JP2003500072A - Cancer-related drugs and their use - Google Patents

Abstract

  (57) [Summary] Cancer-associated antigens were identified by autologous antibody screening of a library of nucleic acids expressed in breast cancer and / or T cell leukemia cells and prostate cancer cells using antisera from cancer patients. The present invention relates to nucleic acids and encoded polypeptides that are cancer-associated antigens expressed in patients with cancer. The invention provides, inter alia, isolated nucleic acid molecules, expression vectors containing those molecules, and host cells transfected with those molecules. The invention also provides isolated proteins and peptides, antibodies to those proteins and peptides, and cytotoxic T lymphocytes that recognize the proteins and peptides. Fragments of the above, including functional fragments and variants are also provided. Kits containing the molecules described above are additionally provided. The molecules provided by the invention can be used for diagnosing, monitoring, searching for or treating conditions characterized by the expression of one or more cancer-associated antigens.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to nucleic acids and encoded polypeptides that are cancer-associated antigens expressed in patients with breast cancer, T-cell leukemia.

The invention also relates to agents that bind nucleic acids or polypeptides. Nucleic acid molecules, polypeptides encoded by such molecules and peptides derived therefrom, and related antibodies and cytolytic T cells are especially useful in diagnostic therapeutic contexts.

BACKGROUND OF THE INVENTION The mechanism by which T cells recognize foreign substances has been implicated in cancer. A number of cytolytic T lymphocyte (CTL) clones directed against autologous melanoma antigens, testis antigens and melanocyte differentiation antigens have been described. Often, the antigens recognized by these clones have been characterized.

[0004] The use of autologous CTL to identify tumor antigens has led to the target cells expressing the antigen in
Autologous CT that can be cultured in vitro and recognizes antigen-expressing cells
It requires that a stable strain of the L clone be isolated and capable of expansion.

This approach has worked well with melanoma antigens, while other types of tumors, such as epithelial cancers including breast and colon cancer, have proven refractory to this approach. .

More recently, another approach to this problem has been proposed by Sahin et al. (Proc. Natl. Acad.
Sci. USA 92: 11810-11813, 1995). According to this approach, autologous antisera are used to identify immunogenic protein antigens expressed in cancer cells by screening an expression library constructed from tumor cell cDNA. The antigen-encoding clones so identified were found to elicit a high titer humoral immune response in patients for whom antisera were obtained. Such a high titer IgG response means helper T cell recognition of the detected antigen. These tumor antigens can then be screened for the presence of MHC / HLA class I and class II motifs and reactivity with CTL.

Since currently known individual tumor antigens can only be expressed in tumor fractions,
The availability of yet another tumor antigen can significantly increase the proportion of patients who may be eligible for therapeutic intervention. Therefore, there is currently a need for additional tumor antigens for the development of therapeutics and diagnostics that are more applicable to cancer patients with various cancers.

[0008]   The present invention is further detailed in the following disclosure.

SUMMARY OF THE INVENTION Autologous antibody screening is currently being applied to breast cancer and T-cell leukemia using antisera from cancer patients. Many cancer-associated antigens have been identified. The present invention is
In particular, isolated nucleic acid molecules, expression vectors containing those molecules and host cells transfected with those molecules are provided. The invention also provides isolated proteins and peptides, antibodies to those proteins and peptides, and CTLs that recognize the proteins and peptides. Fragments, including functional fragments and variants of the above, are also provided. Kits additionally containing the molecules described above are also provided. The above can be used for the diagnosis, monitoring, detection or treatment of conditions characterized by the expression of one or more cancer-associated antigens.

Prior to the present invention, only a few cancer-related genes have been identified in the last 20 years. The present invention involves the surprising discovery of several genes, some of which are previously known and some are previously unknown, which are expressed in individuals with cancer. All of these individuals have serum antibodies to the proteins (or fragments thereof) encoded by these genes. Thus, aberrantly expressed genes may be recognized by the host's immune system and thus form the basis for diagnosis, monitoring and therapy.

The present invention includes the use of single materials, multiple different materials, and even large panels and combinations of materials. For example, a single gene, a single protein encoded by a gene, a single functional fragment thereof, a single antibody against it, etc. can be used in the methods and products of the present invention. Similarly, pairs, groups, and even panels of these substances
And optionally other cancer-associated antigen genes and / or gene products are diagnostic,
It can be used for monitoring and treatment. 2 pairs, groups or panels
It may comprise 3, 4, 5 or more genes, gene products, fragments thereof or agents that recognize such substances. Not only are multiple such agents useful for monitoring, typing, characterizing, and diagnosing cells that abnormally express such genes, but multiple such agents are therapeutically Can also be used. One example of the use of multiple such agents to prevent, delay the onset, ameliorate, etc. of cancer cells that express or will express such genes is prophylactic or short-term. Any and all combinations of genes, gene products, and agents that recognize genes and gene products can be tested and identified for use according to the invention. It would be too verbose to list all such combinations.
A person of ordinary skill in the art can readily determine in what circumstances the combination is most appropriate, especially in light of the teachings contained herein.

As will be apparent from the discussion below, the present invention has in vivo and in vitro applications, including for therapeutic, diagnostic, monitoring and research purposes. One aspect of the present invention is the ability to fingerprint cells expressing a large number of genes identified according to the present invention, for example by quantifying the expression of such gene products.

Such fingerprints characterize, for example, the stage of the cancer, the nature of the cancer type, or even the effect in animal models of treatment on the cancer. Cells can be further screened to determine whether such cells aberrantly express the genes identified by the present invention.

The invention in one aspect is a method of diagnosing a disorder characterized by the expression of a cancer-associated antigen precursor encoded by a nucleic acid molecule. The method comprises contacting a biological sample isolated from a subject with an agent that specifically binds a nucleic acid molecule, its expression product or a fragment of its expression product complexed with MHC, preferably an HLA molecule. Thus, in this case, the nucleic acid molecule is a NA group 1 nucleic acid molecule, and comprises the step of determining the interaction between the agent and the nucleic acid molecule, expression product or fragment of the expression product as a disorder determination.

