CN1164238A - Monoclonal antibodies against human colon carcinoma-associated antigens and uses therefor - Google Patents

Abstract

The invention discloses monoclonal antibodies, in particular 33.28 and 31.1, and chimeric antibodies, in particular mouse/human chimeric Chi #1 specific for glycoprotein antigens of colon carcinoma-associated antigens which are immunogenic in humans, are disclosed. Such antibodies, and fragments and derivatives thereof, are useful in immunodiagnosis and immunotherapy of human colon, breast, and ovarian cancer, and for purification of antigens which can serve as immunotherapeutic agents. Methods of detecting the colon carcinoma-associated antigen in a sample, and methods for treating subjects having colon, breast, and ovarian carcinomas are disclosed.

Description

Monoclonal antibody against human colon cancer-associated antigen and its application

This application is a continuation-in-part of US Patent Application Serial No. 08/159,836 (filed November 30, 1993). And No.08/159,836 is a part of the continuation application of the U.S. patent application serial number N08/117,430 (applied on September 7, 1993), and No.08/117,430 is the U.S. patent application serial number No.07/670,816 (1991 filed on March 18, 1988), and No.07/670,816 is a partial continuation application of US Patent Application Serial No. 07/176,337 (applied on March 31, 1988), and the latter three patents are currently Already gave up.

background of the invention

field of invention

This invention involves two fields of immunology and medicine. It mainly refers to a new type of hybridoma cell line, the monoclonal antibody secreted by this type of cell line can specifically bind to the so-called colon cancer-associated antigen clinically. Such monoclonal antibodies are useful for in vivo immunodetection and immunotherapy. At the same time, it can also be used to detect and purify colon cancer-associated antigens.

technical background

During the development of cancer, a number of cell surface molecules or markers appear on cells. Such tumor-associated markers include: carcinoembryonic proteins, neoglycoproteins, sphingolipids, and modifications of original surface proteins. These newly produced and structurally altered molecules are often shed from the surface of tumor cells and show up in serum or other bodily fluids. The detection of these substances or "tumor markers" can provide a basis for diagnosis or monitoring the occurrence and development of cancerous lesions.

Early animal experiments found that among these tumor markers, a class of cell membrane protein or glycoprotein antigens were immunogenic. Further experiments found that this antigen can effectively prevent the growth of new cancer tissue when the primary tumor of cancer patients is surgically removed.

Hollinshead and Stewart used cancer tissues of lung cancer patients to make relatively pure preparations in an attempt to use relatively similar tumor-associated antigens for clinical treatment (Stewart, T.H.M. et al., Ann.N.Y.Acad.Sci.277:436(1976)). Later, this research work was further carried out to patients with melanoma and colon cancer, and different types of allotype pure preparations were used together with Freund's adjuvant. (Hollinshead, A.C. et al. Cancer 49:1387 (1982); Hollinshead, A.C. et al., Cancer 56:480 (1985)).

The reason for using Freund's adjuvant at the same time is that scientists discovered that the combined use of this adjuvant with normal tissue antigens can cause a strong autoimmune response in animal recipients. On the contrary, if this adjuvant is not used at the same time, no side effects occur. Therefore, scientists believe that adjuvants can promote antigen processing by host macrophages and prolong the reaction time of antigens at the site of action (see Roitt, I., Essential Immunology, 6th Ed., Blackwell Scientific Publication, Oxford (1988)).

It is precisely because of the above experimental results that scientists have begun to try to use specific human tumor cell membrane proteins and glycoproteins as tumor vaccines in clinical practice. Subsequently, various tumor-associated antigens that have been isolated can prolong patient survival and even inhibit the growth of metastatic tumors in some experiments.

With the generation and improvement of monoclonal antibody technology, scientists can obtain purified antibodies, and use them to purify and define different tumor marker molecules and tumor-associated antigens, and finally use these antibodies for immunodiagnosis and immunotherapy. Many monoclonal antibodies show different degrees of selectivity to tumor antigens, some of which can appear on many different types of tumors at the same time, while others show strong tumor specificity, some of which are introduced below:

Herlyn and his colleagues (Proc.Natl.Acad.Sci.USA 76:1438 (1979)) obtained two kinds of monoclonal antibodies after immunizing mice with human colorectal cancer (CRC) cells, and these two kinds of monoclonal antibodies can be selected Sexually interacts with human CRC cells. It has now been shown that one of the mAbs, 1083-17 (the precursor of 17-1A), reacts with the 41KD glycoprotein.

Herlyn et al. (J. Clin. Immunol. 2: 135 (1982)) obtained a monoclonal antibody against the membrane antigen of SW116 cell line, which can detect CRC-associated antigens in the systemic circulation. Monosialoganglioside antigens (Magnani, J.L., etc., Science 212:55 (1981) can be recognized by monoclonal antibodies 19-9 and 52a, and can be clinically combined with 8 types of 12 cell lines, a gastric cancer cell line, A pancreatic cancer cell reaction. Monoclonal antibody C414 can react with 4/6 cultured CRC cells and gastric cancer cells. Moreover, the combination of monoclonal antibodies 19-9 and 52a with tumor cells will be inhibited by the serum of CRC patients. However, gastric cancer Sera from patients and pancreatic cancer patients were more likely to inhibit mAb binding to tumor cells than sera from patients with CRC.

In 1986, Girardet and his companions (J.Im munol.136:1497-1503 (1986)) discovered a monoclonal antibody against human colon cancer. The L-D1 monoclonal antibody can react with a 41KD glycoprotein, which can The protein may be the antigen recognized by mAb 1083-17-1A (Herlyn et al, 1979, supra). Monoclonal antibody L-C5 can precipitate proteins derived from LoVo colon cancer cells with molecular weights of 43, 45, 47 and 53KD, respectively. L-D1 can also react with cervical cancer cell lines and L-C5 breast cancer cell line, but how they react with pancreatic cancer cells is not yet known.

In 1987, Greiner and his colleagues (Science 235:895-898 (1987)) discovered the monoclonal antibody 06.2, which can react with a glycoprotein of 90 KD. This glycoprotein is found in 75-80% of breast cancer patients and more than 90% of colon cancer patients.

Sakamoto and colleagues discovered a series of monoclonal antibodies that can diagnose and treat human colon cancer by other means (US Patent 4,579,827 (4/1/86)). However, none of these monoclonal antibodies can react with the human colon cancer-associated antigen protein with a molecular weight of 61 or 72 KD. It is this property that makes it different from the antibodies we invented. Moreover, Sakamoto's monoclonal antibody does not have any specificity in common with our invented monoclonal antibody.

In 1986, Delaloye and colleagues (J.Clin.Invest.77:301 (1986)) used CEA-specific monoclonal antibodies to detect colorectal cancer in vivo, specifically by means of ¹²³ I-labeled fragments and computer simulation of their release The topology of the isotopes. However, this mAb has nothing to do with the mAb we invented.

In 1986, Douillard and colleagues (Hybridoma 5, Suppl.1: S139 (1986)) discovered the monoclonal antibody 17-1A and its toxicity to gastric-small intestinal adenocarcinoma cells in vitro, and to some extent, monoclonal antibody Anti-17-1A has been successful in immunotherapy trials. Mab 17-1A can recognize 38-41 KD protein and has a wide range of reactivity, but it is not specific to colon cancer tumors, which shows that it is significantly different from the antibody we invented.

The monoclonal antibody disclosed by Scannon et al. US Patent No. 4,590,071 (5/20/86) is combined with toxic proteins such as ricinA chain, and these binding components have the function of treating melanoma, and this monoclonal antibody can specifically recognize melanoma antigen. However, no mAbs against colon cancer antigens and antibodies have been found, nor the use of such mAbs.

Hollinshead et al. in 1972 (Science 177:887-889 (1972)) have reported relatively pure preparations of antigens containing immunogenic colon cancer cell membrane antigens that our antibodies recognize and combined parts.

How to evaluate the value of the above-mentioned antigen preparation clinically has also been reported by Hollinshead et al. (see the aforementioned 1985), mainly including the immunogenicity of the antigen preparation and prolonging the survival period of patients by enhancing specific active immunity.

Intl.Symp.Biotech.in Clin.Med., Rome, Italy, April 13-15, 1987, Tsang et al. described the work of the present invention briefly in the article "Anti-Human Colon Cancer-Associated Antigen Monoclonal Antigen". The publication of this reference occurred less than one year after the date of the underlying original patent application for this patent application (US patent application Ser. No. 07/176,337).

Summary of the invention

The inventors of the present invention have produced murine monoclonal antibodies and murine-human chimeric antibodies directed against colon cancer-associated antigens, which are believed to be immunogenic in humans. These antigens isolated in the inventor's laboratory are different from the colon cancer antigens mentioned above for the following reasons: 1. The epitope recognized by the monoclonal antibody is not the sugar part of the tumor-associated glycoprotein, but the protein part; 2. This antigen is not expressed in normal tissues; 3. These antigens are tumor-specific and exist in tumor tissues of colon cancer, breast cancer and ovarian cancer; 4. These antigens can pass both cellular and humoral responses To enhance the host's anti-tumor immunity, so they are immunologically active to humans; 5. The immunogenicity of antigens is specific to humans, because only patients with colon cancer, breast cancer and ovarian cancer show Immunological specificity for these antigens, but not for other tumors, has been confirmed by immunological experiments in vivo and in vitro.

The monoclonal antibody and chimeric antibody of the present invention can be applied to the diagnosis and treatment of colon cancer, breast cancer and ovarian cancer. For example: delineate tumor metastasis; deliver cytotoxic components to tumor sites; activate host effector mechanisms such as antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) to directly kill tumor cells.

The present invention is a monoclonal antibody directed at colon cancer-associated antigen protein, and this colon cancer-associated antigen has specific immunogenicity to human beings, and can be detected in normal human tissue or other tumors (except colon cancer, breast cancer and ovarian cancer) These antigens are not found in cancer. In addition, the present invention also includes antigen-binding fragments or derivatives of antibodies.

Another part of the invention is a chimeric antibody specific for a human colon cancer-associated protein antigen which is not found in normal human tissue or other tumors (except colon cancer).

In one embodiment, the antibody is specific to a CCAA protein with a molecular weight of about 61KD. In another embodiment, the antibody is specific to a CCAA protein with a molecular weight of 72KD. In a preferred embodiment, the antibody is mouse monoclonal antibody 33.8 or 31.1, or an antibody that specifically binds to the same colon cancer-associated antigen as 33.28 and 31.1. In another preferred embodiment, the antibody is a mouse/human chimeric antibody Chi#1, which specifically binds to the same colon cancer-associated epitope as 31.1.

The antibody in the present invention can be immobilized on a solid phase.

The present invention includes the above-mentioned labeled and detectable antibodies, such as isotope-labeled antibodies.

In additional embodiments, the above-mentioned antibodies can also bind to cytotoxic radionuclides, drugs, or proteins.

In yet another embodiment, the present invention also includes monoclonal antibodies capable of resisting the above-mentioned antibodies, that is, monoclonal antibodies produced for the second time.

In a further embodiment, the present invention also includes the monoclonal antibody produced for the third time, that is, the monoclonal antibody against the above-mentioned secondary antibody.

The colon cancer-associated antigen targeted by the present invention is also unique for the following reasons: 1. The epitope recognized by the monoclonal antibody is not the sugar part of the tumor-associated glycoprotein, but the protein part; 2. This antigen is present in normal tissues 3. These antigens are tumor-specific and exist in tumor tissues of colon cancer, breast cancer and ovarian cancer; 4. These antigens can enhance the host’s anti-tumor immunity through both cellular and humoral responses, Therefore, they are immunologically active to humans; 5. The immunogenicity of antigens is specific to humans, because only patients with colon cancer, breast cancer and ovarian cancer show immunological specificity to these antigens, while It has no specificity for other tumors, which has been confirmed by immunological experiments in vivo and in vitro.

All other purified antigens that have been used so far are unable to elicit cellular or humoral immune responses.

The present invention also provides an effective drug composition for colon cancer, breast cancer and ovarian cancer, which contains a combination of radionuclides with cytotoxicity, drugs with cytotoxicity or proteins with cytotoxicity Antibodies, fragments or derivatives as described above in a suitable excipient.

The present invention also includes an immunoassay method, which can detect the presence of the colon cancer-associated antigen that can bind to the mouse monoclonal antibody 33.28, 31.1 or Chi#1 in the sample. This includes:

(a) contacting the sample with the above-mentioned antibody; and

(b) Detecting the amount of antigen by detecting the bound antibody.

In another preferred embodiment, the present invention also provides an imaging method to detect the presence of colon cancer-associated antigens in the host, which includes:

(a) contacting the labeled antibody with the host; and

(b) Detection of antigens.

The present invention includes a method of killing cells carrying colon cancer-associated antigens, comprising:

(a) delivering said antibody and a cytotoxic effector to the cell; and

(b) Killing cells.

Components that perform the task of killing cells can be complement, or activated effector cells in ADCC. It can also be a labeled antibody that has bound cytotoxic components (radionuclide, drug, protein) to exert a direct killing effect.

