treatment of the antibody with Traut's Reagent. The antibody (1.8 mg) was dissolved in a 0.05 M triethanolamine buffer containing NaCl and EDTA, according to the procedure suggested by the manufacturer. To this solution was added 1.8 μl of Traut's Reagent. After 2 hours at 22 °C, the solution was dialyzed against PBS (pH 7.0). All solutions used in this preparation had been saturated with nitrogen gas in order to eliminate the possibility of oxidation by dissolved oxygen in the solution. To the sulfhydryl- containing antibody was added 2.0 mg of maleimide-activated KLH (Pierce Chemical Co.). The reaction was continued for 24 hours at 4°C with gentle mixing. The product was dialyzed for 24 hours at 4°C against sterile 0.15 M PBS. The 2.5 ml of solution contained the complex at a concentration of 720 μg of antibody per ml. Example 3 Antiidiotypic Antibodies

Five 8-week old C57BL female mice were inoculated intraperitoneally with 200 μl of an adjuvant-PBS emulsion containing, in each injection, either 50 μg of AE3 antibody (day 0) or 100 μg of AE-3 antibody (days 16 and 30) , as a KLH complex. Injections were performed on days 0, 16 and 30, with hyperimmunizations at day 30. Mice were bled prior to each injection, and relative antibody titres of the sera were compared in the five mice. Figure 1 shows the results of the immunization at day 30.

Fusion with of the spleen cells from an immunized mouse with mouse myeloma cells (CRL-1580-P3865, F8896, obtained from the American Type Culture Collection) was performed on day 34 by modification of the method of Galfre and Milstein (Nature 256:495 (1975)). Briefly, myeloma cells were removed and hybridomas were cloned, subcloned, grown in culture, and, finally, grown in ascites form in commercially available strain CByB6Fl mice. As growth medium for hybridoma cells high glucose Dulbecco's Modified Eagle's Medium, with added L-glutamine (0.5 g/liter) , pyruvate, and antibiotics, was used. The medium also contained 10% Fetal Clone I (HyClone, Inc.) and 5% Hybridoma Growth Factor (Igen, Inc.). During the initial few weeks of growth, the medium contained added hypoxanthine, thymidine, and a inopterin (HAT) . Cells were grown in a 5% CO-, atmosphere at 37 °c. Several positive clones were detected. Two of the most promising clones, E-5 and C8F-2, were selected.

Example 4 Anti-Antiidiotvpic Antibodies Four rabbits were inoculated with 300 to 400 μl of an adjuvant-PBS emulsion containing, in each injection, 100 μg of the C8F2 antibody as a KLH complex. Injection of the C8F2-KLH complex was intramuscular. Injections were performed on days 0, 28 and 55. Rabbits were bled at approximately 2-3 week intervals, and relative antibody titres of the sera were compared for the rabbits.

Four mice each of the C57BL and Balb/c strains were inoculated intraperitoneally with 200 μl of an adjuvant-PBS emulsion containing, in each injection, 30 to 50 μg of C8F2 antibody as a KLH complex. Inoculations were performed on days 0, 28 and 55, with hyperimmunizations 4 to 6 weeks later. Mice were bled prior to each injection, and relative antibody titres of the sera were compared for the mice. Figure 2 shows the results of the immunization at day 50. The results revealed that a significantly higher antibody titre was obtained in the C57BL mice.

Fusions were made using spleen cells from each immunized mouse strain. Fusions were performed with mouse myeloma cells (CRL-1580-P3865 , F8896, obtained from the American Type Culture Collection) 4-5 days following hyperimmunizations by modification of the method of Galfre and Milstein (Nature 256:495 (1975)). Briefly, myeloma cells were removed and hybridomas were cloned, subcloned, grown in culture, and, finally, grown in ascites form in commercially available strain CByB6Fl mice. As growth medium for hybridoma cells high glucose Dulbecco's Modified Eagle's Medium, with added L-glutamine (0.5 g/liter), pyruvate, and antibiotics, was used. The medium also contained 10% Fetal Clone I (HyClone, Inc.) and 5% Hybridoma Growth Factor (Igen, Inc.). Cells were grown in a 5% CO atmosphere at 37 °C. For the initial several weeks, the medium contained added hypoxanthine, thymidine, and aminopterin. The Balb/c mice produced few viable hybridomas. The C57BL mice, by contrast, gave excellent results. Several positive clones were isolated from fusions involving the C57BL mice. Examole 5 Preparation of Monoclonal Antibodies

The polyethylene glycol used in the fusions was obtained from Boehringer-Mannheim. Approximately 400 wells were filled after each fusion with about 150 μl/well. The most active cultures, as determined on immunoplates by ELISA-related methodology, were cloned by infinite dilution. The cells were grown in 6-well plates (Corning) and finally in flasks (25 cm²) . Cultured cells were grown intraperitoneally in CByB6Fl mice. The ascitic fluid was placed in an equal volume of saturated ammonium sulfate. The precipitate was used for antibody production.