In one embodiment, the agent is (a) a nucleic acid molecule comprising a NA group 1 nucleic acid molecule or fragment thereof, (b) a nucleic acid molecule comprising a NA group 3 nucleic acid molecule or a fragment thereof, (c) an NA group 5 nucleic acid. Nucleic acid molecule containing molecule or fragment thereof, (d) antibody that binds expression product of NA group 1 nucleic acid or fragment thereof, (e) antibody that binds expression product of NA group 3 nucleic acid or fragment thereof, (f) group NA An antibody that binds to the expression product of 5 nucleic acids or a fragment thereof, (g)
MHC, preferably an agent that binds to a complex of a fragment of an HLA molecule and an expression product of an NA group 1 nucleic acid, (h) an agent that binds to a complex of MHC, preferably an HLA molecule and a fragment of an expression product of an NA group 3 nucleic acid. , And (i) an agent that binds to a complex of MHC, preferably an HLA molecule and a fragment of an expression product of an NA group 5 nucleic acid.

Diseases can be characterized by the expression of multiple human cancer-associated antigen precursors. Accordingly, the diagnostic method involves the use of a plurality of agents, each of which is specific for a different human cancer-associated antigen precursor, including at least one of the cancer-associated antigen precursors disclosed herein. However, in this case the plurality of agents is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 such agents.

In each of the above embodiments, the agent can be specific for human cancer-associated antigen precursors, such as the breast cancer disclosed herein, and T cell leukemia-associated antigen precursors.

In another aspect, the invention is a method of determining regression, progression or onset of a condition characterized by the expression of abnormal levels of a protein encoded by a nucleic acid molecule that is a NA group 1 molecule. The method comprises providing a sample from a patient having or suspected of having the symptoms of (i) protein, (ii) protein-derived peptide, (ii)
of the above conditions with respect to a parameter selected from the group consisting of i) an antibody that selectively binds a protein or peptide, and (iv) a cytolytic T cell specific for a complex of peptide and MHC molecule derived from the protein. Monitoring as a determination of regression, progression or onset. In one embodiment, the sample is bodily fluid,
Body exudates or tissues.

In another embodiment, the step of monitoring comprises (a) an antibody that selectively binds a protein of (i) or a peptide of (ii), (b) a protein or peptide that binds to an antibody of (iii). And peptides (c) (iv) and MHC
Contacting the sample with a detectable agent selected from the group consisting of cells that present a complex of molecules. In a preferred embodiment, the antibody, protein, peptide or cell is labeled with a radioactive label or enzyme. The sample in the preferred embodiment is assayed for peptides.

According to another embodiment, the nucleic acid molecule is one of the following: NA group 3 molecule or NA group 5 molecule. In yet another embodiment, the protein is a plurality of proteins and the parameter is a plurality of parameters, each of the plurality of parameters being specific to a different one of the plurality of proteins.

[0021]   The invention, in another aspect, is a pharmaceutical formulation for a human subject.

The pharmaceutical formulation comprises an agent that selectively enriches for the presence of a complex of HLA molecule and human cancer associated antigen when administered to a subject, and a pharmaceutically acceptable carrier,
In this case, the human cancer-associated antigen is a fragment of human cancer-associated antigen precursor encoded by a nucleic acid molecule containing NA group 1 molecule. In one embodiment, the nucleic acid molecule is a NA group 3 nucleic acid molecule.

The agent, in one embodiment, comprises a plurality of agents, each of which selectively enriches a complex of HLA molecules and different human cancer-associated antigens in a subject. Preferably, the plurality is at least 2, at least 3, at least 4 or at least 5 different such agents.

[0024] In another embodiment, the agent is for expressing (1) an isolated polypeptide comprising a human cancer-associated antigen or a functional variant thereof, (2) an isolated polypeptide or a functional variant thereof. An isolated nucleic acid operably linked to a promoter, (3) a host cell expressing the isolated polypeptide or a functional variant thereof, and (4) a polypeptide or a functional variant thereof and an HLA molecule. It is selected from the group consisting of isolated complexes.

The agent can be a cell expressing the isolated polypeptide. In one embodiment, the agent is
A cell expressing an isolated polypeptide comprising a human cancer-associated antigen or a functional variant thereof. In another embodiment, the agent is a cell expressing an isolated polypeptide comprising a human cancer-associated antigen or a functional variant thereof, wherein the cell expresses an HLA molecule that binds the polypeptide.

The cell may recombinantly express one or both of the polypeptide and HLA molecule. In a preferred embodiment, the cells are non-proliferative. In yet another embodiment, the agent is at least 2, at least 3, each representing a different human cancer-associated antigen,
At least 4 or at least 5 different polypeptides, or functional variants thereof.

The agent is, in one embodiment, a PP group 2 polypeptide. In other embodiments, the agent is a PP group 3 polypeptide or PP group 4 polypeptide.

In one embodiment, each of the pharmaceutical formulations described herein also comprises an adjuvant.

According to another aspect of the invention, there is provided a composition comprising an isolated agent that selectively binds a PP group 1 polypeptide. In another embodiment, the agent is: PP group 2
It selectively binds to a polypeptide selected from a polypeptide, a PP group 3 polypeptide, a PP group 4 polypeptide and a PP group 5 polypeptide. In other embodiments, the agents are different agents that selectively bind at least 2, at least 3, at least 4 or at least 5 different such polypeptides.

[0030]   In each of the above embodiments, the agent can be an antibody.

In another aspect, the invention is a composition of matter comprising a complex of an agent of the above composition of the invention and a therapeutic or diagnostic agent. Preferably, the complex of agent and therapeutic or diagnostic agent is anti-neoplastic.

The present invention, in another aspect, comprises an isolated nucleic acid molecule selected from the group consisting of (1) NA group 1 molecule and (2) NA group 2 molecule, and a pharmaceutically acceptable carrier. It is a pharmaceutical composition. In one embodiment, the isolated nucleic acid molecule comprises NA group 3 molecules or NA group 4 molecules. In another embodiment, the isolated nucleic acid molecule comprises at least two isolated nucleic acid molecules encoding two different polypeptides, each polypeptide comprising a different human cancer associated antigen.