The present invention also provides a method of treatment for people who are suspected to have colon cancer, breast cancer, ovarian cancer, these people often carry the 33.28, 31.1 monoclonal antibody, Chi#1 Antigens, this treatment may use effective doses of the pharmaceutical compositions as described above.

In addition, a method for producing immunogenic components for clinical immunotherapy of colon cancer, breast cancer, and ovarian cancer is provided, which includes:

(a) preparing a cell membrane extract of a tumor or cell line containing an antigen that binds to 33.28 mAb or 31.1 mAb or Chi#1 antibody; and

(b) Using the above-mentioned antibody to isolate the antigen by means of affinity purification. This enables the production of immunogenic components.

In another embodiment, the present invention also includes using the above-mentioned antigens to prepare vaccines.

The present invention also includes a method for detecting and diagnosing colon cancer, breast cancer, and ovarian cancer, specifically by labeling and binding biological monoclonal antibodies or chimeric antibodies on human colon cancer-associated antigens.

In another embodiment, the present invention also includes a kit for selectively identifying colon cancer, breast cancer, and ovarian cancer.

Brief description of the chart

Figure 1 is a record of the characteristic image of the HPLC eluate of a Hollinshead "vaccine", which refers to a partially purified colon cancer cell membrane extract.

Fig. 2 is a record of HPLC eluate of a colon cancer-associated antigen obtained by affinity purification of the substance in peak 4 in Fig. 1 .

Figure 3 is a graph depicting the biodistribution of mAb 31.1 in nude mice xenografted with human tumor LS-174T.

Figure 4 is the biodistribution image of mAb 33.28 in nude mice transplanted with human tumor LS-174T.

Description of the preferred embodiment

The present invention provides antibodies, including monoclonal antibodies and chimeric antibodies, which specifically bind immunogenic human colon cancer-associated antigens (CCAA), which are natural proteins. These antibodies are used in the diagnosis and treatment of patients who have or are developing colon, breast, and ovarian cancer.

This invention not only provides a mouse monoclonal antibody, but also a chimeric antibody composed of the V region of the same monoclonal antibody, which can also recognize the CCAA segment.

The "segment" here refers to the part of the molecule that can be recognized and bound by the antibody. Usually, segments are composed of chemically active interfacial molecular clusters, such as amino acids and sugar chains, which have special three-dimensional structures and special charge characteristics. The segments of interest in the present invention are composed of amino acids.

"Antigen" here refers to a molecule or part of a molecule that can be bound by an antibody, and which can also induce an animal to produce antibodies that can bind to its own segment. An antigen may contain one or more segments. The above-mentioned reaction is highly selective, and the antigen can only react with the relevant antibody.

The "antibody" here includes the whole immunoglobulin molecule, its fragments and derivatives, such as Fab, Fab', F(ab')2, Fv, which can bind to the antigen. These antibody fragments lack the Fc fragment of an intact antibody, are cleared more quickly from the blood, and have less nonspecific tissue binding than other intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). These fragments are all cut from the complete antibody by well-established methods, such as papain and pepsin to obtain Fab and F(ab')2 fragments, respectively.

A "derivative" of an antibody comprises chemical components that are not structural proteins. Covalent modification of proteins is encompassed by this invention. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatives capable of interacting with selected side chains or terminal residues. For example, derivatization of bifunctional drugs is useful for linking antibodies or fragments to water-insoluble supports or other macromolecular carriers.

"Vaccine" refers to a class of drugs used to stimulate the immune system of organisms to provide immune protection against future infectious drugs. The vaccines contemplated by the present invention can be administered to patients using any known or standard techniques. These include oral, intestinal intubation or broncho-nasal spray. Other methods of administration, such as intravenous injection to allow the microorganisms to reach the bloodstream of humans or animals, are acceptable when the microbial vector cannot replicate.

The antibodies of the invention are novel. They are the first known monoclonal antibodies (mAbs) and are CCAA-specific chimeric antibodies immunogenic against tumor antigens in humans. The antigens recognized by the antibodies of the present invention can induce immune responses in patients with colon, breast and ovarian cancers, but not in patients with other types of cancer. The immunogenicity of these antigens is primarily manifested by cell-mediated immunity and can be detected by in vivo delayed skin allergy assays ("skin tests") or by analysis of in vitro responses to various specific lymphocytes, such as lymphocyte Proliferation or metastasis inhibition assays. For general principles of immunogenicity and analysis of various specific immune responses see the following references: Roitt, I., Essential Immunology, 6th Ed., Blackwell Scientific Publications, Oxford (1988); Roitt, I. et al., Immunology , C.V.Mosby Co., St.Louis, MO91985); Klain, J., Immunology, Blackwell Scientific Publications, Inc., Cambridge, MA (1990); Klain, J., Immunology; The Science of Self-Nonself Discrimination, John Wiley & Sons, New York, NY (1982); Paterson, P.Y., Textbook of Immunopathology, Grune and Stratton, New York, (1986).

In a preferred embodiment, the antibody of the invention is a murine monoclonal antibody designated 33.28. In another preferred embodiment, the antibody is a murine monoclonal antibody designated 31.1. In yet another preferred embodiment, the antibody is a chimeric antibody recognizing the epitope identified by 33.28. In yet another embodiment, the antibody is a chimeric antibody that recognizes the epitope identified by 31.1.

Chimeric antibodies of the invention comprise heavy (H) and light (L) chains of different immunoglobulins (Ig). The chimeric H chain comprises an antigen binding region derived from the H chain of a non-human antibody specific for the epitope recognized by 33.28 or 31.1, linked to at least a portion of the human H chain C region ( _CH ).

An optimal chimeric L chain comprises an antigen binding region from the L chain of the monoclonal antibody 33.28 or 31.1 linked to at least a portion of the human L chain C region ( _CL ).

Alternatively, an optimal chimeric H chain comprises an antigen-binding region from the L chain of the 33.28 or 31.1 monoclonal antibody linked to at least a portion of the human L chain C region ( _CL ).

The term "antigen-binding region" herein refers to the amino acid residue portion of an antibody molecule that interacts with an antigen and provides antibody specificity and antigen affinity. Antibody regions include the "framework" amino acid residues necessary to maintain the proper configuration of the antigen-binding residues.

The term "chimeric antibody" herein includes monovalent, bivalent or multivalent immunoglobulins. A monovalent chimeric antibody is a dimer composed of chimeric H and L chains linked by disulfide bonds. A bivalent chimeric antibody is a tetramer composed of two HL dimers linked by at least one disulfide bond. Multivalent chimeric antibodies can also be obtained, eg, by polymerization of the H chain C region (eg, IgM H or μ chain).

The invention also provides derivatives of mAbs or chimeric antibodies. It includes proteins encoded by genes that are truncated or modified to produce a type of molecule that functions like an Ig fragment. Such modifications include, but are not limited to, the addition of gene sequences encoding cytotoxic proteins such as plant and bacterial toxins. These fragments and derivatives can be derived from prokaryotic and eukaryotic hosts, similar to the recombinant means described herein. They may be derived from chemical processes such as protein cleavage of intact Ig molecules, or other chemical modifications or derivatizations described in the literature. Such derivatized components can enhance solubility, absorption rate, biological half-life, and other beneficial properties, as well as optionally eliminate or attenuate any deleterious side effects of the antibody protein. Components capable of such effects have been reported. (see literature Remington's Pharmaceutical Sciences, 16 th ed., Hack Publishing Co., Easton, PA (1980)).

Antibodies, fragments or derivatives of chimeric H and L chains with the same or different V region binding specificities can be prepared by linking different polypeptide chains (see literature Sear et al., Proc.Natl.Acad.Sci.USA 72:353 -357(1975)). By this method, the host expressing the chimeric H chain (or its derivatives) is cultured separately from the host expressing the chimeric L chain (or its derivatives), the chains are spontaneously joined in the culture medium, and then the assembled Ig, fragments or derivatives.

Mouse hybridoma cells that produce mAbs specific to CCAA, such as the mAbs 33.28 and 31.1 of the present invention, are formed by fusion of mouse fusion partner cells, such as SP2/0 cells, with spleen cells of CCAA-immunized mice .

Mice can be immunized with a crude or semi-pure preparation containing the antigen of interest, eg Hollinsheed "vaccine" is a semi-purified colon cancer cell membrane preparation. To immunize mice, a variety of traditional methods can be used. For example, mice can receive primary and boosting immunizations with antigen preparations.

Cell fusion can be accomplished by standard procedures well known in the field of immunization (see Kohler and Milstain, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110; Hartlow, E. et al, supra; Campbell, A., "Monoclonal Antibody Technology″In: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R., et al, eds.), Elsevier, Amsterdam (1984); Kennett et al., Monoclonal Antibodies (Kennett et al., eds.pp. 365-367, Plenum Press, NY, 1980); da St. Groth, S.F.. et al., J. Immunol. Meth. 35; 1-21 (1980); Galfre, G., et al., Methods Enzymol. 73:3-46 (1981); Goding, J.W. 1987, Monoclonal Antibodies; Principles and Practice, 2nd ed. Academic Press, London, 1987).

Fusion partner cell lines and methods for fusion, selection of hybridoma cells, and screening of mAbs are well established. (see literature Hartlow, E.et al., Supra; Kawamoto, T.etal, Meth.Enzymol.121:266-277 (1986); Keamey, J.F.et al., J.Immunol.123:1548-1550 (1979 ); Kilmartin, J.V.et al, J.Cell Biol.93:576-582 (1982); Kohler, G.et al., Eur.J.Immunol.6:292-295 (1976); Lane, D.P.et al ., J.Immunol.Meth.47:303-307 (1981); Mueller, U.W.et al., J.Immunol.Meth.87:193-196 (1986); Pontacorvo, G., Somatic Cell Genet.1: 397-400(1975); Sharo.J., et al., Proc.Natl.Acad.Sci.USA 76:1420-1424(1979); SHulman, M.et al, Nature276:269-270(1978); Springer, T.A. (ed), Hybridoma Technology in the Biosciences and Medicine, Plenum Press, New York, 1985; and Taggart, R.T. et al., Science 219:1228-1230 (1982)).

The mAbs of the present invention can be produced in large quantities by intraperitoneally injecting hybridoma cells secreting antibodies into mice, collecting ascites fluid containing high-titer mAbs after an appropriate time, and isolating the mAbs. Alternatively, mAbs can be isolated by culturing hybridoma cells in vitro and from the cell culture medium.

The human gene encoding the C region of the chimeric antibody of the present invention is derived from cells expressing and producing human Ig. The human H chain C region may be derived from any known species or isotype of human H chain, including γ, μ, α, δ or θ. Since the H chain type is responsible for multiple effector functions of antibodies, the selection of the H chain C region should be guided by the effector function of interest, such as complement fixation or antibody-dependent cytotoxicity. The ideal H chain C region is derived from γ1 (IgG1), γ3 (IgG3), γ4 (IgG4) and μ (IgM).

The human L chain C region can be derived from human isotype L chain, kappa or lambda.

The gene encoding the human IgC region can be obtained from human cells by standard cloning techniques (Sambrook. J. et al, Molecular cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor, Press, Cold Spring Haxbor, NY (1989)) . Human C region genes exist in gene clones expressing two types of L chains and five types of H chains. Chimeric antibody fragments, such as F(ab')2 and Fab, can be obtained by designing appropriately truncated chimeric H-chain genes, for example, a chimeric gene encoding the H portion of the F(ab')2 fragment includes a gene encoding CH1 region and H hinge region, followed by a translation stop codon to generate a truncated molecule.

Usually, the chimeric antibody of the existing invention is prepared by cloning the DNA fragments encoding the H and L chain antigen-binding regions of CCAA-specific antibodies, preferably non-human genes, and the best is the genes in 33.28 or 31.1, and then these The fragments are connected with DNA fragments encoding human H chain C regions and L chain C regions to prepare genes encoding chimeric Ig.

Therefore, in a preferred embodiment, the created fusion gene encodes at least one Ag-binding region of non-human origin, such as a functionally rearranged V region with a connecting segment (J), and is at least compatible with one coding part of the human C region. The second DNA fragment is ligated. This fusion can be accomplished using PCR as reported by Temando et al. (see Miami Symp. Short Reports 3:88, 1993).

The DNA encoding the antibody binding region may be genomic DNA or cDNA. A convenient choice for selecting chromosomal gene fragments as the DNA source of the antigen-binding fragments in the coding mouse V region is to use cDNA to construct chimeric Ig genes (see literature Liu et al., Proc.Natl.Acad.Sci.USA 84: 3439( 1987) and J. Immunology 139:3521 (1987)). The application of cDNA requires that the gene expression elements suitable for the host cell should be combined with the gene to obtain the synthesis of the target protein. The use of cDNA sequences is advantageous over genomic sequences (including introns) because cDNA sequences can be expressed in bacteria or other hosts that lack an appropriate RNA splicing system.

Therefore, in the way of using the cDNA encoding the V region of the antibody, the method for preparing the chimeric antibody can be summarized as the following steps:

1. Isolation of mRNA from the mAb-producing cell line from which the clones and cDNA products are derived.

2. Prepare a full-length cDNA library with purified mRNA, from which the appropriate L and H chain V region gene fragments can: (I) identify with appropriate probes; (II) sequence; (III) match with the C region gene fragments.