Example 6 Antibody Purification

The precipitate in the ammonium sulfate was removed by centrifugation and dialyzed against PBS. The immunoglobulins were bound to the solid support in an

AvidChrome column and were released by lowering the pH.

After dialyzing against a Tris buffer at pH 8.6, the antibody was purified by passage through a DEAE 5W column

(Waters) . The purity and class of the antibodies were evaluated by polyacryla ide gel electrophoresis under reducing conditions. In contrast to AE-3 antibody (Ab-l) , the E-5 and F-2 antibodies were of the IgG class.

Example 7 Evaluation of Murine Antiidiotypic Antibodies and Murine Anti-Antiidiotvpic

Antibodies With A Competitive Binding Assay Immunoassays were used to determine antibody class and antibody activity (titre) . Competitive binding assays have been particularly useful. The particular procedure employed in obtaining proof of the identity of the antibodies as antiidiotypic and anti-antiidiotypic antibodies follows: 1. Coating of wells. Plate containing 100 μl of coating substrate per well (100 ng/ml) was incubated at 4°C overnight. Wells were washed with PBS-Tween20 (0.05%).

2. Blocking of wells. Plate containing 220 μl of unreactive protein (Superblock; Pierce Chemical Co.) per well was incubated for 60 minutes. Wells were washed with PBS-Tween20 (0.05%).

3. Competitive reaction. 50 μl of inhibitor solution and 50 μl of primary antibody solution per well were incubated overnight at 4°C. Wells were washed with PBS-Tween20 (0.05%).

4. Binding of enzyme-labeled antibody to primary antibody bound to well. 100 μl of labeled antibody solution per well was incubated for 1-2 hours. Antibody was labeled with alkaline phosphatase or horseradish peroxidase.

5. Color formation. 100 μl/well of enzyme substrate solution was incubated until color developed. Optical density at 405 nm was read in a plate reader, 6. Concentration of inhibitor. When a standard is available, a standard curve is plotted from dilutions of the standard run with each plate. Concentrations of the inhibitors are then calculated from the curve.

The entire procedure was performed in a 96 well Nunc Maxisorp Immuno plate. Table 1 sets forth some of the particular components which were used in the procedure in establishing the character of the antibodies.

TABLE 1

Coat Plated With Binder Inhibitor

EPGN AB-3 (AB-1) AB-2

EPGN AB-3 AB-2

EPGN AB-3 EPGN

AB-1 AB-2 AB-3

AB-1 AB-2 AB-1

AB-2 AB-3 EPGN

EPGN = epiglycanin; AB-1 = an antibody raised against epiglycanin (e.g., AE-3); AB-2 = an antibody raised against AE-3 (e.g., C8F2); and AB-3 = an antibody raised against C8F2.

The results from this experiment demonstrate that the specificity of the C8F2 antibody is for the hypervariable region of the AE-3 antibody. The results also establish that the C8F2 antibody mimics the same structure present in the unique epitope on epiglycanin and HCA that is recognized by AE-3. It follows then that Ab-2 antibodies, as exemplified by the C8F-2 antibody, behave immunologically like epiglycanin.

The results from this experiment further demonstrate that the Ab-3 antibodies are specific for the hypervariable region of the C8F-2 antibody. Therefore, it follows that AB-3 antibodies have properties similar to those of the AE- 3 antibody.

Example 8 Evaluation of Rabbit Anti-Antiidiotvpic

Antibodies With A Competitive Binding Assay To demonstrate that the antibody produced in rabbit by immunization with mouse anti-idiotypic antibodies is a true anti-antiidiotypic antibody, the competitive binding assay described in Example 7 was used. The entire procedure was performed in a 96 well Nunc Maxisorp Immuno plate. Table 2 sets forth some of the particular components which were used in the assay. TABLE 2

Coat Plated With Binder Inhibitor

EPGN AB-2 (E-5) Rabbit Anti-E5

EPGN Rabbit Anti-E5 AB-2 (E-5)

EPGN Rabbit Anti-E5 AB-2 (F-2)

EPGN = epiglycanin;

AB-2 = an antibody raised against AE-3 (e.g., E-5 and F-2); Rabbit Anti-E5 = antibody raised against F-2.