Preferably, the pharmaceutical composition also comprises an expression vector having a promoter operably linked to the isolated nucleic acid molecule. In another embodiment, the pharmaceutical composition also comprises a host cell that recombinantly expresses the isolated nucleic acid molecule.

According to another aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises an isolated polypeptide comprising a PP group 1 or PP group 2 polypeptide as well as a pharmaceutically acceptable carrier. In one embodiment, the isolated polypeptide is PP group 3 or PP group 4
Contains a polypeptide.

In another embodiment, the isolated polypeptide comprises at least two different human cancer-associated antigens, at least one of which is encoded by a NA group 1 molecule as described herein. Contains a polypeptide. In another embodiment, the isolated polypeptide is selected from the following PP group 3 polypeptides or HLA binding fragments thereof, and PP group 5 polypeptides or HLA binding fragments thereof.

In one embodiment, each of the pharmaceutical compositions described herein also includes an adjuvant.

In another aspect, the invention is an isolated nucleic acid molecule comprising a NA group 3 molecule. In another aspect, the invention is an isolated nucleic acid molecule comprising a NA group 4 molecule.

The invention, in another aspect, comprises: (a) a sequence unique within the human genome.
of a nucleic acid molecule consisting of SEQ ID NOs: 1, 3, 4, 6, 7, 9, and 16 that is of sufficient length to present a nucleic acid encoding a human cancer-associated antigen precursor A fragment of a nucleic acid selected from the group, (b) an isolated nucleic acid molecule selected from the group consisting of the complements of (a), provided that the fragment has (1) the sequence having the GenBank accession number in Table 4. , (2) the complement of (1), and (3) a sequence of contiguous nucleotides that is not identical to any sequence selected from the group of sequences consisting of the fragments of (1) and (2).

In one embodiment, the sequence of contiguous nucleotides is (1) at least two contiguous nucleotides that are not identical to the sequence in Table 4, (2) at least three contiguous nucleotides that are not identical to the sequence in Table 4, (3 ) At least 4 contiguous nucleotides not identical to the sequence in Table 4, (4) at least 5 contiguous nucleotides not identical to the sequence in Table 4, (5) at least 6 contiguous nucleotides not identical to the sequence in Table 4, or (6) selected from the group consisting of at least 7 contiguous nucleotides that are not identical to the sequences in Table 4.

In another embodiment, the fragment is at least 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, 24 nucleotides,
26 nucleotides, 28 nucleotides, 30 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides,
It has a size selected from the group consisting of 1000 nucleotides and any integer length nucleotides in between.

In yet another embodiment, the molecule encodes a polypeptide or fragment thereof that binds the human HLA receptor or human antibody.

Another aspect of the invention is an expression vector comprising the above isolated nucleic acid molecule of the invention operably linked to a promoter.

According to one aspect, the invention is an expression vector comprising a nucleic acid operably linked to a promoter, wherein the nucleic acid is NA group 1 or group 2 molecule. In another aspect, the invention is an expression vector comprising a NA group 1 or group 2 molecule and a nucleic acid encoding an MHC, preferably an HLA molecule.

In yet another aspect, the invention is a host cell transformed or transfected with an expression vector of the invention as described above.

In another aspect, the present invention is an expression vector comprising the above isolated nucleic acid molecule of the present invention operably linked to a promoter, or an expression vector comprising a nucleic acid operably linked to a promoter. Thus, the host cell is a host cell in which the nucleic acid is NA group 1 molecule or 2 molecule and is transformed or transfected with an expression vector further containing a nucleic acid encoding HLA.

According to another aspect of the invention, there is provided an isolated polypeptide encoded by the isolated nucleic acid molecule of the invention described above. These include PP group 1-5 polypeptides.
The invention also includes fragments of the polypeptide that are immunogenic. In one embodiment, the fragment or a portion of the fragment binds HLA or human antibody.

The invention in another aspect is an isolated fragment of a human cancer-associated antigen precursor that binds HLA or human antibody, or a portion thereof, wherein the precursor is a NA group 1 molecule. Fragment or part thereof.

In one embodiment, the fragment is part of a complex with HLA. In another embodiment, the fragment is 8-12 amino acids in length. In another embodiment, the invention provides an isolated polypeptide comprising a fragment of the polypeptide that identifies a polypeptide that is a human cancer-associated antigen precursor, of sufficient length to exhibit sequence uniqueness within the human genome. Including.

According to another aspect of the invention, a kit for detecting the presence of the expression of a cancer-associated antigen precursor is provided. The kits each comprise (a) a 12-32 nucleotide contiguous segment of the nucleotide sequence of any of the NA group 1 molecules, and (b) ("a
)) Comprising a pair of isolated nucleic acid molecules consisting essentially of a molecule selected from the group consisting of complements, wherein the consecutive segments are non-overlapping. Pairs are constructed and arranged to selectively amplify isolated nucleic acid molecules that are NA group 3 molecules, preferably the pairs amplify human NA group 3 molecules.

According to another aspect of the invention, there is provided a method of treating a subject having a disorder characterized by the expression of human cancer-associated antigen precursors. The method is a method comprising administering to the subject an amount of an agent that selectively enriches in the subject the presence of a complex of HLA molecule and human cancer-associated antigen effective to ameliorate the disorder, The human cancer-related antigen is (a) N
A nucleic acid molecule containing an A group 1 nucleic acid molecule, (b) a nucleic acid molecule containing an NA group 3 nucleic acid molecule, (c
) A fragment of a human cancer-associated antigen precursor encoded by a nucleic acid molecule selected from the group consisting of nucleic acid molecules including NA group 5 nucleic acid molecules.

In one embodiment, the disorder is characterized by the expression of multiple human cancer-associated antigen precursors, wherein the agent is each subject to the presence of an HLA molecule and a complex of different human cancer-associated antigens. A plurality of agents selectively enriched with, preferably a plurality of
At least 2, at least 3, at least 4, or at least 5 such agents.

In another embodiment, the agent is PP group 1, PP group 2, PP group 3, PP group 4 and P
An isolated polypeptide selected from the group consisting of P group 5 polypeptides.