3. Preparation of C region gene fragments from cDNA preparations and cloning.

4. As described above, by linking the cloned specific V region gene and the cloned human C region gene, the sequence encoding the complete H or L chain can be constructed.

5. Expression and production of chimeric L and H chains in selected hosts, including prokaryotic and eukaryotic cells.

A common feature of all IgH and L genes and the mRNAs they encode is the J region. The J regions of the H and L chains have different sequences, but the sequence homology is high (>80%), especially around the C region. This homology is exploited in this approach, and the identical sequences of the J regions of the H and L chains can be used to design oligonucleotide primers for introducing useful restriction sites in the J region for subsequent V region Ligation of fragments to human body C region fragments.

The C-region cDNA vector prepared from human cells can add restriction sites at similar positions in the human sequence through site-directed mutagenesis. For example, one can clone the complete human kappa chain C region and the complete human gamma-1 C region. In this case, other methods using genomic C-region cloning as a source of C-region vectors do not allow expression of these genes in bacterial systems lacking the enzymes necessary to remove the inserted sequence. Cloned V region fragments are cut out and ligated into L or H chain C region vectors. Alternatively, the human γ-1 C region can be modified by introducing a stop codon, thereby generating a gene sequence encoding the H chain portion of the Fab molecule. In this way, the coding sequences with V and C regions can be transferred into appropriate expression vectors for expression in suitable hosts such as prokaryotic and eukaryotic cells.

Two DNA coding sequences are said to be "operably linked" if the linkage results in a continuously translatable sequence without altering or interrupting the reading frame of the triplet. A DNA coding sequence is operably linked to a gene expression element if the linkage results in the normal function of the gene expression element such that the DNA coding sequence is expressed.

Expression vectors include plasmids or other vectors. Preferred among these is a vector carrying a functionally complete human H or L chain C region sequence with properly inserted restriction sites so that any H or L chain V region sequence with appropriate cohesive ends can be easily inserted. Thus, a vector containing the human H or L chain C region sequence serves as an intermediate for expression of any desired complete H or L chain in any suitable host.

Typically, mouse-human antibodies are synthesized from genes driven by the promoters of the chromosomal genes of the native mouse H and L chain V regions in the construct; splicing usually occurs at the splice donor site of the mouse J region and the human C region Between the pre-splicing acceptors; it can also appear between the splicing regions in the C region of the human H chain; polyadenylation and transcription termination occur downstream of the coding region of the human natural chromosome.

Gene expression elements useful for cDNA gene expression include: (a) viral transcriptional promoters and their enhancer elements, such as the SV40 early promoter ((see document OkayamaH.et al, Mol.CellBiol.3: 280 (1983)) , Roux sarcoma virus LTR (see literature Gorman.C, etc., Proc.Natl.Acad.Sci.USA 79:6777 (1982)) and Moloney mouse leukemia virus LTR (see literature Grosschedd, R.et al, Cell 41 : 885 (1985)). (b) Splice region and polyadenylation site derived from the late region of SV40 (see literature Okayama et al., see above). (c) Polyadenylation site, such as within SV40 (see Literature Okayama et al., see supra).

The Ig cDNA gene can be expressed as such (see literature Liu et al., see above, and Weidle et al., Gene 51:21 (1987))): with the SV40 early promoter and its enhancer, the enhancer of the mouse IgH chain promoter, SV40 Late region mRNA splicing, rabbit β-globin insertion sequence, Ig and rabbit β-globin polyadenylation site and SV40 polyadenylation site as expression elements. For Ig genes consisting of part cDNA and part genomic DNA (see literature Whittle et al., Protein Engineering 1:499 (1987)), the transcriptional promoter can be human "CMV", derived from CMV and mouse/human Ig promoters Enhancers, or mRNA splicing and polyadenylation regions derived from native chromosomal Ig sequences.

In one embodiment, for expression of cDNA in rodent cells, the transcriptional promoter is a viral LTR sequence, the enhancer of the transcriptional promoter is either a mouse IgH chain enhancer, or a viral LTR enhancer, and the splicing region comprises more than The 31 bp intron, and the polyadenylation and transcription termination regions are from the native chromosomal sequence corresponding to the synthetic Ig chain. In other forms, cDNA sequences encoding other proteins are combined with the expression elements described above to obtain protein expression in mammalian cells.

Each fusion gene is assembled or inserted into an expression vector, and then a gene encoding chimeric H or L chain is used to individually transfect recipient cells capable of expressing chimeric Ig chain gene products; or a gene encoding chimeric H and L Chained genes were co-transfected. Transfected recipient cells are cultured under conditions permissive for expression of the incorporated gene, and expressed Ig chains, whole antibodies or fragments are recovered from culture. In an embodiment, the fusion gene or part thereof encoding the intercalated H and L chains is assembled in different expression vectors and used to co-transfect recipient cells.

Each vector may contain two selection genes, one for selection in bacterial systems and the other for selection in eukaryotic systems, so each vector has a different pair of genes. This strategy results in directed production of fusion genes in bacterial systems, vectors that allow amplification. Furthermore, such genes prepared and amplified in bacterial hosts can be used to co-transfect eukaryotic cells and allow selection of co-transfected cells carrying the transfected gene of interest.

Genes that can be selected for use in bacterial systems are the ampicillin resistance gene and the chloramphenicol resistance gene. The genes selected in eukaryotic transfection are preferably xanthine-guanine phosphoribosyltransferase gene (gpt) and Tn5 phosphotransferase gene (neo). The basis for the selection of cells expressing gpt is that the enzyme encoded by this gene synthesizes purine nucleotides using xanthine as a substrate, while the similar endogenous enzyme does not. On the medium containing mycophenolic acid, hypoxanthine monophosphate cannot be converted into xanthine monophosphate and xanthine, and only cells expressing gpt can survive. The product of neo blocks the inhibitory effect of G418 and neomycin antibiotics on protein synthesis.

These two methods can be selected simultaneously or carried out sequentially so that the immunoglobulin chain genes can be expressed in eukaryotic cells mediated by two different DNA vectors. But eukaryotic cells do not require a different selectable marker. One H-chain vector and one L-chain vector each have the same selectable marker and can be transfected at the same time. After selection of untransfected cells, the majority of clones will contain intact copies of the vector containing the H and L chains.

Alternatively, fusion genes encoding chimeric H and L chains may be assembled on the same expression vector.

For transfection of expression vectors and production of chimeric antibodies, the transfected cell line is preferably myeloma cells. Myeloma cells synthesize, aggregate, and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess mechanisms to glycosylate immunoglobulins. A particularly preferred transfected cell line is the Ig-non-producing myeloma cell SP2/0 (ATCC #CRL 8287). SP 2/0 produces only the immunoglobulin encoded by the transfected gene. Myeloma cells can be cultured, or grown in the peritoneal cavity of mice, and the secreted immunoglobulin can be obtained from ascites. Other suitable transfected cells include lymphocytes such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells.

The expression vector carrying the chimeric antibody of the present invention can be introduced into suitable host cells by various methods. These methods include biochemical methods, such as transformation, transfection, ligation, protoplast fusion, calcium phosphate precipitation method, polyvalent cationic reagent method, such as diethylaminoethyl dextran (DEAE), and other physical and mechanical methods, such as Electroporation, direct microinjection, and microprojectile bombardment (Johnston et al, Scence 240: 1538 (1988)). The preferred method for introducing DNA into lymphocytes is electroporation (Dotter et al, Proc. Natl. Acad. Sci. USA 81:7161 (1984); Yoshikawa, K et al, Jan. J. Cancer Res. 77:1122-1133). During this process, DNA is incorporated into transfected cells under electrical pulses. Generally, after transfection, cells are recovered in complete medium for 24 hours, and then seeded in selective medium in 96-well culture plates. The concentration of G418 selection was 0.4-0.8 mg/ml. Mycophenolic acid screening uses a concentration of 6ug/ml plus 0.25mg/ml xanthine. The electroporation technique allows the transfection frequency of SP2/0 cells to be 10 ^-5 to 10 ^-4 . In the protoplast fusion method, lysozyme is used to strip the mucositised cell wall of the recombinant plasmid containing the chimeric antibody gene. Under the action of polyethylene glycol, spheroplasts fuse with myeloma cells.

The chimeric immunoglobulin genes of the present invention can also be expressed in mammalian non-lymphocytes or other eukaryotic cells, such as yeast, or in prokaryotic cells, in certain bacteria.

Yeast has important advantages for the production of immunoglobulin H and L chains. It performs post-translational peptide modification reactions, including glycosylation. Many recombinant DNA methods have emerged that utilize enhanced promoter sequences and high copy number plasmids so that desired proteins can be obtained in yeast. Yeast can recognize the leader sequence of cloned mammalian gene products and secrete a peptide containing the leader sequence (eg: propeptide] (Hitziman, et al, 11th International Conference on Yeast, Genetics and Molecular Biology, Montpeller, France, September 13-17 , 1982)

Generally, the quality of the yeast gene expression system can be judged from the level of the product, the situation of secretion, the stable level of chimeric H and L chain proteins and chimeric antibodies. Any yeast gene expression system that combines promoter genes and terminator factors can be used. These promoter genes and terminators are derived from actively expressed genes encoding glycolytic enzymes produced by yeast cultured in large quantities in a glucose-rich medium. Known glycolytic genes may also provide efficient translational control signals, for example, the initiator and terminator of the gene for phosphoglycerol kinase (CPK) may be used. A number of means are available for judging the selection of optimal expression plasmids for immunoglobulin cDNA cloned in yeast. (See Glover, D.M.ed., DNA Cloning, Vol II pp.45-66, IRL Press, 1985)

Bacterial strains can be used as hosts for the production of antibody molecules or antibody fragments described herein. E. Coli K12 strains such as E. Coli W3110 (ATCC 27325) and other Enterobacteriaceae such as Selmonella typhimurium or Serratia Marcascery and various Pseudomonas species are available.

Plasmid vectors containing replicons and control sequences from species compatible with the host cell are used for ligation with these bacterial hosts. Vectors carry replication sites and specific genes that provide phenotypic selection capabilities. Evaluation of expression plasmids for the production of chimeric antibodies or antibody chains encoded by cloned immunoglobulin cDNAs in bacteria can be accomplished by a number of methods. (See Glover, D.M.ed.; DNA Cloning, Vol I, IRL Press, 1985).

Other preferred hosts are mammalian cells that can be grown in vivo or in vitro. Mammalian cells provide post-translational modifications to immunoglobulin molecules, including: leader peptide removal, folding, assembly of H-chain and L-chain r-glycosylated antibody molecules, and secretion of functional antibody proteins.

In addition to the above-mentioned lymphoid-derived cells, mammalian cells that can be used as hosts to produce antibody proteins include some fibroblast-derived cells, such as Vero. (ATCC CRL 81) CHO-Ki (ATCC CRL 61).

There are many vector systems for expressing cloned H chain and L chain in mammalian cells. (See Glover, DMed., DNA Cloning. Vol. II pp. 143-238, IRL Press, 1985) Complete _H2L2 antibodies can be obtained _by different routes. As mentioned above, it is completely possible to co-express the H chain and the L chain in the same cell and combine them in the cell to obtain a complete tetrameric H ₂ L ₂ antibody. In the same host cell, no matter using the same or different plasmids, they can be co-expressed. The genes encoding H chain and L chain can be placed in a plasmid, then transfected into cells, and finally expressed. Alternatively, a plasmid encoding one chain, such as the L chain, is first transfected into cells, and these cells are then transfected with a second specific marker encoding the H chain. No matter which way is used, H ₂ L ₂ molecules can be obtained. In addition, if some additional factors are encoded on the plasmid, the obtained H ₂ L ₂ molecule will also have characteristics such as high yield and good stability.

In addition to monoclonal antibodies and chimeric antibodies, the present invention can also produce anti-Id antibodies specifically directed against the variable region (V) of monoclonal antibodies and chimeric antibodies. An anti-Id antibody is an antibody that specifically recognizes a specific determinant related to the antigen-binding region of another antibody, such as the anti-CCAA antibody code-named 33.28, which is called an idiotype or Id antibody. Anti-Id antibodies can be obtained by immunizing animals of the same species and genotype with Id antibodies or antigen conjugates. It is also possible to further immunize another animal with an anti-Id antibody as an immunogen, thereby obtaining an anti-anti-Id antibody animal. In fact, this anti-anti-Id antibody is identical to the original induced anti-Id antibody. Thus, by using antibodies against idiotopes of the mAb, it is possible to distinguish other clones expressing antibodies of the same specificity.

Therefore, the mAbs and chimeric antibodies of the invention can be used to induce anti-Id antibodies in suitable animals, such as BALB/C mice. Splenocytes from these immunized mice can be used to generate anti-Id hybridomas that secrete anti-Id monoclonal antibodies. Then the anti-Id monoclonal antibody is integrated on a carrier (such as KIH) and used to immunize other BALB/c mice; finally, the serum of this mouse contains anti-anti-Id antibody, which can specifically Conjugated to the CCAA segment of the original mAb.