Figure 8 shows that rabbit anti-E5 is inhibited by the E-5 antibody and the F-2 antibody. Figure 9 shows that E-5 antibody is inhibited by rabbit anti-E5 serum.

The results of this experiment show that rabbit anti- E5 antibodies are specific for the hypervariable region of the E-5 antibody and the F-2 antibody, indicating that an antibody produced in a rabbit by immunization with mouse anti-idiotypic antibodies is a true anti-antiidiotypic antibody. Therefore, it follows that rabbit anti-E5 antibodies have properties similar to those of the AE-3 antibody.

Example 9 A Competitive Binding Assay fthe COD Test) A competitive binding assay (CBA) particularly useful for serum samples was developed for the determination of the concentration of the HCA in human serum. The following materials are employed:

1. Coated and blocked plate (Epigen) .

Alternatively, a Nunc Maxisorp 96 well plate is coated by incubating 14-18 hours at 4°C with 100 μl of a phosphate buffered saline (PBS) containing epiglycanin (100 ng/ml) .

The plate is blocked with Superblock (Pierce Chemical Co.). 2. Wash Concentrate (Epigen), diluted 5 times with water for washing wells. Alternatively, a PBS (pH 7.5) solution with 0.05% Tween-20 is used for washing wells.

3. Monoclonal antibody (AE3) at 400 ng/ml (Epigen). Alternatively, a solution of AE3 can be prepared in PBS as described above.

4. Substrate solution (Epigen). Alternatively, ABTS One Step (Pierce Chemical Co.) is used as the substrate solution.

The following procedure is employed:

1. Epiglycanin Standards

Reconstitute Epigen lyophilized standards with 1.0 ml of pure (sterile, if possible) water. Use concentrations of 2000, 1000, 500, 250, 100 and 50 ng/ml. (The standards have been lyophilized with Superblock.) Standards are placed on the wells in triplicate, at 50 μl/well, and directly from the vial, without dilution.

2. Serum Samples Serum samples are prepared as follows:

One vial containing frozen serum is removed from the freezer and placed on dry ice. When the time for setting up the plate is nigh, the vial is placed in a small vessel with water and allowed to thaw rapidly. As soon as the serum is thawed, the vial is placed in ice. Vials should not be kept at 0°C for more than 60 minutes before the sera are placed on the wells. Before adding to the wells, each serum sample is diluted with an equal volume of Sample Dilution Buffer. (Alternatively, dilution may be made with Superblock.)

Three control wells should contain Sample Dilution Buffer or (Superblock) only. These represent 0 ng/ml of epiglycanin.

Three wells should contain no AE3 antibody. These represent the blank. 4. Antibody

After placing inhibitor in the wells (50 μl epiglycanin standard or 50 μl serum sample) , 50 μl per well of AE3 monoclonal antibody (Epigen antibody + 5.5 ml water) is placed in the wells. The plate is then wrapped with plastic sheet, placed on a shaker in the cold room (0-4°C) and allowed to incubate for 14-18 hours with gentle shaking. The wells are washed two times with Wash Concentrate (1:5) (or PBS- Tween-20) .

5. Goat-Anti-Mouse Antibody

To each well is added 100 μl goat anti-mouse IgM labeled with horseradish peroxidase (HRP) (Epigen) . Alternatively, the Jackson or Pierce products at a dilution of about 1:5000 is used. The plate is wrapped with plastic and incubated on a rotary shaker at about 22 °C for 60-70 minutes. The plate is washed four times with Wash Concentrate (1:5).

6. Detection Step To each well is added 100 μl of Substrate (Epigen label) . Alternatively, the Pierce ABTS One Step is used as the substrate. The plate is placed on a rotary shaker at room temperature, and the color development observed. It can be read periodically on the automatic plate reader at 405 nm. Generally, the optimal reading is at about 30 minutes.

7. Concentration of Unknown

Optical density (O.D.) values are recorded. The mean values are recorded, as well as standard deviations. A standard curve is prepared from mean values and plotted on Logit-Log paper. Values for unknowns are found from the curve and multiplied by the dilution in order to obtain ng/ml values or Units.

Equivalents Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWhat is claimed:

1. A method of assaying for the presence of Human Carcinoma Antigen (HCA) in a sample comprising the steps of: a) contacting the sample with a first antibody selected from the group consisting of: i) a monoclonal antibody which binds to HCA; and ii) an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA, in the presence or absence of an antiidiotypic antibody which binds to an antibody which binds HCA, thereby producing a mixture; and b) assaying for binding of HCA to said first antibody, wherein when said antiidiotypic antibody which binds to an antibody which binds HCA is absent, said first antibody is an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA.