[0053]   In yet another embodiment, the disorder is cancer.

According to another aspect, the invention is a method of treating a subject having a condition characterized by expression of a human cancer-associated antigen precursor in cells of the subject. This method involves removing (i) a sample containing immunoreactive cells from a subject, and (ii) transforming into host cells under conditions that favor the production of cytolytic T cells against human cancer-associated antigens that are fragments of precursors. Contacting a sample containing immunoreactive cells, and (iii) introducing cytolytic T cells into the subject in an amount effective to lyse cells expressing human cancer-associated antigens, wherein the host cells are Is transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operably linked to a promoter, wherein the isolated nucleic acid molecule is NA group 1, NA group 2, NA group 3, NA group 4, NA group. Selected from the group of 5 nucleic acid molecules.

In one embodiment, the host cell recombinantly expresses an HLA molecule that binds a human cancer-associated antigen. In another embodiment, the host cell is HL that binds a human cancer-associated antigen.
The A molecule is expressed endogenously.

The invention, in another aspect, is a method of treating a subject having a condition characterized by the presence of a cancer-associated antigen precursor in cells of the subject. The method comprises: (i) identifying a nucleic acid molecule expressed by cells associated with the above conditions, wherein the nucleic acid molecule is NA group 1 molecule; and (ii) (a) identified. Nucleic acid molecule, (b
) A fragment of the identified nucleic acid containing a segment encoding a cancer-associated antigen, (c)
Deletion, substitution or addition to (a) or (b), and (d) (a), (
transfecting a host cell with a nucleic acid selected from the group consisting of b) or (c) degeneracy, and (iii) culturing the transfected host cell described above to express the transfected nucleic acid molecule. And (iv) introducing into the subject an amount of the above-described host cells or an extract thereof that is effective to increase the immune response to the cells of the subject associated with the condition.

[0057]   Preferably the antigen is a human antigen and the subject is human.

In one embodiment, the method comprises: (a) MH displaying a portion of the expression product of the nucleic acid molecule.
Also included is the step of identifying the C molecule, in which case the host cell expresses the same MHC molecule identified in (a) and the host cell exhibits the MHC binding portion of the expression product of the nucleic acid molecule.

In another embodiment, the method also comprises treating the host cells so that they are non-proliferative.

In yet another embodiment, the immune response comprises a B cell response or a T cell response.
Preferably, the response is a T cell response that includes the generation of cytolytic T cells specific for host cells that express a portion of the expression product of the nucleic acid molecule or cells of interest that express human cancer associated antigens.

[0061]   In another embodiment, the nucleic acid molecule is a NA group 3 molecule.

Another aspect of the invention is for treating, diagnosing, or monitoring a subject having a condition characterized by the expression of an abnormal amount of protein encoded by a nucleic acid molecule that is a NA group 1 molecule. Is the way. This method comprises the step of administering to a subject an antibody that specifically binds to a protein or a peptide derived therefrom and that is bound to a therapeutically useful agent in an amount effective to treat the condition. Including.

In one embodiment, the antibody is a monoclonal antibody. Preferably, the monoclonal antibody is a chimeric antibody or a humanized antibody.

In another aspect, the invention is a method of treating a condition characterized by expression in a subject of an abnormal amount of a protein encoded by a nucleic acid molecule that is an NA Group 1 nucleic acid molecule. The method comprises the step of administering to the subject at least one of the pharmaceutical compositions of the invention described above in an amount effective to prevent, delay or suppress the onset of symptoms in the subject. In one embodiment, the condition is cancer. In another embodiment, the method comprises first identifying that the subject expresses an abnormal amount of protein in the tissue.

The invention, in another aspect, is a method of treating a subject having a condition characterized by the expression of an abnormal amount of a protein encoded by a nucleic acid molecule that is an NA Group 1 nucleic acid molecule. The method comprises: (i) identifying cells from a subject that express an abnormal amount of protein; (ii) isolating a sample of cells; (iii) culturing the cells; (iv) Introducing the cells into the subject in an amount effective to elicit an immune response against the cells.

In one embodiment, the invention comprises the step of rendering cells non-proliferative prior to introducing them into a subject.

In another aspect, the invention is a method of treating a pathological cellular condition characterized by aberrant expression of a protein encoded by a nucleic acid molecule that is a NA group 1 nucleic acid molecule. The method comprises the step of administering to a subject in need thereof an effective amount of an agent that suppresses protein expression or activity.

In one embodiment, the agent is a suppressor antibody that selectively binds a protein, where the antibody is a monoclonal antibody, chimeric antibody, humanized antibody or fragment thereof. In another embodiment, the agent is an antisense nucleic acid molecule that selectively binds to a nucleic acid molecule that encodes a protein. In yet another important embodiment, the nucleic acid molecule is a NA group 3 nucleic acid molecule.

The invention includes, in another aspect, a composition of matter useful for stimulating an immune response to a plurality of proteins encoded by nucleic acid molecules that are NA group 1 molecules. The composition is a plurality of peptides derived from the amino acid sequence of a protein, where the peptides are displayed on the surface of cells expressing an abnormal amount of the protein 1.
It binds to one or more MHC molecules.

[0070] In one embodiment, at least a portion of the plurality of peptides binds to an MHC molecule,
It elicits a cytolytic response to it. In another embodiment, the composition of matter comprises an adjuvant. In another embodiment, the adjuvant is saponin, GM-CS.
F or interleukin. In yet another embodiment, the composition also comprises at least one peptide useful in stimulating an immune response to at least one protein that is not encoded by a nucleic acid molecule that is a NA group 1 molecule, but in this case at least one peptide. The peptide binds to one or more MHC molecules.

According to another aspect, the present invention provides (i) a peptide derived from a protein encoded by a nucleic acid molecule that is a NA group 1 molecule, and (ii) an MHC that binds to the peptide to form a complex. An isolated antibody that selectively binds to a complex of molecules, wherein the isolated antibody does not bind (i) or (ii) alone.

In one embodiment, the antibody is SEQ ID NO: 14. In other embodiments, functional variants of peptides are used.