The antibodies of the present invention and their antigen-binding fragments and derivatives can have various functions of diagnosing, monitoring and treating cancer, including colon cancer, breast cancer and ovarian cancer. These uses are summarized by Schlom, J. in Cancer Res. 46:3225-3238 (1986).

When they are used as a diagnosis, the antibodies of the present invention can be used in immunodiagnosis to examine body fluids such as serum, urine, cerebrospinal fluid, sputum, and exudate for the presence or absence of CCAA. These antibodies can also be used for scanning or immunoimaging to determine the location of the primary cancer and metastases, and even the lymph nodes where the cancer cells have metastasized, if labeled with the appropriate immune markers.

The antibodies of the invention are also suitable for immunopathological analysis. For example, the diagnosis of tumor differentiation and the analysis of tumor subtypes based on CCAA expression are very important for predicting the possibility of tumor metastasis, treatment outcome and prognosis.

It is particularly worth mentioning that due to the specificity of the mAbs and chimeric antibodies of the present invention, it is possible to determine the different types of cells in a growing heterogeneous tumor. These antibodies can be applied by means of flow cytometry to determine the type and cross-pairing of tumor cells that make up a tumor. This is done before surgery and treatment. Using the antibody of the present invention and other monoclonal antibodies to determine the subtype of tumor cells refers to: A which antigen preparation is most suitable for immunotherapy with high specific activity. B Which monoclonal antibody or chimeric antibody is effective for ADCC. C Which antibody or combination of monoclonal antibodies is most suitable for predicting whether there will be recurrence and metastasis in patients after treatment.

In addition to diagnostic applications, the antibodies of the invention are also suitable for monitoring the progression of disease by monitoring the presence of CCAA in body fluids, radioimaging of tumors, or by detecting lymph node and bone marrow biopsies, body fluid cells, metastatic exhaled cells.

The comprehensive therapeutic approach of the antibody of the present invention includes 1. direct cytotoxicity pathway, which includes two pathways through complement CDC or effector cell ADCC; 2. linkage with antitumor drugs, toxins, and radionuclides. In in vitro experiments, these antibodies were able to eliminate tumor cells in the blood circulation and bone marrow.

All these methods will be described in detail in the following sections. One of ordinary practice will also be able to know how to use the antibodies of the invention in diagnostic, monitoring and practical therapeutic practice by studying and mastering the method description provided here.

Animals to which the present invention is most applicable are mammals, which refers here to organisms of the class Mammalia. Especially for humans.

The so-called "treatment" here refers to the use of our antibody fragments or derivatives for the prevention, alleviation and cure of colon cancer, breast cancer and ovarian cancer.

All the pharmaceutical components of the antibody of the present invention are helpful to achieve certain medical purposes. Dosage and treatment regimens of these antibody fragments and derivatives can be mastered with routine clinical skills in the treatment of colon cancer, breast cancer and ovarian cancer and related diseases. For example, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal and buccal administration can be used. In addition, they can be taken orally alone or at the same time, and the dosage depends on factors such as the patient's age, health status, body weight, whether there are concomitant medications, treatment frequency, and expected curative effect.

The composition of the scope of the present invention is determined by the combination of all these antibody fragments and derivatives, which depends on the therapeutic purpose to be achieved. When an individual patient adjusts the composition of the drug, the optimum range of the amount of each active ingredient can be determined within the technically permitted range. The effective dosage is a combination of the presence and nature of the other therapeutic ingredients to which the particular chimeric or monoclonal antibody is combined, and the clinical condition of the patient. This effective dose can vary within the range of 10 ng/kg-100 mg/Kg body weight. Preferably in the range of 0.1-10mg/Kg body weight.

In addition to pharmacologically active ingredients, new medicinal chemical compositions may include appropriate medicinal chemical carriers, including excipients and adjuvants. Because these excipients and adjuvants can accelerate the formulation process of active ingredients. The content of active ingredients and excipients in the preparation is in the range of 0.01%-99%, preferably 20%-75%.

Preparations of antibodies, antibody fragments, and derivatives suitable for enteral administration of the present invention, whether in a detectably labeled form, in a bound form or in a non-bound form, may include sterile aqueous solutions, non-aqueous solutions, suspensions Liquids and Emulsions. Aqueous solutions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol/water solutions, emulsions or suspensions, all of which contain saline, buffer media The carrier of parenteral route includes NaCl solution, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution or fixed oil, and the carrier of intravenous injection mainly includes liquid and nutritional supplement They are often made on the basis of dextrose Ringer's solution. Of course, preservatives and other additives are also essential, such as antimicrobial ingredients, antioxidants, chelating ingredients, inert gases, etc. For details, see Remington's Pharmaceutical Science, 16th ed., Mack Publishing Co., Easton.PA.1980.

Antibodies, antibody fragments and derivatives thereof of the present invention are also helpful in the treatment of existing or developing colon cancer, breast cancer and ovarian cancer, and this treatment is administered by parenteral route with single or multiple doses of antibodies and antibody fragments Or derivatives or conjugates.

The reason why the antibodies of the present invention can exert therapeutic effect is that they can fight against cells with CCAA on their surface, and the specific effect is accomplished by mediating ADCC and/or CDC. Whether endogenous or exogenous effector cells (for ADCC) or complement components (for CDC) can be the means by which antibodies exert their effects.

Antibodies, antibody fragments, and derivatives of the present invention can also be used as immune conjugates to exert therapeutic effects. (See Dillman, R.O., Ann. Int Med. 111:592-603 (1989)). They can be coupled to cytotoxic proteins including, but not limited to, Ricin-A, Pseudomonas toxin, diphtheria toxin and tumor necrosis factor. Techniques for coupling toxins to antibodies or other ligands are available (see Olsnes, S. et al., Immunol. Today 10:291-295 (1989)). Plant and bacterial toxins are the most capable of disrupting protein synthesis, thereby killing cells.

Antibodies of the invention can also be conjugated to other types of therapeutically useful substances. These substances include, but are not limited to, diagnostic radioisotopes, cytotoxic components (eg, cytotoxic radionuclides, drugs, and proteins). Radionuclides that can be coupled to antibodies and transported to the site of action in vivo include, for example, ²¹² Bi, ¹³¹ I, ¹⁸⁶ Re, and ⁹⁰ Y (these are not exhaustively listed). Radionuclides can locally irradiate cells with radiation to achieve their cytotoxic effects. This is the general mechanism of radiation therapy.

The aforementioned cytotoxic drugs capable of binding to antibodies include daunorubicin, doxombicin, methotrexate and mitomycin C, but are not limited to these. Cytotoxic drugs can affect many important activities, such as DNA, RNA and protein synthesis, as detailed in Goodman, A.C., et al, Goodman and Gilman's THE PHARMACOLOGY BASIS OF THERAPEUTICS, 7th Ed., Macmillan Publishing Co., 1985.

Another advantage of the antibody of the present invention is that it can be used in combination with other monoclonal antibodies, chimeric antibodies, lymphokines, erythropoietic growth factors, etc., and these substances can increase the viability and number of effector cells.

The antibodies, fragments and derivatives of the present invention are attached to a solid support and are capable of removing soluble colon cancer-associated antigens from body fluid tissues and cell extracts. In a preferred embodiment, they are used to remove soluble tumor antigens from blood and plasma. In another preferred embodiment, the antibody is used in an in vitro immunosorbent device (see Seminars in Hematology, Vol. 26(2 Suppl. 1) (1989)). After the patient's blood and other body fluids pass through the adsorbed antibodies, CCAA (free or immune complexes) in the circulation can be partially or completely removed, including cells carrying CCAA, and then these body fluids can be returned to the body. This immunoadsorption method can be performed in a continuous flow state, with or without a cytocentrifugation step. See for example Terman. D. S. et al., J. Immunol. 117:1971-1975 (1976).

The present invention also provides the above antibodies, fragments or derivatives, all detectably labeled, as described below.

The antibody of the present invention can be used in immunoassay to detect or quantify CCAA in samples or cells. Such immunoassays generally consist of incubating a biological sample with an antibody of the invention that is detectably labeled to recognize a tumor antigen; and detecting the labeled antibody bound to the sample.

Therefore, considering the present invention from this aspect, biological samples can be treated with nitrocellulose or other solid supports or carriers capable of fixing cells, cell debris, or soluble proteins and glycoproteins, washed with an appropriate buffer, and then added to the present invention. Labeled antibody; wash the stationary phase support again with buffer to remove unbound antibody. The amount of antibody bound to the stationary phase support can be determined by conventional methods.

A stationary phase support or carrier refers to any substance that can bind to an antigen or antibody. Commonly used are glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural or artificial cellulose, polyvinylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble to some extent. As long as the linked molecules can bind to CCAA or CCAA-specific antibodies, the material of the support can be in any possible structural shape. Thus, it may be spherical like a ball, cylindrical like the inside of a test tube, outside like a rod, planar like a paper or dipstick, and so on. A more suitable support is polystyrene beads. The skilled artisan will also know of other suitable carriers for binding the antibody or antigen, or can use routine experimentation to determine which carrier is suitable.

The binding activity of the same batch of antibodies can be determined by some common methods. Skilled technicians can judge the operation method and optimal analysis conditions of each index through routine experiments.

One of the methods of comparing the antibodies of the present invention is to link the antibodies to an enzyme, which is detected by an enzyme assay (EIA) or enzyme-linked immunosorbent assay (ELISA), when the enzyme contacts a substrate, a The reaction produces a chemical species that can be detected, for example, by spectrophotometry, fluorometry, or visible light. In a supplementary embodiment the enzyme delabels the ligand of an antibody of the invention. Such ligands may be the constant and variable regions of an antibody of the invention, such as a heterologous anti-mouse immunoglobulin antibody. Alternatively, the binding partner may be a non-antibody protein, such as staphylococcal protein A or streptococcal protein G, that binds to the antibody of the present invention.

CCAA-specific antibodies that can be used to label the antibodies of the invention, or enzymes that bind ligands for these antibodies, can include those listed below, but are not limited to: malate dehydrogenase, staphylococcal nuclease, delta-5-steroid Isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-hemi Lactosylase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glycoamylase, and acetylcholinesterase.

If the antibody or binding ligand of the present invention is radiolabeled, CCAA can be detected by radioimmunoassay (see Work, T.S. et al, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y. (1978)). Radioisotopes It can be detected by γ-counter or scintillation counter or autoradiography. The isotopes used for the antibodies of the present invention are those that are commonly used.

Fluorescent complexes can also be used to label antibodies or binding ligands. When a fluorescently labeled antibody is exposed to light of a certain wavelength, its presence can be detected by fluorescence. The most commonly used fluorescent labeling complexes are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, isophycocyanin, o-phthalaldehyde, and fluorescamine.

Antibodies can also be labeled with fluorescent metals, such as ¹⁵² Eu, or other lanthanides. These metals can be attached to the antibody using metal chelating agents such as DTPA or EDTA.

Antibodies of the invention may also be labeled with conjugated chemiluminescent complexes. Labeled antibodies emit light during the chemical reaction and are detected. Some chemiluminescent labels that are particularly commonly used are luminol, luminol isomers, imidazoles, acridinium salts, and oxalates.

Likewise, bioluminescent substances may be used to label the antibodies of the invention, whether fragments or derivatives. A bioluminescent substance is a chemiluminescent substance found in a biological system in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by the light it emits. Important bioluminescent compounds for labeling are luciferin, luciferase and sequorin.

Detection of antibody fragments or derivatives can be accomplished by scintillation counting if the label is a radioactive gamma emitter; by fluorimetry if the label is a fluorescent substance; in enzyme-labeled methods, by colorimetry To complete the detection, this method requires an enzyme substrate, or by comparing the degree of enzyme reaction with a pre-prepared standard and substrate.

In situ detection requires obtaining a histological specimen from the patient and preparing the specimen with either labeled antibody, or unlabeled antibody plus a labeled binding ligand. Through such a process, not only the presence of the antigen can be detected, but also its distribution in the tested tissue can be understood. Using the present invention, a person of ordinary skill can easily find that many histological methods (such as staining methods) can be modified for in situ detection. These methods include, for example, immunohistochemical staining. In a preferred embodiment, avidin-biotin immunoperoxidase staining can be used, a kit of which is under development.

The kit made with the monoclonal antibody or chimeric antibody of the present invention can be used to evaluate the immune tissue characteristics of colon cancer, breast cancer and ovarian cancer. The results of the histological studies were used to evaluate tumor cell subpopulations expressing the antigens of mAbs 31.1 and 33.28.

The Colon Cancer Detection Kit contains the following reagents for immunohistochemical analysis:

A. Monoclonal antibody 31.1, 33.28 or mouse/human chimeric antibody Chi#1, and monoclonal antibody to carcinoembryonic antigen (CEA), which is the standard monoclonal for colon tissue immunohistochemistry.

B. Immunoperoxidase reagent, (blocking reagent) such as goat serum; secondary antibody, such as goat anti-mouse antibody.

C, immunoperoxidase and.