2. The method of Claim 1 wherein in step b) , assaying for binding of HCA to said first antibody is by an immunoassay procedure selected from the group consisting of: a competitive immunoassay and a sandwich immunoassay.

3. The method of Claim 1 wherein antiidiotypic antibody which binds to an antibody which binds HCA is present.

4. The method of Claim 3 wherein said antiidiotypic antibody is immobilized on a solid surface and said first antibody is labeled with a detectable marker.

5. The method of Claim 3 wherein said first antibody is immobilized on a solid surface and said antiidiotypic antibody is labeled with a detectable marker.

6. The method of Claim 3 wherein step b) comprises: i) determining the level of binding of said first antibody to said antiidiotypic antibody; and ii) comparing the level determined in step i) with a control level, wherein if the level determined in step i) is less than the control level, binding of HCA has been determined.

7. A method of assaying for the presence of Human Carcinoma Antigen (HCA) in a sample comprising the steps of: a) contacting the sample with an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA, in the presence or absence of an antiidiotypic antibody which binds to an antibody which binds

HCA, thereby producing a mixture; and b) assaying for binding of HCA to said anti- antiidiotypic antibody.

8. The method of Claim 7 wherein said anti-antiidiotypic antibody is immobilized on a solid surface and wherein step b) comprises: i) contacting a labeled antibody which binds to HCA with the mixture of step a) ; and ii) determining the level of binding of said antibody to HCA.

9. A method of assaying for the presence of a Human Carcinoma Antigen (HCA) in a sample comprising the steps of: a) contacting the sample with the following: i) a solid-phase immunoadsorbent containing immobilized antiidiotypic antibody which binds to an antibody which binds HCA; and ii) a labeled anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA; b) determining the level of labeled anti- antiidiotypic antibody bound to said solid-phase immunoadsorbent; and c) comparing the level determined in step b) with a control level, wherein if the level determined in step b) is less than the control level, binding of HCA has been determined.

10. A method of assaying for the presence of a Human Carcinoma Antigen (HCA) in a sample comprising the steps of: a) contacting the sample with the following: i) a solid-phase immunoadsorbent containing immobilized anti-antiidiotypic antibody which binds to a member of the group consisting of:

A) epiglycanin;

B) HCA; and C) an antiidiotypic antibody which binds to an antibody which binds to HCA; and ii) a labeled antiidiotypic antibody which binds to an antibody which binds HCA; b) determining the level of labeled antiidiotypic antibody bound to said solid-phase immunoadsorbent; and c) comparing the level determined in step b) with a control level, wherein if the level determined in step b) is less than the control level, binding of HCA has been determined.

11. A method of assaying for the presence of Human

Carcinoma Antigen (HCA) in a sample comprising the steps of: a) contacting the sample with the following: i) a solid-phase immunoadsorbent containing immobilized anti-antiidiotypic antibody which binds to a member of the group consisting of: A) epiglycanin;

B) HCA; and

C) an antiidiotypic antibody which binds to an antibody which binds to HCA; and ii) a labeled monoclonal antibody which binds HCA, thereby producing a mixture; and b) assaying for binding of labeled monoclonal antibody to said solid-phase immunoadsorbent to determine binding of HCA.

12. The method of Claim 11 wherein in step a), the sample is contacted simultaneously with the solid-phase immunoadsorbent of subpart i) and the labeled monoclonal antibody of subpart ii) .

13. The method of Claim 11 wherein in step a), the sample is first contacted with the labeled monoclonal antibody of subpart ii) under conditions and for a period of time sufficient for formation of an immunocomplex between HCA and the labeled monoclonal antibody, and second contacted with the solid-phase immunoadsorbent of subpart i) .

14. The method of Claim 11 wherein in step a), the sample is first contacted with the solid-phase immunoadsorbent of subpart i) under conditions and for a period of time sufficient for formation of an immunocomplex between HCA and the immobilized anti- antiidiotypic antibody, and second contacted with the labeled monoclonal antibody of subpart ii) .

15. The method of Claim 11 wherein step b) comprises: i) determining the level of labeled monoclonal antibody bound to said solid-phase immunoadsorbent; and ii) comparing the level determined in step i) with a control level, wherein if the level determined in step i) is less than the control level, binding of HCA is determined.

16. A method of in vivo imaging of a tumor in a patient comprising administering to said patient an anti- antiidiotypic antibody which binds to a member of the group consisting of: 1) epiglycanin; ii) Human Carcinoma Antigen (HCA) ; and 111) an antiidiotypic antibody which binds to an antibody which binds to HCA, coupled to an imaging agent.