The present invention also includes the use of genes, gene products, fragments thereof, agents that bind to them, etc. in the preparation of drugs. The particular agent is for treating cancer, and the further particular agent is for treating breast cancer, kidney cancer, T cell leukemia, colon cancer, head and neck cancer, and / or ovarian cancer. is there.

In certain embodiments, the method comprises using one or more of the following cancerous tissues with some of the genes, gene products, fragments thereof and / or agents that bind to them. : Renal cancer, adrenal adenoma, colon adenocarcinoma, pancreatic cancer, uterine adenocarcinoma, germ cell tumor, colon adenocarcinoma, ovarian cancer, Wilms tumor, squamous cell carcinoma, glioblastoma, anaplastic oligodendroglioma, It is preferably not performed for B cell chronic lymphocytic leukemia, larynx, germinal center B cells and breast tumors.

Still other embodiments and aspects of the present invention will become apparent in connection with the following description of the invention.

[0076] [Detailed Description of the Invention]   In the summary and detailed description above, a list of sequences is provided.

The list consists of two separate single sequences, each of which forms part of the same gene.
Two or more sequences that are related to up to that number of different genes, including the total number on the list, so that each and every combination is listed separately and specifically, together with one or more sequences Are intended to include any combination of the sequences. Similarly, when describing fragment sizes, the minimum referring to the total length of each (and less than one nucleotide or amino acid to be a fragment) sequence intended for each and every fragment length to be specifically listed. A range is intended to include fragments. Thus, if a fragment can be 10-15 in length, it is 10, 11, 12, 13,
Clearly intended to mean 14 or 15.

The summary and claims describe the antigen precursors and antigens. As used in the summary and claims, the precursor is a substantially full-length protein encoded by the coding region of isolated DNA, and the antigen is complexed with MHC, preferably HLA, and the complex Are peptides that are involved in the immune response as part of Such antigens are typically 9 amino acids long, but this can vary slightly.

As used herein, a subject is a human, non-human primate, cow, horse, pig,
It is a sheep, goat, dog, cat or rodent. In all embodiments, human cancer antigens and human subjects are preferred. The invention includes in one aspect the cloning of a cDNA encoding a human cancer-associated antigen using an autologous antiserum of a subject having breast cancer, T-cell leukemia. The sequences of clones representing the genes identified by the methods described herein are set forth in the attached sequence listing. Of the above, it can be seen that some of the clones are considered completely new, as no coding regions are found in the searched database. The other clones are novel, but have some nucleotide or amino acid homology with the sequences deposited in the database (primarily EST sequences). Nevertheless, the entire gene sequence was previously unknown. In some cases the features are not guessed, and in others
Even when the function was inferred, it was not known that the gene was associated with cancer.
In all cases, it was not known or speculated that the gene encodes a cancer antigen that reacts with antibodies from autologous serum.

Analysis of the clone sequences by comparison with nucleic acid and protein databases determined that yet other clones were surprisingly closely related to other previously cloned genes. The sequences of these related genes are also shown in the sequence listing.

The nature of the above genes when encoding an antigen recognized by the immune system of cancer patients is, of course, not expected.

The invention thus provides, in one aspect, cancer-associated antigenic polypeptides, genes encoding those polypeptides, functional modifications and variants of the above, useful fragments of the above, and diagnostics associated therewith. And treatment.

Homologues and alleles of the cancer-associated antigenic nucleic acids of the invention can be identified by conventional techniques. Therefore, one aspect of the invention is a nucleic acid sequence encoding a cancer-associated antigen precursor. This application contains so many sequences that the following charts are provided to identify the sequences of the various groups discussed in the claims and the summary: Nucleic acid sequences NA group 1. (A) a cancer-associated antigen that hybridizes under stringent conditions with a molecule consisting of a nucleic acid sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 1, 3, 4, 6, 7, 9, and 16; A nucleic acid molecule encoding the precursor, (b) deletions, additions and substitutions encoding the respective cancer-associated antigen precursors, (c) a (a) or (b) in the codon sequence due to the degeneracy of the genetic code A nucleic acid molecule different from the nucleic acid molecule, and the complement of (d) (a), (b) or (c).

NA group 2. A fragment of NA group 1 that encodes a polypeptide or a portion thereof that binds an MHC molecule to form a complex recognized by autologous antibodies or lymphocytes.

NA group 3. The nucleotide sequences are as follows: (a) for example, a previously unknown human nucleic acid that encodes a human cancer-associated antigen precursor, (b) a deletion, addition and substitution that encodes each human cancer-associated antigen precursor, (c) Selected from the group consisting of a nucleic acid molecule that differs from the nucleic acid molecule of (a) or (b) in the codon sequence due to the degeneracy of the genetic code, and (d) the complement of (a), (b) or (c) NA group 1 subset.

NA group 4. A fragment of NA group 3 that encodes a polypeptide or a part thereof that binds an MHC molecule to form a complex recognized by autologous antibodies or lymphocytes.

NA group 5. A subset of NA group 1 containing human cancer-associated antigens that react with allogeneic cancer antisera.

Polypeptide Sequence PP Group 1. A polypeptide encoded by NA group 1. PP group 2. A polypeptide encoded by NA group 2. PP group 3. A polypeptide encoded by NA group 3. PP group 4. A polypeptide encoded by NA group 4. PP group 5. A polypeptide encoded by NA group 5.

[0089]   The term "stringent conditions" is used herein.
As used herein, it refers to parameters that are well known to those of ordinary skill in the art. Nucleic acid hybrid
Dization parameters can be found in references that edit such methods, such as Mo.
lecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Yo
rk, 1989, or Current Protocols in Molecular Biology, F.M.Ausubel, et
Al., eds., John Wiley & Sons, Inc., New York. In particular
The stringent conditions used in the present specification include, for example, hybridization.
Ization buffer (3.5xSSC, 0.02% Ficoll, 0.02% poly
Vinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH_TwoPO _Four Hybridization at 65 ° C. in (pH 7), 0.5% SDS, 2 mM EDTA)
Refers to dization. SSC is 0.15M sodium chloride / 0.15M queer
Sodium phosphate, pH 7, SDS is sodium dodecyl sulfate, and
And EDTA is ethylenediaminetetraacetic acid. After hybridization, DN
The film on which A is transferred is, for example, in 2 × SSC at room temperature, and then 0 at room temperature to 68 ° C.
． Wash in 1-0.5xSSC / 0.1xSDS.