D. Brown chromogenic reagent.

Similar kits are available for immunohistochemical analysis of breast and ovarian cancers. The immunoperoxidase technique was from Sternberger. The primary antibody (mAb or chimeric antibody) can be combined with multiple secondary antibodies as an antigen. The secondary antibody forms a "bridge" between the primary antibody and the horseradish peroxidase-antiperoxidase complex.

The kit presented here can be used to study changes in the colon, cancer, polyps to determine the degree of malignancy, to study whether transformation sites in benign polyps have been overlooked, to study enteritis to check for any undetected changes. Similar kits are available for research on breast and ovarian cancers.

Another kit, similar to the above, is planned, using five monoclonal antibodies for colon cancer, lesion typing and crossmatching for all tumor subpopulations.

The antibodies, fragments or derivatives of the present invention are suitable for use in immunoassays, also known as "double layer" assays or "sandwich" assays. In general immunoassay analysis, a certain amount of unlabeled antibody (or antibody fragments) is linked to a solid support, which is insoluble in the liquid to be tested, and labeled soluble antibodies are added, so that through the solid phase antibody, The detection of the complex formed by the three substances of antigen and labeled antibody can determine or quantitatively determine the analyte.

Typical and optimal immunoassay assays include "forward" assays. The antibody bound to the solid phase is first mixed with the sample to be tested, and the tumor antigen in the sample first forms a solid-phase antibody-CCAA complex with the antibody; after a period of stabilization, the solid phase support is washed to remove the remaining sample, including untreated Combined antigen; then add a solution containing an unknown amount of labeled antibody (as a reporter molecule); after stabilization, the solid phase support combined with the labeled antibody and CCAA is bound together by the unlabeled antibody, and the solid phase support is washed again, Remove unreacted labeled antibody. This method can simply determine the presence or absence of CCAA, or it can be quantified by comparing the measurement of the labeled antibody with a standard sample of known antigen quantity. This "two-layer" analysis method and "sandwich" analysis method can be found in the description of Wide (Radioimmune Assay Method, Kirkham, ed., E. & S. Livingstone, Edinburgh, 1970, pp.199-206).

Another "sandwich" assay that can be used to determine CCAA is known as the "simultaneous" and "reverse" assay. The simultaneous method includes an incubation process, that is, the antibody is bound to the solid support and the labeled antibody is also added to the sample to be tested at the same time. When incubation is complete, the solid support is washed to remove remaining liquid sample and unbound labeled antibody. As in the "forward" method described above, the labeled antibody bound to the solid support is measured.

In the reverse method, the labeled antibody is first added to the liquid sample, followed by the unlabeled antibody which, after a period of time, binds to the solid support. After the second incubation, the stationary phase is washed to remove the remaining sample to be tested and unbound labeled antibodies, and the combined labeled antibodies are determined by "synchronous" and "forward" analysis methods. In one example, the combined use of the present invention Antibody-specific isolated epitopes allow for the construction of a more sensitive three-position immunoradiometric assay.

To use the antibody of the present invention to detect colon cancer, breast cancer and ovarian cancer in vivo, ordinary skilled artisans can use different markers and different labeling methods. The types of labels that can be used for the antibodies of the invention are, for example, radioactive isotopes, paramagnetic isotopes, and PET (Positron Emission Tomography) imageable complexes. Those of ordinary skill will know of other labeling methods or will determine one by routine experimentation. Alternatively, conjugation labeling can be performed by the skilled artisan by standard techniques.

For in vivo diagnostic purposes, the choice of radionuclide depends on the available detection equipment. The decay of the selected radionuclide must be detectable by the available instruments. In general, any conventional method for visualizing diagnostic images can be used in the present invention.

Another important factor in the selection of diagnostic radionuclides for in vivo imaging systems is that the half-life of the radionuclide should be long enough so that it can still be detected when the target tissue has reached its maximum uptake; Radiation hazards are minimized. In a specific experiment, the radionuclides used for in vivo imaging do not emit particles, but generate a large number of photons in the 140-200 KeV range, which are easily detected with a gamma camera.

For in vivo diagnostic purposes, radionuclides can be directly or indirectly linked to antibodies through an intermediary functional group. The intermediary functional groups commonly used to bind radioisotopes in the form of metal ions are chelating agents, diethylene Triaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA). Metal ions that can be bound to the antibody of ^the present invention are, for example, ^99mTc , ^123I , ^111In , 131I, ^97Ru , ^67Cu , ^67Ga , ^125I , ^68Ga , ^72As , ^89Zr and ^201Tl .

mAbs 33.28 and 31.1, and the chimeric antibody Chi#1 can be used to expedite the generation of other mAbs that bind to the same antigen or immunologically cross-reactive colon cancer-associated antigens. First, these antibodies can be attached to a chromatography column and used to immunopurify colon cancer-associated antigens. These purified antigens in turn stimulated an immune response in some animals. Second, splenocytes from reactive animals can be fused with immortalized cells, and the resulting hybridomas can be screened for secreted antibodies that bind to purified antigens, and/or antibodies to colon cancer-associated antigens , which were competitively inhibited by 33.28 or 31.1 antibody or chimeric antibody Chi#1.

The above is a general description of our invention. Our invention can be further understood through some specific examples. These illustrations are only for the purpose of illustration and do not limit the scope of the present invention.

Embodiment 1

Preparation and Characterization of Colon Cancer-Associated Antigen

Antigen preparations were obtained from pooled colon carcinoma membranes as described by Hollinshead (Cancer, 56; 480 (1985)). This antigenic material is purified so that the membrane no longer contains HL-A antigen and most of the non-immunogenic glycoproteins are removed. The final state of this antigen preparation should be shown to induce delayed hypersensitivity reactions only in patients with active colon, breast and ovarian cancer.

Tumor cells from freshly removed specimens were made into saline suspension. Obtain a single cell suspension according to the traditional method, centrifuge at 400xg for 10 minutes, save the supernatant, resuspend the cell pellet, and centrifuge. Electron microscopy was used to confirm that only membrane substances existed instead of intact cells, and the contained proteins were determined by Lowry's method.

The membrane material is then sonicated at low frequency to make it a membrane protein suspension. The solution was separated by Sephadex-6200 gel filtration. Collect a tube of 2ml and record the absorption rate from 220 to 280um. Fractions from individual peak proteins were pooled and concentrated by Diafle ultrafiltration. IB and IIA were separated using Sephadex-G200, as defined by Hollinshead et al. (see supra); and further purified by gradient polyacrylamide gel electrophoresis (PAGE). The skin test was performed on patients with colon cancer to test whether the protein fragments prepared above could induce positive delayed hypersensitivity reactions. These immunogenic fragments contain colon cancer-associated antigen (CCAA) and are used as immunogens and screening materials for the preparation of mAbs.

After gradient PAGE, it was determined that it is related to carcinoembryonic antigen (Gold, P.et al., J.Exp.Med.122:467-481(1965); Hollinshead, A.et al., Cancer 56:480(1985)) The different double-banded antigens were separated, and tracer staining of the bands comprising this antigen showed lengths of 6.3 and 6.6 cm. Biochemical analysis of the antigen proved it to be a glycoprotein. The molecular weight of the antigen can be estimated from the electrophoretic mobility (6.4-6.5 cm) of transferrin with a molecular weight of 76.5 KDa.

Example II

             Monoclonal Antibody Preparation and Screening

BALB/C mice were immunized with CCAA as described above, splenocytes were dissected, fused and hybridized with mouse myeloma cells SP2/0-Ag14, and anti-human colon cancer-associated antigen (CCAA) antibodies could be obtained from their clones and products. Monoclonal antibodies.

Five hybrid clones were established, named 31.2, 31.1, 77, 33.23 and 33.28, as described below. All five mAbs reacted strongly to CCAA and two colon cancer cell lines (SW480 and SW620) by ELISA analysis, among which 31.1 and 33.28 were studied in detail.

A. Immunization and cell fusion

According to the description of Hollonshead's clinical trial (Hollinshead. et al,), BALB/C mice were taken and injected intraperitoneally with 50 μg CCAA emulsified in complete adjuvant. Ten days later, these mice were boosted with intravenous injection of the same dose of CCAA salt solution. Three days later, they were sacrificed and spleen cells were collected. 5×10 ⁷ mouse splenocytes and 10 ⁷ SP2/)Ag ¹⁴ myeloma cells were incubated in 40% polyethylene glycol (MW-1500) to fuse the cells.

B. Screening of hybrid clones

To detect hybridoma clones producing CCAA-specific antibodies, Tsang's enzyme-linked immunosorbent assay (Tsang et al, JNCI 77: 1175 (1986)) can be used. CCAA was immobilized on polystyrene microplates (100 ng/well). Antibodies in the supernatant bind to the immobilized antigen, plus a secondary antibody specific for mouse immunoglobulin conjugated to peroxidase to detect the conjugated murine mAb. This is followed by the addition of D-phenylenediamine, the chromogenic substrate for peroxidase. Wells with absorbance of chromogenic reaction ≥0.500 units were considered positive. An absorbance in the range of 0.01 to 0.09 is considered negative.

The positive hybridomas determined by ELISA were then screened by indirect immunofluorescence. The different tumor cells and normal cells that have been identified are listed in Table 1 below. All tumor cell lines were obtained from ATCC. Cells and cultured hybridoma cell supernatant were diluted with phosphate buffer at a ratio of 1:2 and co-incubated at 4° C. for 1 hour. Cells were washed and incubated with fluorescently labeled goat anti-mouse immunoglobulin antibody. Cells were washed three times with PBS and visualized by fluorescence microscopy. The results are listed in Table 1. Table 1 Reactivity of anti-colon cancer-associated antigen monoclonal antibodies and indirect immunofluorescence reactive cells of human cultured cells

31.2 77 31.1 33.28 33.23 The tumor cell line SW948 (color)-+--hct116 (color) ---+-widr (color)+++++color 320 (color)+++--hs619 (color)-- ----HS853 (color)------CACO-2 (color)++------SK-CO-1 (color)++-++HT-29 (color)++-++++SW1116 (coll )+-+++SW480 (color)+++++SW620 (color)++++231 (br)---CAMA-1 (br)--pan-(PAN) (PAN) -+-Mia (PAN)-+-HS766T (PAN)----M-14 (MEL)-----HT1080 (FIB) --- -LM (OS)---- - -TE-85(OS) - - - - - - - -

Normal dermal fibroblasts - - - - - - -

Bone marrow cells - - - - - - Normal human peripheral blood mononuclear cells - - - - - - -

A. The cultured cell suspension was diluted 1:2 with PBS

B. Determine positive (+) or negative (-) according to the membrane fluorescence intensity obtained from the control.

C.COL: colon cancer; BR: breast cancer; PAN: pancreatic cancer; FIB: fibrosarcoma

Example III

Analysis of monoclonal antibodies and their reactivity

The production and testing of the anti-CCAA monoclonal antibody described above also uses fresh human tissue to test its reactivity. The tissue types listed in Table 2 were placed in a cryostat, fixed with 3.5% formaldehyde phosphate buffer, and washed three times with PBS. In indirect immunofluorescence studies, tissues are incubated with mAbs and then stained with fluorescently-conjugated secondary antibodies.

As shown in Table 2, mAbs 31.1 and 33.28 are highly specific to colon cancer cells. This shows that the antigen used (CCAA) is highly specific to colon cancer, and it is an immunogen for colon cancer patients (DH reaction positive), and it is also a good immunogen for mouse phycoerythrin excitability test. The priming zone was established with cells positively detected against 90° light scatter. As shown in Table 4, both 31.1 and 33.28 mAbs can bind to colon cancer cells. The connection between the two and PBMC cells is not obvious. Table 2 Detection of anti-CCAA monoclonal antibody reactivity by indirect immunofluorescence on fresh human tissue

Reactivity of monoclonal antibodies

Organization 33.28 31.1

the tumor

Colon cancer 3/3 3/3

Pancreatic cancer 0/2 0/2

Melanoma 0/2 0/1

Breast cancer 0/2 0/1

normal tissue

Placenta 0/1 0/1

Liver 0/1 0/1

Colon 0/3 0/3

Spleen 0/1 0/1

Thymus 0/1 0/1

Muscle 0/1 0/1

a. Ascites was diluted 1:50 with PBS. Cryosections (4-6 μM thick) were fixed with 3.5% formalin in phosphate buffer for 10 minutes, and then washed three times with PBS. If not used immediately, store slices at -70°C.

The results are expressed as the number of positive test results/number of negative test results.

Table 3 lists the immunosorbent analysis results of monoclonal antibodies. Three colon cancer cell lines (HT-29, WIDR and SW620) and one osteosarcoma cell line (LM) were used to adsorb mAbs 31.1 and 33.28 conjugated to FITC. Ascites of mice inoculated with hybridomas was taken, diluted 1:50, and added to the adsorbed cells. The mixture was incubated at 4°C for 1 hour, and 2×10 ⁷ cells (A in Table 3) or 10 ⁴ cells (B in Table 3) were used to absorb the antibody (Table 3). Osteosarcoma cell lines had no activity of adsorbing 31.1 or 33.28, while colon cancer cells did.