17. The method of Claim 16 wherein said imaging agent is a radioisotope.

18. The method of Claim 16 wherein said anti-antiidiotypic coupled to said imaging agent is infused into said patient intravenously.

19. A vaccine comprising a member of the group consisting of epiglycanin, HCA, an antiidiotypic antibody or an antigenic fragment thereof which binds to an antibody which binds HCA, and a pharmaceutically acceptable carrier.

20. A vaccine comprising a vector comprising a nucleic acid molecule which encodes an antigen selected from the group consisting of epiglycanin, HCA, an antiidiotypic antibody or an antigenic fragment thereof which binds to an antibody which binds Human Carcinoma Antigen.

21. A pharmaceutical composition comprising an anti- antiidiotypic antibody which binds to a member of the group consisting of: i) epiglycanin; ii) Human Carcinoma Antigen (HCA) ; iii) and an antiidiotypic antibody which binds to an antibody which binds to HCA, and a pharmaceutically acceptable carrier.

22. A method for treating or preventing a carcinoma in a patient in need thereof comprising administering to said patient an effective amount of a vaccine of Claim 19.

23. A method for treating or preventing a carcinoma in a patient in need thereof comprising administering to said patient an effective amount of a vaccine of Claim 20.

24. A method of Claim 23 wherein the antiidiotypic antibody is selected from the group consisting of: a chimeric antibody, a humanized antibody and a resurfaced antibody or fragment thereof.

25. A method for treating or preventing a carcinoma in a patient in need thereof comprising administering to said patient an anti-antiidiotypic antibody which binds to a member of the group consisting of: i) epiglycanin; ii) Human Carcinoma Antigen (HCA) ; iii) and an antiidiotypic antibody which binds to an antibody which binds to HCA, in a therapeutically effective amount.

26. A method of Claim 25 wherein the anti-anti-idiotypic antibody is selected from the group consisting of: a chimeric antibody, a humanized antibody and a resurfaced antibody or fragment thereof.

27. In an assay for carcinoma which employs epiglycanin and an antibody which binds epiglycanin, the improvement comprising substituting for epiglycanin an antiidiotypic antibody which binds to an antibody which binds human carcinoma antigen.

28. An antiidiotypic antibody which binds to an antibody which binds Human Carcinoma Antigen (HCA) .

29. The antiidiotypic antibody of Claim 28 wherein the antiidiotypic antibody binds to an antibody which binds to epiglycanin and HCA.

30. The antiidiotypic antibody of Claim 29 further comprising a label.

31. The anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, Human Carcinoma Antigen (HCA) and an antiidiotypic antibody which binds to an antibody which binds to HCA.

32. The anti-antiidiotypic antibody of Claim 31 wherein the antiidiotypic antibody binds to epiglycanin and HCA.

33. The anti-antiidiotypic antibody of Claim 32 which is of the IgGl isotype.

34. The anti-antiidiotypic antibody of Claim 33 further comprising a label.

35. A kit for detecting human carcinoma antigen comprising:

(a) a first antibody selected from the group consisting of: i) a monoclonal antibody which binds to HCA; and ii) an anti-antiidiotypic antibody which binds to a member of the group consisting of epiglycanin, HCA and an antiidiotypic antibody which binds to an antibody which binds to HCA, (b) an inhibitor selected from the group consisting of: i) epiglycanin, a mutant or a fragment thereof which binds to said first antibody; ii) HCA, a mutant or fragment thereof which binds to said first antibody; and iii) an antiidiotypic antibody or a fragment thereof which binds to said first antibody; and wherein one of said first antibody and said inhibitor is labeled.

36. The kit of Claim 35 further comprising a reagent which immobilizes said first antibody or said inhibitor on a solid support.

37. The kit of Claim 35 wherein said first antibody or said inhibitor is immobilized upon a solid support.

38. The kit of Claim 37 wherein said inhibitor is immobilized upon said support and said first antibody is labeled.

39. The kit of Claim 37 wherein said first antibody is immobilized upon said support and said inhibitor.

40. A method of assaying for the presence of Human

Carcinoma Antigen (HCA) in a sample comprising the steps of: a) contacting the sample with the following: i) a solid-phase immunoadsorbent containing immobilized monoclonal antibody which binds HCA ; and ii) a labeled anti-antiidiotypic antibody which binds to a member of the group consisting of:

A) epiglycanin; 5 B) HCA; and

C) an antiidiotypic antibody which binds to an antibody which binds to HCA, thereby producing a mixture; and b) assaying for binding of labeled anti- 10 antiidiotypic antibody to said solid-phase immunoadsorbent to determine binding of HCA.