There are other conditions, reagents, etc. that can be used and these produce comparable stringency. The person skilled in the art is familiar with such conditions and therefore they are not shown here. However, it will be appreciated by one of skill in the art that the conditions can be manipulated (eg, using low stringency conditions) in a manner that allows unambiguous identification of homologues and alleles of the cancer-associated antigenic nucleic acids of the invention. Those of skill in the art are well aware of methods for screening cells and libraries for expression of such molecules, which are then routinely isolated, followed by isolation and sequencing of related nucleic acid molecules.

In general, homologues and alleles typically share at least 80% nucleotide identity and / or at least 90% amino acid identity with the sequences of cancer-associated antigen nucleic acids and polypeptides, respectively, and In some cases share at least 90% nucleotide identity and / or at least 95% amino acid identity, and in still other cases at least 95% nucleotide identity and / or at least 99% amino acid. Share the same. Homology is obtained from the Internet (ftp: /ncbi.nlm.nih.gov/pub/) National Cen
ter for Biotechnology Information (Bethesda, Maryland) can be calculated using various publicly available software tools. An example of the tool is http: //www.ncbi.nlm.preferably using the default settings.
The BLAST system available at nih.gov. Pairwise and Clustal
W alignment (BLOSUM30 matrix setting) and Kyte-Doolitt
le Hydrotherapy Analysis is a MacVector Sequence Analysis Software (Oxford Molecular Group
) Is obtained.

[0092] Watson-Crick complements of the above nucleic acids are also included in the invention.

For example, alleles include HOM-TALL1-5 and MO-BC203.
Examples include genes encoding clones. As disclosed herein, these clones are isolated from different individuals, but share about 95-99% nucleotide identity in their overlapping regions. Therefore, these clones appear to represent allelic variants of the same gene present in different individuals. Allelic variants of the nucleic acid molecules (and polypeptides) disclosed herein, such as these, are encompassed by the present invention.

For screening for cancer-associated antigen genes, Southern blots can be performed using the above conditions with radioactive probes. After washing the membrane to which the DNA was last transferred, the membrane was placed against X-ray film and the radioactive signal could be detected. Screening for expression of cancer-associated antigen nucleic acid involves screening for a sample taken from a breast cancer, T-cell leukemia patient or subject suspected of having a condition characterized by expression of the cancer-associated antigen gene disclosed herein,
Northern blot hybridization using the conditions described above can be performed. Amplification protocols such as the polymerase chain reaction with primers that hybridize to the presented sequences can also be used for the detection or expression of cancer-associated antigen genes.

Breast cancer, T-cell leukemia and / or ovarian cancer-related genes are represented by SEQ ID NOs: 1, 3, 4
, 6, 7, 9, and 16. Preferred breast cancer and / or T cell leukemia-associated antigens for the methods of diagnosis disclosed herein are those found to react with allogeneic cancer antisera (ie NA group 5). Encoded polypeptides (eg proteins), peptides and antisera thereto are also preferred for diagnosis.

As used herein with respect to nucleic acids, the term “isolated” refers to (i)
Amplified in vitro by, for example, polymerase chain reaction (PCR), or (ii
) Means recombinantly produced by cloning, (iii) purified by eg cleavage and gel separation, or (iv) synthesized eg by chemical synthesis. An isolated nucleic acid is one that can be easily manipulated by recombinant DNA technology well known in the art. Thus, a nucleotide sequence whose 5'and 3'restriction sites are known or contained in a vector in which a polymerase chain reaction (PCR) primer sequence has been disclosed is considered to be isolated, but not its native. Nucleic acid sequences that are present in their native state in the host are not. An isolated nucleic acid can be, but need not be, substantially purified. For example, a nucleic acid isolated in a cloning or expression vector is not pure in that it may contain only a minimal percentage of the material in the cell in which it is present. However, such nucleic acids are isolated, as the term is used herein, as it is readily manipulated by standard techniques known to those of skill in the art. Isolated nucleic acid, as used herein, is not a natural chromosome.

As used herein with respect to a polypeptide, “isolated” means separated from its native environment and present in an amount sufficient to allow its identification or use. . Isolated, when referring to a protein or polypeptide, means, for example, (i) selectively produced by expression cloning, or (ii) purified, for example by chromatography or electrophoresis. The isolated protein or polypeptide may, but need not be, substantially pure. The term "substantially pure" refers to other proteins or polypeptides in which they are found, either naturally or in vivo, to the extent practical and suitable for their intended use. It means essentially free of substances. Substantially pure polypeptides can be produced by techniques well known in the art. The isolated protein can be mixed in a pharmaceutical formulation with a pharmaceutically acceptable carrier so that the protein may only comprise a small percentage by weight of the preparation. A protein is nevertheless isolated, ie, isolated from other proteins, when it is separated from substances that may be associated with the living system.

The present invention also includes degenerate nucleic acids containing alternative codons for those present in native material. For example, serine residues are codons TCA, AGT, TCC, TCG,
Coded by TCT and AGC. Each of the six codons is equivalent for purposes of encoding a serine residue. Therefore, in
It will be apparent to those skilled in the art that either serine-encoding nucleotide triplets can be used to direct the protein synthesizer to incorporate serine residues in vitro or in vivo. Similarly, nucleotide sequence triplets encoding other amino acid residues include CCA, CCC, CCG and C.
CT (proline codon); CGA, CGC, CGG, CGT, AGA and AG
G (arginine codon); ACA, ACC, ACG and ACT (threonine codon); AAC and AAT (asparagine codon); and ATA, ATC
And ATT (isoleucine codon).
Other amino acid residues are similarly encoded by the multiple nucleotide sequence. Thus, the invention includes degenerate nucleic acids that differ from the biologically isolated nucleic acid in the codon sequence due to the degeneracy of the genetic code.