Table 3 Immunosorbent analysis of monoclonal antibody

Suction Attached Cell Monoclonal Antibody HT-29 WIDR SW620 LM

A B A B A B A B33.28 - + + - + + - + + + +31.1 - + + - + - + +

The binding status of antibody 31.1, 33.28 and control monoclonal antibody to tumor cells HT29, WIDR and SW480 and peripheral blood mononuclear cells (PBMCs) can be obtained by cytofluorescence assay. Autofluorescence of Ortho Spectrum III cells with an argon laser to activate fluorescein and phycoerythrin. The priming zone was established with cells positively detected against 90° light scatter. As shown in Table 4, both 31.1 and 33.28 antibodies can bind to colon cancer cells, while monoclonal antibodies cannot bind to PBMC.

Table 4 Cytofluorescence analysis results

Monoclonal antibody staining percentage cells 33.28 31.1 control

HT29 51.0 53.9 9.2

WIDR 21.0 ND 8.1SW48037.0 32.0 3.8

PBMC 2.1 2.1 ND

The type of heavy and light chains of monoclonal antibodies can be determined by immunodiffusion. The 31.1 mAb is IgG1 with Kappa light chain, and the 33.28 mAb is IgG2a with Kappa light chain (Table 5). This is completely different from the past 19.9 monoclonal antibody (Herlyn, M. et al, J. Biol. Chem. 257:14365-14369 (1982)), which belongs to the IgG1 class (Herlyn D. et al, Proc. Natl. Acad. Sci. USA 79:4761-4765 (1982)). Importantly, antibodies of the IgG2a type are more effective for immunotherapy. (Cilcher, D. et al, Proc. Natl. Acad. Sci. USA. 78: 3199-3203 (1981)). 19.9 Monoclonal antibody is also reactive to colon tumors, but it is immunized with pancreatic cancer as an antigen , is cross-reactive to the colon. This is somewhat similar to the B72.3 monoclonal antibody (【citation】), which is an antibody obtained from breast cancer tissue as an immunogen, and is also reactive to the colon.

Table 5 Isotype Culture Suspension of Monoclonal Antibody IgG1 IgG2a IgG2b IgM Light Chain

Kabbah λ

31.2 - + + - - - + + -

31.1 + + - - - - + + -

77 - - - + + - - + + -

33.23 - + + - - - + + -

33.28 - + + - - - + + -

Example IV

Characterization of colon cancer-associated antigens

The molecular weight of the antigen bound by the above mAbs was determined by western blot analysis of soluble proteins from the SW480 and SW620 colon cancer cell lines. It was found that the antigens with molecular weights of 61.1KDa and 72KDa in these two cell lines could react with 33.28 and 31.1 mAbs. These mAbs did not react with human peripheral blood mononuclear cells or tumor cell lines from other tissue types in Western blot analysis.

In order to better determine the specificity of the monoclonal antibody of the present invention to the CCAA immune response, and establish whether the monoclonal antibody will react with the immunogen components in the cell membrane preparations that have been used in clinical treatment (Hollinshead et al, supra), the above The immunogenic preparations will be analyzed by high performance liquid chromatography (HPLC).

The analysis showed 4 more significant peaks, and the immunoreactivity (induced with DH) of each peak was observed by skin test in colon cancer patients. Of the 10 patients tested, only the protein from peak 4 was skin tested positive. The antigen of peak 4 reacted with 33.28 mAbs, while 31.1 mAbs reacted with the antigen of peak 3, which was the second most significant peak.

The references cited above are consistent with the results here.

Example V

Affinity purification of colon cancer-associated antigens

Antigens extracted from HT-29 cell line were separated by affinity chromatography with 33.28 mAb. 5 mg of purified 33.28 IgG was bound to CNBr-activated agarose gel electrophoresis 4B. The column was pre-eluted with 0.05M diethylamine at pH 11.5, and then equilibrated with 0.14M NaCl/0.01M Tris (pH8.0). The CCAA preparation was injected into the column, and then eluted with diethylamine at pH 11.5 0.05M. The eluted fraction was neutralized by adding IM Tris-HCl at pH 8.0.

The material bound to the 33.28 affinity matrix and eluted was then processed by HPLC. The eluted CCAA preparation was adjusted with a starting buffer (0.01M potassium phosphate buffer, pH7.0), added to a Synchropak Wax Weak anion exchange HPLC column (250×4.6mm), and prepared with 0.01M potassium phosphate buffer. - Gradient elution with 1M NaCl solution, the elution rate is 1ml/min. Anion exchange chromatography was performed using Hewlett-Packard HPLC (HP1090, HP, Arondals, PA)

The results are shown in Figure 2. The peak produced by the antigenic substance isolated from HT-29 cells with 33.28 mAb coincides with the fourth peak, and the immunoreactivity to humans is also similar, indicating that 33.28 mAb can be used to isolate the antigenic substance causing human infection. Immunized colon cancer preparations.

Example VI

    ADCC activity of monoclonal antibodies 33.28 and 31.1

To be therapeutically useful, an immunogenic tumor antigen-specific monoclonal antibody should possess the following characteristics: (A) high specificity for tumor tissue; (B) no cross-reactivity for normal human tissue; (C) Tumor-related biological activities, such as antibody-dependent cytotoxicity.

The ADCC activities of mAbs 33.28 and 31.1 were detected using the colon cancer cell line WIDR as target cells. The melanoma cell line M-14 was used as a specificity control. Analyzing ADCC adopts the traditional 51Cr 4-hour release analysis method, using human PBMC as effector cells, and the results are expressed by isotope release percentage (decomposition %) (Table 6). The base number of decomposition was 8.3%. When the ratio of effector cells to target cells was 100:1, 33.28 mAb led to tumor cell decomposition rate of 40.3%, while 31.1 mAb was 51.8%.

The ADCC activity of table 6 monoclonal antibody 33.28 and 31.1

Target cell release percentage (E:T ratio)

WIDR M-14 antibody or control 25 50 100 25 50 100

33.28 23.1 40.3 45.3 6.9 8.4 9.0

31.1 14.3 26.7 51.8 7.5 6.4 8.7

OSA1 10.0 9.2 12.2 11.4 14.8 10.9

NMS 12.2 11.7 13.1 14.2 15.0 11.1

PBS 8.2 5.1 7.6 11.0 14.2 10.5 ADCC was analyzed by ⁵¹ Cr 4 hour release assay. The base for ⁵¹ Cr release is 8.3%. The E:T ratio represents the ratio of effector cells to target cells. Monoclonal antibody or serum was tested after dilution of 1:100; OSA1-monoclonal antibody to osteosarcoma-associated antigen; NMS-normal mouse serum; WIDR-colon cancer cell line; M14-melanoma cell line.

Example VII

Detection of CCAA with mAbs 33.28 and 31.1 in circulating blood

Detection of circulating CCAA in 79 cases of unknown serum samples (Table 7) can be used to detect the monoclonal antibody of the present invention. This analysis is based on the principle that serum inhibits the binding of mAbs to CCAA in an ELISA assay. None of the 50 normal sera showed false positives, 9 of the 10 active colon cancer patients had positive sera, and none of the colon cancer cases were positive after one year of cure.

Table 7 Detection of circulating colon cancer-associated antigens

(the amount of serum inhibiting the binding of monoclonal antibody to CCAA)

                                                                       

Donor Samples ＜15% ＞15% ＜15% ＞15%

(Negative) (Positive) (Negative) (Positive) Colon cancer 10 7 3 2 8 Colon cancer (resection) 4 4 0 0 4

Breast cancer 9 9 0 9 0 melanoma 5 0 5 0 prostate cancer 1 0 1 0 normal serum 50 50 0 50 0 0

Colon cancer-associated antigens were detected by ELISA. 100 μl of serum was taken for each analysis.

Example VIII

Comparison of the specificity of other mAbs reactive to colon cancer

Tumor specificity was further investigated by ELISA method (Table 8). Mab 31.1 was compared with CC49, a colorectal cancer-specific monoclonal antibody purified from B-72.3, and mouse myeloma protein was used as a control. The 31.1 mAb had a smaller range of reactivity with colorectal cancer than CC49, but it was more specific and had less or no cross-reactivity with gastric tumors or normal colon tissue. Table 8 ELISA results of normal and tumor tissues using mAb 31.1, CC49 and MOPC-21

Organization 31.1 CC49 MOPC-21

colorectal cancer

1. COCA2A - +++ -

2. COCA2 - +++ -

3. COCA3 + + ++ ++ -

4. COCA4 +++ +++ -

5.G820 ± ++ ++ -

6.G853 +++ ++ ++ -

7.G817 +++ +++ -

8.G781 - - - -

other cancer

1. Breast cancer 1 - - - -

2. Breast cancer 2 - - - -

3. Lung cancer 1 - - - -

4. Lung cancer 2 - - - -

5. Ovarian cancer D106 - - - -

6. Ovarian cancer 5 - - - -

7. Ovarian cancer V5 - - - -

8. Ovarian cancer V45 - - - -

9. Ovarian cancer V43 - - - -

10. Gastric cancer 14A ++ +++ -

11. Gastric cancer 12A - +++ -

12. Gastric cancer 15A - - - -

other normal tissues

1. Endometrium E21 - - - -2. Endometrium EC19 - - - -3. Endometrium EC17 - + -4. Endometrium EC18 - - - - -

(RBC)

5. Red blood cell 1 - - - -

6. Red blood cell 2 - - - -

7. Red blood cell 3 - - - -

8. Erythrocyte 4 - - - -

9. Erythrocyte 5 - - - -

10. Red blood cell 6 - - - -

11. Red blood cell 7 - - - -

12. Red blood cells 8 - - - -

13. Red blood cell 9 - - - -

14. Red blood cells 10 - - - -

15. Erythrocytes 11 - - - -

16. Granulocytes - - - -

17.385 - - - -

18.386 - - - -

19. Normal spleen 3 - - - -20.392 (normal spleen) - - - -21.395 (normal liver) - - - -22.387 (normal kidney) - - - -23.398 (normal spleen) - - - liver ) - 24.390 (normal

25. Normal Spleen #1 - - - -

26. Normal Spleen #2 - - - -27.800 (Normal Colon) - ++ -28. Normal Colon (GW) - ++ -29. Normal Colon (Meloy) - - - -

30. Normal colon - - - -

31.G1155B (normal colon) - - - -

32.G1164B (normal colon) - - - -

33. Normal colon ± - -

34. Normal stomach A - - - -

35. Normal stomach B - - - -

36. Normal stomach C - - - -

37. Normal lung - - - -

38. Normal liver - - - -

CC49-NCI Colorectal Monoclonal Antibody

MOPC-21-Myeloma Protein Negative Control

All monoclonal antibodies are 40ng/well, POGAM dilution is 1:3000

Example IX

Distribution of mAbs 33.28 and 31.1 at tumor sites in vivo

The distribution behavior of the monoclonal antibody of the present invention in vivo is obtained after studying the pharmacokinetics of ^{the 125} I-labeled monoclonal antibody and athymic nude mice transplanted with colon cancer LS-174T. Mice implanted with melanin A375 served as controls. Radiation site index (concentration of radioactive marker in tumor site/concentration of radioactive marker in surrounding tissue) indicates the relative concentration of monoclonal antibody in the tumor relative to surrounding tissues (liver and spleen). Table-9 is the biodistribution of ¹²⁵ I-labeled mAbs 31.1 and 33.28. It was found that the monoclonal antibody was significantly concentrated in the tumor site, much higher than the distribution in normal tissues (liver and spleen), which was 6 times that of normal tissues at 96 hours and 12 times at 168 hours. Figure 3 and Figure 4 are the radiation distribution indexes of monoclonal antibody in tumor tissue, blood and liver. Table 9 Biodistribution of ¹²⁵ I-labeled monoclonal antibody in tumor-bearing athymic nude mice

Organization 96 hours 168 hours

LS174T A375 LS1745T A375A.mAb 31.1

Blood 7.30 NA 4.67 4.42

Tumor 21.92 NA 25.43 2.91

Liver 3.74 NA 2.16 1.24

Spleen 3.68 NA 2.41 1.32B.mAb 33.28

Blood 7.80 NA 5.58 3.95

Tumor 13.12 NA 15.50 2.24

Liver 2.55 NA 1.74 1.68

Spleen 2.31 NA 1.70 1.92 The results are expressed by injected dose/tissue weight (grams) LS174T-colon cancer; A375-melanoma

Example X

  Immunohistochemical studies

Using the ability of specific mAbs to selectively define tumor populations, tumor markers can be identified in serum by immunohistochemistry to delineate the neoplastic process and characterize the neoplastic cell population.

Mabs 31.1 and 33.28 were tested with more than 50 colon cancers using immunoperoxidase staining. The experiments found that the two mAbs had high reactivity to colonic neoplasia and low reactivity to adjacent normal tissue. When checking for monoclonal antibody reactivity to polyps, benign sites such as villous-tubular adenomas were unresponsive to the unit. The reaction with monoclonal antibody appeared only in the already malignant adenoma site. When similar tissues were examined with common carbohydrate-antigen-induced monoclonal antibodies, it was found that both normal and neonatal tissues reacted with this monoclonal antibody. In the experiment of mAb 3.11 and 33.28, it was found that the two mAbs reacted with different populations of cells in the tumor tissue respectively. This suggests that different oncogenes produce different cell surface antigens.

Example XI

Selectively binds epithelial subpopulations of paraffin-embedded benign and malignant breast tissue

Mabs 3.11 and 33.28 were studied by avidin-biotin staining in 41 formalin-fixed, paraffin-embedded benign and malignant breast specimens. Both antibodies stained positively on the cell surface and in the cytoplasm before enzyme treatment. Monoclonal antibody 31.1 was positive for 33% of ductal carcinogens and 25% of benign breast disease specimens; monoclonal antibody 33.28 was positive for 48% of ductal carcinomas and 35% of benign breast disease specimens. 10-75% of fine and medium groups stain positive. These results indicate that the antigens defined by mAbs 3.11 and 33.28 are selectively expressed in a subset of women with breast disease, which will help in the diagnosis of this disease.

Example XII

Selectively binds subpopulations of epithelial cells from fresh-frozen benign and malignant ovarian tumors

MAbs 31.1 and 33.28 were studied in fresh-frozen biopsies from 21 ovarian tumors using an avidin-biotin indirect immunoperoxidase assay. As a result, 4/7 and papillary mucinous adenomas, 1/1 mucin-stimulated adenomas, and 1/2 endometrioid adenomas had focal positive staining. And no non-epithelial ovarian tumors were positive for monoclonal antibody staining. These results indicate that the antigens defined by mAbs 31.1 and 33.28 are selectively expressed in a subset of women with ovarian cancer, which will also help in the diagnosis of this disease.

Example XIII

Immunoreactivity of human colon cancer tissues and cell lines to monoclonal antibodies 31.1 and 33.28

Mabs 31.1 and 33.28 were used to examine the immunoreactivity of cell lines including colon adenocarcinoma, lymphoma, leukemia and neuroblastoma.

Using avidin-biotin immunoperoxidase staining, it was found that monoclonal antibodies 31.1 and 33.28 could strongly bind to colon adenocarcinoma cell lines, WIDR and HT-29, while in cell lines KGI-a, HL-60 , Molt-3 and JUKRAT have no response. Both antibodies reacted weakly to the lymphoma cell line JY. Monoclonal antibody 33.28 also had a weak reaction with leukemia cell line K562 and neuroblastoma cell line U87, MG. These results are consistent with those of flow cytometry and immunofluorescence reactions described previously. The monoclonal antibody can only react with colon adenocarcinoma cell lines, but not with other tumor cell lines, but not with SKBR-3 breast cancer cells.

Both monoclonal antibodies showed the characteristic of reacting only with colon adenocarcinoma tissue (mab 33.28 reacted with 84% cells, monoclonal antibody 31.1 reacted with 64% cells), but with normal tissue or lymphoma, leukemia and adult Neuroblastomas did not respond. These results demonstrate that these antibodies can be used as tools for investigating tumor markers and cell biology studies.

Example XIV

Expression and characterization of chimeric antibodies against human colorectal cancer-associated antigens

The chimeric mouse/human heavy chain gene was obtained by splicing the exons of the variable region of the heavy chain of antibody 31.1 to the constant region of the human gammal chain by PCR. Subsequently, the chimeric antibody gene of 31.1 was cloned into a retrovirus expression vector plgpCXII and transfected into cell line PA317. Transfected cell PA317H was cultured with another cell, PA317, containing an unrelated mouse/chimeric light chain gene expressed in the retroviral expression vector plneoCXII and in SP2/00-Ag14 cells. Transduced SP2/-A14 cells were able to produce intact chimeric antibody CH#1, which reacted with horseradish peroxidase-conjugated goat anti-human 1gGFc fragment in an enzyme-linked reaction, which demonstrated the constant expression of Ch#1 Districts are human species. Cytofluorometric analysis showed that Ch#1 stained human colorectal cancer cell lines HT-29 and LS174T, but not human lung cancer cell lines. Antibody-dependent cell-mediated cytotoxicity assay (ADCC) showed that Ch#1 was able to lyse Ls174T cells. These results indicate that Ch#1 has the ability to bind to the specific antigen of mouse 31.1 mAb, which further indicates that this chimeric antibody can play a role in the prognosis of colon cancer.

We have described the invention in detail above, and this technique can be used within the same parameters, concentrations and conditions that have been experimentally confirmed.

While this invention has been described as limited to specific embodiments, it should also be understood that it can be modified. The important features mentioned in the application of the invention, and the scope of the claims set forth below, are intended to cover actions which would change the variation, use and scope of the invention.

Claims (112)