The present invention also provides modified nucleic acid molecules that include additions, substitutions and deletions of one or more nucleotides. In a preferred embodiment, these modified nucleic acid molecules and / or
Alternatively, the polypeptides they encode may be associated with at least one activity or function of an unmodified nucleic acid molecule and / or polypeptide, such as antigenicity, enzymatic activity, receptor binding, binding of peptides by MHC class I and class II molecules. Retains complex formation and the like. In certain embodiments, the modified nucleic acid molecule encodes a modified polypeptide, preferably a polypeptide having conservative amino acid substitutions as described elsewhere herein. The modified nucleic acid molecule is structurally related to the unmodified nucleic acid molecule, and in a preferred embodiment, the modified and unmodified nucleic acid molecule are hybridized to the unmodified nucleic acid molecule such that they hybridize under stringent conditions known to those of skill in the art. Fully structurally related.

For example, modified nucleic acid molecules can be prepared that encode a polypeptide having a single amino acid change. Each of these nucleic acid molecules is compatible with nucleotide changes corresponding to the degeneracy of the genetic code as described herein 1, 2, or 3
It may have one nucleotide substitution. Similarly, modified nucleic acid molecules can be prepared which encode a polypeptide having two amino acid changes, eg, 2
It has ~ 6 nucleotide changes. For example amino acids 2 and 3, 2 and 5,2
Numerous modified nucleic acid molecules similar to these are readily contemplated by those of skill in the art, including substitution of nucleotides at codons encoding and etc. In the above example, each combination of two amino acids is included in the modified nucleic acid molecule set as well as in every nucleotide substitution that encodes an amino acid substitution. Encodes a polypeptide with additional substitutions (ie, 3 or more), additions or deletions (eg, by the introduction of stop codons or splice site (s)), as readily contemplated by one of skill in the art. Additional nucleic acid molecules can be prepared and are included in the present invention. Any of the above nucleic acids or polypeptides can be tested by routine experimentation for retention of structural association or activity with the nucleic acids and / or polypeptides disclosed herein.

The present invention also provides an isolated unique fragment of a cancer-associated antigen nucleic acid sequence or its complement. Unique fragments are fragments that are the "feature" for large nucleic acids. It is of sufficient length to ensure, for example, that its exact sequence is not found in molecules within the human genome (and human alleles) outside the cancer-associated antigenic nucleic acids described above. One of ordinary skill in the art can only apply routine techniques to determine whether a fragment is unique within the human genome. The unique fragment, however, may contain nucleotide sequences of any of the GenBank deposit numbers listed in Table 1 or priority binding data for sequences listed in the respective priority documents or overlapping sequences of the present invention. Exclude fragments that consist entirely of other previously published sequences similar to those of the binding data of this application for the sequences listed for the first time.

A fragment consisting entirely of the sequence set forth in the GenBank Deposit above is a fragment that does not contain any of the nucleotides unique to the sequence of the invention. Therefore, a unique fragment must contain a nucleotide sequence or fragments thereof other than the exact sequence of GenBank. The differences can be additions, deletions or substitutions with respect to the GenBank sequences, or they can be sequences that are completely separate from the GenBank sequences.

Unique fragments can be used as probes in Southern and Northern blot assays to identify such nucleic acids, or can be used in amplification assays, such as PCR-based assays. As is known to those of skill in the art, large probes such as 200, 250, 300 or more nucleotides are preferred for certain applications, such as Southern and Northern blots,
On the other hand, small fragments are preferred for applications such as PCR. The unique fragment can be used to generate antibodies or to determine binding of polypeptide fragments, or to produce fusion proteins to produce immunoassay components. Similarly, unique fragments can be used, for example, for preparing antibodies and for producing non-fusion fragments of cancer-associated antigen polypeptides useful in immunoassays. The unique fragment can further be used as an antisense molecule for suppressing the expression of cancer-associated antigen nucleic acids and polypeptides, especially for therapeutic purposes as described in more detail below.

As will be appreciated by those in the art, the size of the unique fragment depends on its conservation in the genetic code. Thus, some regions of cancer-associated antigenic sequences and their complements require longer segments to be unique, while others
Up to the full length of the disclosed sequences, typically between 12 and 32 nucleotides (eg,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
Short segments (24, 25, 26, 27, 28, 29, 30, 31 and 32 or more base length) are required. As noted above, the present disclosure begins with the first nucleotide and extends to the second nucleotide, ..., And with a shortness up to the last eight nucleotides, from the eighth, ninth, ten ... It is intended to include each and every fragment of each sequence, ending in place, provided the sequence is unique as described above.

Every segment of the polypeptide coding region of the novel cancer-associated antigenic nucleic acid or its complement that is 25 or more nucleotides in length is virtually unique. Those skilled in the art will typically base all such sequences on known database sequences based on the ability of the unique fragment to selectively distinguish that sequence from other sequences in the human genome of that sequence. Although well familiar with the method of selecting, in vitro confirmatory hybridization and sequencing analysis may be performed.

Particularly preferred are nucleic acids encoding a series of epitopes known as "polytopes". The epitope is a natural flanking sequence,
Alternatively, or not, they can be arranged in a sequential or overlapping manner (eg, Thomson et al., Proc. Natl. Acad. Sci. USA 92: 5845-5849).
, 1995, Gilbert et al., Nature Biotechnol. 15: 1280-1284, 1997) and optionally separated by unrelated linker sequences. Polytopes are processed to produce individual epitopes recognized by the immune system for the generation of immune responses.