1. a specificity to the monoclonal antibody of human colon cancer-associated protein antigen, described antigen has following characteristics: (1). The monoclonal antibody recognizes the epitope of the protein component of the antigen, rather than the epitope of the sugar component of the antigen; (2). The antigen cannot be found in normal human tissues; (3). The antigen cannot be found on human cancer cells other than colon cancer cells; and (4). The antigen is specifically immunogenic to humans. 2. The antibody according to claim 1, which is mouse monoclonal antibody 33.28 or an antibody which specifically binds to the same colon cancer-associated epitope as 33.28. 3. The antibody according to claim 2, said colon cancer-associated antigen is a protein having a molecular weight of about 61.1 kD. 4. The antibody according to claim 1, which is mouse monoclonal antibody 31.1 or an antibody that specifically binds to the same colon cancer-associated epitope as mouse monoclonal antibody 31.1. 5. The antibody according to claim 4, said colon cancer-associated antigen is a protein with a molecular weight of about 72KD, said colon cancer-associated antigen is a glycoprotein, and its protein component has a molecular weight of 61.1KD. 6. The antibody according to claim 1, immobilized on a solid phase. 7. The antibody according to claim 1, which is a labeled detectable monoclonal antibody. 8. The antibody according to claim 7, said detectable label being an isotopic label. 9. The antibody according to claim 1, conjugated to a cytotoxic radionuclide. 10. The antibody according to claim 1, conjugated to a cytotoxic drug. 11. The antibody according to claim 1, which binds to a cytotoxic protein. 12. A pharmaceutical composition for immunotherapy of human colon cancer, breast cancer and ovarian cancer, comprising an antibody according to claim 9, combined with a pharmaceutically acceptable carrier. 13. A drug composition for immunotherapy of human colon cancer, breast cancer and ovarian cancer, comprising a therapeutically effective amount of an antibody according to claim 10, combined with a pharmaceutically acceptable carrier. 14. A drug composition for immunotherapy of human colon cancer, breast cancer and ovarian cancer, comprising a therapeutically effective amount of an antibody according to claim 11, combined with a pharmaceutically acceptable carrier. 15. A human colon cancer-associated protein antigen having the following properties: (1). The epitope of the protein component of the antigen can produce antibodies in the lactating host, while the epitope of the sugar component of the antigen cannot produce the antibody; (2). The antigen cannot be found in normal human tissues; (3). The antigen cannot be found on human cancer cells other than colon cancer cells; and (4). The antigen is specifically immunogenic to humans. 16. An antigen according to claim 15 which is a protein having a molecular weight of about 61 kD. 17. The antigen of claim 15 which is a protein having a molecular weight of about 72 kD. 18. The antigen according to claim 16, which specifically binds to the mouse monoclonal antibody 33.28, or an antibody that specifically binds to the same colon cancer-associated epitope as the mouse monoclonal antibody 33.28 combined. 19. The antigen according to claim 17, which can specifically bind to the mouse monoclonal antibody 31.1, or can specifically bind to the same colon cancer-associated epitope as the mouse monoclonal antibody 31.1 antibody binding. 20. A monoclonal antibody raised against the monoclonal antibody of claim 1. 21. A monoclonal antibody raised against the monoclonal antibody of claim 2. 22. A monoclonal antibody raised against the monoclonal antibody of claim 3. 23. A monoclonal antibody raised against the monoclonal antibody of claim 4. 24. A monoclonal antibody raised against the monoclonal antibody of claim 5. 25. An immunoassay method for detecting colon cancer-associated antigens capable of binding to mouse monoclonal antibody 33.28 in a sample comprising: (1). Contacting said sample with an antibody according to claim 1; and (2). The antigen is detected by detecting the bound antibody. 26. An immunoassay method for detecting a colon cancer-associated antigen capable of binding to mouse monoclonal antibody 31.1 in a sample comprising: (1). Contacting said sample with an antibody according to claim 1; and (2). The antigen is detected by detecting the bound antibody. 27. An imaging method for detecting a colon cancer-associated antigen in a host comprising: (1) contacting said animal with a labeled antibody according to claim 7; and (2). Detection of the antigen. 28. A method of killing cells bearing a colon cancer-associated antigen comprising delivering to said cells an effective amount of the antibody of claim 1 and a cytotoxic effector agent. 29. A method according to claim 28, said effector agent being selected from a group of effector cells containing complement and having antibody-dependent cytotoxicity. 30. A method of killing cells bearing a colon cancer-associated antigen comprising delivering an antibody according to claim 9 to said cells. 31. A method of killing cells bearing a colon cancer-associated antigen comprising delivering an antibody according to claim 10 to said cells. 32. A method of killing cells bearing a colon cancer-associated antigen comprising delivering an antibody according to claim 11 to said cells. 33. A method of treating a patient suspected of having colon cancer bearing an antigen capable of binding to mouse monoclonal antibody 33.28 or 31.1 comprising administering to said patient an effective amount of the composition of claim 12. 34. A method of treating a patient suspected of having colon cancer bearing an antigen capable of binding to mouse monoclonal antibody 33.28 or 31.1 comprising administering to said patient an effective amount of the composition of claim 13. 35. A method of treating a patient suspected of having colon cancer bearing an antigen capable of binding to mouse monoclonal antibody 33.28 or 31.1 comprising administering to said patient an effective amount of the composition of claim 14. 36. A method of producing an immunogenic component for clinical immunotherapy of human colon cancer, comprising: (a) preparing a cell membrane extract of a tumor or cell line containing an antigen that binds to mouse monoclonal antibody 33.28 or 31.1; and (b) isolating said antigen by affinity purification using the antibody according to claim 6, so that said fraction can be produced. 37. An immune vaccine against human colon cancer, comprising an effective amount of the antigen according to claim 15 or its active fragment coupled with conventional vaccine vectors. 38. An immune vaccine against human colon cancer, comprising an effective amount of human colon cancer-associated antigen or its active fragment combined with traditional vaccine carrier and capable of binding to mouse monoclonal antibody 33.28 or 31.1. 39. An immunotherapy method for human colon cancer, comprising giving the patient a vaccine containing an effective dose of the antigen according to claim 15 or its active fragment linked with a traditional vaccine carrier as needed. 40. An immunotherapy method for human colon cancer, comprising giving the patient a vaccine containing an effective dose of human colon cancer-associated antigen or its vaccine combined with a traditional vaccine carrier and capable of binding to mouse monoclonal antibody 33.28 or 31.1 as needed active fragment. 41. The vaccine according to claim 37, the dosage effective range is about 0.001-100 mg antigen/kg body weight. 42. The vaccine according to claim 38, the dosage effective range is about 0.001-100 mg antigen/kg body weight. 43. A method of diagnosing human colon cancer comprising: (a) a histological specimen taken from a patient suspected of having colon cancer bearing an antigen according to claim 15; (b) contacting the specimen with a monoclonal antibody that recognizes said antigen; (c) Immunohistochemical staining of the specimen; and (d) Detection of the presence of antigen-antibody complexes by staining. 44. A method of diagnosing human colon cancer comprising: (a) a histological specimen taken from a patient suspected of having colon cancer with an antigen that binds to mouse monoclonal antibody 33.28 or 31.1; (b) contacting the specimen with a mouse monoclonal antibody that recognizes the antigen; (c) subjecting the specimen to immunohistochemical staining; and (d) Detecting the presence of antigen-antibody complexes. 45. The method of claim 43, wherein said staining is an avidin-biotin immunoperoxidase staining. 46. The method of claim 44, wherein said staining is an avidin-biotin immunoperoxidase staining. 47. A kit for immunohistochemical detection of colon cancer comprising: (a) mouse monoclonal antibodies 33.28 and 31.1 and monoclonal antibodies to carcinoembryonic antigen; (b) immunoperoxidase and secondary antibody reagents; (c) immunoperoxidase; and (d) Stains. 48. A kit for immunohistochemical detection of colon cancer comprising: (a) mouse monoclonal antibodies 31.1, 31.2, 33.23, 33.28 and 77 and monoclonal antibodies to carcinoembryonic antigen; (b) immunoperoxidase and secondary antibody reagents; (c) immunoperoxidase; and (d) Stains. 49. A compartmentalized kit for the detection of an antigen according to claim 15, said kit comprising: a first container containing an antibody or an active ingredient of said antigen and two containers containing said antigen. A second container for the antibody or active ingredient, said secondary antibody being labeled with a reporter molecule giving a detectable signal. 50. The kit according to claim 49, wherein said reporter molecule is a radioisotope, an enzyme, a fluorescent molecule, a chemiluminescent molecule or a bioluminescent molecule. 51. The kit of claim 49, said reporter molecule being an enzyme. 52. A kit according to claim 49, said kit further comprising a third container containing an enzyme-containing substrate. 53. A monoclonal antibody specific to human colon cancer-associated protein antigen, said antigen having the following characteristics: (i) The monoclonal antibody recognizes the epitope of the protein component of the antigen, rather than the epitope of the sugar component of the antigen. (ii) the antigen cannot be found in normal human tissue; (iii) the antigen is not found on human cancer cells other than breast cancer cells, and (iv) The antigen is specifically immunogenic to humans. 54. The antibody according to claim 53 which is mouse monoclonal antibody 33.28 or an antibody which specifically binds to the same colon cancer-associated epitope as 33.28. 55. The antibody according to claim 54, said colon cancer associated antigen is a protein having a molecular weight of about 61.1 kD. 56. The antibody according to claim 53, which is mouse monoclonal antibody 31.1 or an antibody that specifically binds to the same colon cancer-associated epitope as 31.1. 57. The antibody according to claim 53, said colon cancer-associated antigen is a protein with a molecular weight of about 72KD, said colon cancer-associated antigen is a glycoprotein, and the molecular weight of its protein component is 61.1KD. 58. A pharmaceutical composition for immunotherapy of human breast cancer, comprising a therapeutically effective amount of an antibody according to claim 53 combined with a pharmaceutically acceptable carrier. 59. A human colon cancer-associated protein antigen having the following characteristics: (i) the epitope of the protein component of the antigen can produce antibodies in a lactating host, while the epitope of the sugar component of the antigen cannot produce the antibody; (ii) the antigen cannot be found in normal human tissue; (iii) said antigen is not found on cells of human cancer groups other than breast cancer cells; and (iv) The antigen is specifically immunogenic to humans. 60. The antigen of claim 59 which is a protein having a molecular weight of about 61 kD. 61. The antigen of claim 59 which is a protein having a molecular weight of about 72 kD. 62. The antigen according to claim 60, said antigen specifically binding to mouse monoclonal antibody 33.28 or an antibody specifically binding to the same colon cancer-associated epitope as mouse monoclonal antibody 33.28 combined. 63. The antigen according to claim 61, which is an antibody that specifically binds to the mouse monoclonal antibody 31.1 or can specifically bind to the same colon cancer-associated epitope as the mouse monoclonal antibody 31.1 combined. 64. A monoclonal antibody against the monoclonal antibody according to claim 53. 65. An immunoassay for detecting colon cancer-associated antigens capable of binding to mouse monoclonal antibody 33.28 in a sample comprising: (a) contacting said sample with an antibody according to claim 53; and (b) detecting the antigen by detecting antibody binding. 66. An immunoassay for detecting colon cancer-associated antigens capable of binding to mouse monoclonal antibody 31.1 in a sample comprising: (a) contacting said sample with an antibody according to claim 53; and (b) detecting the antigen by detecting antibody binding. 67. A method of killing cells bearing a colon cancer-associated antigen comprising delivering to said cells an effective amount of an antibody according to claim 53 and a cytotoxic effector agent. 68. A method of treating a patient suspected of having breast cancer bearing an antigen capable of binding to mouse clone antibody 33.28 or 31.1 comprising administering to said patient an effective amount of the composition of claim 58. 69. An immune vaccine against human breast cancer, comprising an effective amount of the antigen according to claim 59 or its active fragment linked with a traditional vaccine carrier. 70. An immune vaccine against human breast cancer, comprising an effective amount of human colon cancer-associated antigen or its active fragment combined with traditional vaccine carrier and capable of binding to mouse monoclonal antibody 33.28 or 31.1. 71. An immunotherapy method for human breast cancer, comprising administering to the patient a vaccine containing an effective amount of the antigen according to claim 59 or its active fragment coupled with a traditional vaccine carrier as needed. 72. An immunotherapy method for human breast cancer, comprising giving the patient a vaccine containing an effective amount of human colon cancer-associated antigen or its activity combined with a traditional vaccine carrier and capable of binding to mouse monoclonal antibody 33.28 or 31.1 as needed fragment. 73. A method of diagnosing human breast cancer comprising: (a) a histological specimen taken from a patient suspected of having breast cancer bearing an antigen according to claim 59; (b) contacting the specimen with a monoclonal antibody that recognizes said antigen; (c) Immunohistochemical staining of the specimen; and (d) Detection of the presence of antigen-antibody complexes by staining. 74. A method of diagnosing human breast cancer comprising: (a) a histological specimen taken from a patient suspected of having breast cancer with an antigen that binds to mouse monoclonal antibody 33.28 or 31.1; (b) contacting the specimen with a mouse monoclonal antibody that recognizes the antigen; (c) Immunohistochemical staining of the specimen; and (d) Detecting the presence of antigen-antibody complexes. 75. A kit for immunohistochemical detection of breast cancer, comprising: (a) mouse monoclonal antibodies 31.1 and 33.28 and monoclonal antibodies to carcinoembryonic antigen; (b) immunoperoxidase and secondary antibody reagents; (c) immunoperoxidase; and (d) Stains. 76. A monoclonal antibody specific to human colon cancer-associated protein antigen, said antigen having the following properties: (i) the monoclonal antibody recognizes the epitope of the protein component of the antigen, rather than the sugar component epitope of the antigen; (ii) the antigen cannot be found in normal human tissue; (iii) the antigen is not found on human cancer cells other than ovarian cancer cells; and (iv) The antigen is specifically immunogenic to humans. 77. The antibody according to claim 76 which is mouse monoclonal antibody 33.28 or an antibody which binds specifically to the same colon cancer-associated epitope as 33.28. 78. The antibody according to claim 77, said colon cancer associated antigen is a protein having a molecular weight of about 61.1 kD. 79. The antibody of claim 76, which is mouse monoclonal antibody 31.1 or an antibody that specifically binds to the same colon cancer-associated epitope as mouse monoclonal antibody 31.1. 80. The antibody according to claim 79, said colon cancer-associated antigen is a protein having a molecular weight of about 72KD, said colon cancer-associated antigen is a glycoprotein, and the molecular weight of its protein component is 61.1KD. 81. A pharmaceutical composition for immunotherapy of human ovarian cancer, comprising a therapeutically effective amount of an antibody according to claim 76 combined with a pharmaceutically acceptable carrier. 82. A human colon cancer-associated protein antigen having the following characteristics: (i) the epitope of the protein component of the antigen can produce antibodies in a lactating host, while the epitope of the sugar component of the antigen cannot produce the antibody; (ii) the antigen cannot be found in normal human tissue; (iii) the antigen is not found on human cancer cells other than ovarian cancer cells; and (iv) the antigen is specifically immunogenic to humans. 83. The antigen of claim 82 which is a protein having a molecular weight of about 61 kD. 84. The antigen of claim 82 which is a protein having a molecular weight of about 72 kD. 85. The antigen according to claim 83, which specifically binds to mouse monoclonal antibody 33.28 or an antibody that specifically binds to the same colon cancer-associated epitope as mouse monoclonal antibody 33.28 combined. 86. The antigen according to claim 84, said antigen specifically binding to mouse monoclonal antibody 31.1 or an antibody that specifically binds to the same colon cancer-associated epitope as mouse monoclonal antibody 31.1 combined. 87. A monoclonal antibody raised against the monoclonal antibody of claim 76. 88. An immunoassay for detecting colon cancer-associated antigens capable of binding to mouse monoclonal antibody 33.28 in a sample, comprising: (a) contacting said sample with an antibody according to claim 76; and (b) detecting the antigen by detecting antibody binding. 89. An immunoassay for detecting colon cancer-associated antigens capable of binding to mouse monoclonal antibody 31.1 in a sample, comprising: (a) contacting said sample with an antibody according to claim 76; and (b) detecting the antigen by detecting antibody binding. 90. A method of killing cells bearing a colon cancer-associated antigen comprising delivering to said cells an effective amount of an antibody according to claim 76 and a cytotoxic effector agent. 91. A method of treating a patient suspected of having ovarian cancer bearing an antigen capable of binding to mouse monoclonal antibody 33.28 or 31.1 comprising administering to said patient an effective amount of a composition according to claim 81. 92. An immune vaccine against human ovarian cancer, comprising an effective amount of the antigen according to claim 82 or its active fragment coupled with conventional vaccine vectors. 93. An immune vaccine against human ovarian cancer, comprising an effective amount of human colon cancer-associated antigen or its active fragment combined with traditional vaccine carrier and capable of binding to mouse monoclonal antibody 33.28 or 31.1. 94. An immunotherapy method for human ovarian cancer, comprising administering to the patient as needed a vaccine containing an effective dose of the antigen according to claim 82 or its active fragment linked to a conventional vaccine carrier. 95. An immunotherapy method for human ovarian cancer, comprising giving the patient a vaccine containing an effective dose of human colon cancer-associated antigen or its vaccine combined with a traditional vaccine carrier and capable of binding to mouse monoclonal antibody 33.28 or 31.1 as needed active fragment. 96. A method of diagnosing human ovarian cancer comprising: (a) a histological specimen taken from a patient suspected of having ovarian cancer bearing an antigen according to claim 82; (b) contacting the specimen with a monoclonal antibody that recognizes said antigen; (c) Immunohistochemical staining of the specimen; and (d) Detection of the presence of antigen-antibody complexes by staining. 97. A method of diagnosing human ovarian cancer comprising: (a) a histological specimen taken from a patient suspected of having ovarian cancer bearing an antigen that binds to mouse monoclonal antibody 33.28 or 31.1; (b) contacting the specimen with a mouse monoclonal antibody that recognizes the antigen; (c) Immunohistochemical staining of the specimen; and (d) Detecting the presence of antigen-antibody complexes. 98. A kit for immunohistochemical detection of ovarian cancer, comprising: (a) mouse monoclonal antibodies 31.1 and 33.28 and monoclonal antibodies to carcinoembryonic antigen; (b) immunoperoxidase and secondary antibody reagents; (c) immunoperoxidase; and (d) Stains. 99. A chimeric antibody specific to human colon cancer-associated protein antigen, said antigen having the following characteristics: (i) the monoclonal antibody recognizes an epitope of the protein component of the antigen, rather than an epitope of the carbohydrate component of the antigen; (ii) the antigen cannot be found in normal human tissue; (iii) the antigen is not found on human cancer cells other than colon cancer cells; and (iv) The antigen is specifically immunogenic to humans. 100. A chimeric antibody according to claim 99, which is mouse/human chimeric antibody Chi#1. 101. The chimeric antibody according to claim 100, said colon cancer associated antigen is a protein having a molecular weight of about 72KD. 102. A pharmaceutical composition for immunotherapy of human colon cancer, comprising a therapeutically effective amount of an antibody according to claim 99, combined with a pharmaceutically acceptable carrier. 103. The antigen according to claim 101, wherein said antigen specifically binds to chimeric antibody Chi#1 or a chimeric antibody that specifically binds to the same colon cancer-associated epitope as chimeric antibody Chi#1 antibody binding. 104. A monoclonal antibody raised against the chimeric antibody according to claim 99. 105. An immunoassay for detecting a colon cancer-associated antigen capable of binding to a mouse/human chimeric antibody Chi#1 in a sample, comprising: (a) contacting said sample with an antibody according to claim 99; and (b) detecting the antigen by detecting antibody binding. 106. A method of killing cells bearing a colon cancer-associated antigen comprising delivering to said cells an effective amount of an antibody according to claim 99 and a cytotoxic effector agent. 107. A method of treating a patient suspected of having colon cancer bearing an antigen capable of binding to mouse/human chimeric antibody Chi#1 comprising administering to said patient an effective amount of the composition of claim 102. 108. An immune vaccine against human colon cancer, comprising an effective amount of human colon cancer-associated antigen or its active fragment coupled with a traditional vaccine carrier and capable of binding to mouse/human chimeric antibody Chi#1. 109. An immunotherapy method for human colon cancer, comprising giving the patient a vaccine containing an effective amount of human colon cancer-associated antigen or a vaccine combined with a traditional vaccine carrier and capable of combining with mouse/human chimeric antibody Chi#1 of its active fragment. 110. A method of diagnosing human colon cancer comprising: (a) a histological specimen taken from a patient suspected of having colon cancer bearing an antigen according to claim 15; (b) contacting the specimen with a chimeric antibody that recognizes said antigen; (c) Immunohistochemical staining of the specimen; and (d) Detection of the presence of antigen-antibody complexes by staining. 111. A method of diagnosing colon cancer in a human, comprising: (a) a histological specimen taken from a patient suspected of having colon cancer with an antigen that binds to the mouse/human chimeric antibody Chi#1; (b) contacting the specimen with a mouse/human chimeric antibody that recognizes said antigen; (c) Immunohistochemical staining of the specimen; and (d) Detecting the presence of antigen-antibody complexes. 112. A kit for immunohistochemical detection of colon cancer, comprising: (a) Chimeric antibody of mouse/human chimeric antibody Chi#1 and carcinoembryonic antigen; (b) immunoperoxidase and secondary antibody reagents; (c) immunoperoxidase; and (d) Stains.