Thus, for example, from a polypeptide having an amino acid sequence encoded by one of the nucleic acids disclosed herein (such as the peptide having SEQ ID NOs: 1, 1, 12, 13, 14, and 15). A peptide, which is presented by an MHC molecule and recognized by CTLs or T helper lymphocytes, is combined with a peptide from one or more other cancer-associated antigens (eg, a hybrid nucleic acid or polypeptide). Can be formed) to form a "polytope". The two or more peptides (or nucleic acids encoding the peptides) can be selected from those described herein, or they can be one or more of the conventionally known cancer-associated antigens. It may include the above peptides. Examples of cancer-associated peptide antigens that can be administered to induce or enhance an immune response include tumor-associated genes and encoded proteins such as MAGE-A1, MAGE-A2, M.
AGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-
A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11
, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-
4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9
, BAGE-1, RAGE-1, LB33 / MUM-1, PRAME, NAG,
MAGE-B2, MAGE-B3, MAGE-B4, tyrosinase, brain glycogen phosphorylase, melan-A, MAGE-C1, MAGE-C2, MAGE
-C3, MAGE-C4, MAGE-C5, NY ESO-1, LAGE-1,
SSX-1, SSX-2 (HOM-MEL-40), SSX-4, SSX-5,
Obtained from SCP-1 and CT-7 (eg PCT application publication WO 96/1
0577). Other examples are known to those of skill in the art (eg, Coulie, Stem
Cells 13: 393-403, 1995), and can be used in the present invention in a similar manner as disclosed herein. Those skilled in the art will prepare a polypeptide comprising one or more peptides and one or more of the above cancer-associated peptides, or a nucleic acid encoding such a polypeptide, according to standard techniques of molecular biology. can do.

Polytopes are thus a group of two or more potentially immunogenic or immune response stimulating peptides that can be joined together at different sequences (eg linked, overlapping). Polytopes (or polytope-encoding nucleic acids) can be administered in standard immunization protocols, eg, to animals, to test the effectiveness of polytopes in stimulating, enhancing, and / or eliciting an immune response. it can.

The peptides can be joined together to form a polytope, either directly or through the use of flanking sequences. The use of polytopes as vaccines is well known in the art (eg Thomson et al., Proc. Natl. Acad. Sci. USA 9
2 (13): 5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15 (12): 128.
0-1284, 1997; Thomson et al., J. Immunol. 157 (2): 822-826, 1996; Tam et.
al., J. Exp. Med. 171 (1): 299-306, 1990). For example, Tam is MHC
We have shown that polytopes consisting of both class I and class II binding epitopes successfully generate antibodies and protective immunity in a mouse model. Tam also demonstrated that polytopes containing "rows" of epitopes were processed to produce individual epitopes presented by MHC molecules and recognized by CTLs.
Thus, polytopes containing varying numbers and combinations of epitopes can be prepared and tested for recognition by CTLs and for efficacy in increasing immune response.

It is known that tumors express a set of tumor antigens, only certain subsets of which can be expressed in tumors of any given patient. Polytopes can be prepared that correspond to different combinations of epitopes representing a subset of tumor rejection antigens expressed in a particular patient. Polytopes can be prepared to reflect a broader range of tumor rejection antigens known to be expressed by tumor types. Polytopes can be introduced into patients in need of such treatment as polypeptide structures or through the use of nucleic acid delivery systems known in the art (eg, Allsopp et al., Eur. J. Immunol. 26 (8): 1951-1959, 1996). Adenovirus, poxvirus, Ty-virus-like particle, adeno-associated virus,
Plasmids, bacteria, etc. can be used for such delivery. The delivery system can be tested in a mouse model to determine the efficacy of the polytope delivery system. This system can also be tested in human clinical trials.

When the human HLA class I molecule has a tumor rejection antigen derived from a cancer-associated nucleic acid, the expression vector is a nucleic acid encoding an HLA molecule having any particular tumor rejection antigen derived from these nucleic acids and polypeptides. Sequences may also be included. Alternatively, the nucleic acid molecule encoding such an HLA molecule can be included in a separate expression vector. In the situation where the vector contains both coding sequences, the single vector
Either can be used to transfect cells that do not normally express. If the cancer-associated antigen precursor, and the sequences encoding the HLA molecule carrying it, are contained in separate expression vectors, the expression vectors can be co-transfected.

The cancer-associated antigen precursor coding sequence can be used alone, eg, when the host cell already expresses an HLA molecule that presents a cancer-associated antigen derived from the precursor molecule. Of course, there is no limitation as to the particular host cell that can be used. Since the vector containing the two coding sequences can be used in any antigen presenting cell as desired, the gene for the cancer-associated antigen precursor can be used in host cells that do not express the HLA molecule with the cancer-associated antigen. Furthermore, cell-free transcription systems can be used instead of cells.

As described above, the present invention includes antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a cancer-associated antigen polypeptide and reduce expression of the cancer-associated antigen. This is practically desirable in any medical condition where reduced expression of cancer-associated antigens is desirable, eg in the treatment of cancer. It is also useful to test the effect of reduced expression of one or more cancer-associated antigens in vitro or in vivo.

As used herein, the term “antisense oligonucleotide” or “antisense” hybridizes under physiological conditions to DNA containing a particular gene, or the mRNA transcript of that gene. Then, an oligonucleotide that is an oligoribonucleotide, an oligodeoxyribonucleotide, a modified oligoribonucleotide or a modified oligodeoxyribonucleotide, which thereby suppresses the transcription of the gene or the translation of the mRNA, is described. Antisense molecules are designed to interfere with the transcription or translation of a target gene upon hybridization with the target gene or transcript. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and the degree of complementarity with this target will depend on the particular target chosen, including the sequence of the target and the particular bases containing that sequence. To do.
The antisense oligonucleotide is configured to selectively bind the target under physiological conditions, ie, to substantially hybridize to the target sequence under physiological conditions over any other sequence in the target cell. , Are preferably arranged. Depending on the sequence of the nucleic acid encoding the breast cancer and / or T cell leukemia-associated antigen, or depending on the allele or the corresponding genomic and / or cDNA sequence, one of ordinary skill in the art will appreciate that there are many suitable All of the antisense molecules can be easily selected and synthesized. For example, "gene transfer" involving a series of oligonucleotides of 15 to 30 nucleotides over the length of a cancer-associated antigen.
e walk) ”can be manufactured and then tested for inhibition of cancer-associated antigen expression. Optionally, a gap of 5-10 nucleotides can be left between the oligonucleotides to reduce the number of oligonucleotides synthesized and tested.

In order to be sufficiently selective and efficacious for suppression, such antisense oligonucleotides should have at least 10, and more preferably at least 15 contiguous bases complementary to the target. Should be included, but in some cases,
Modified oligonucleotides as short as 7 bases have been successfully used as antisense oligonucleotides (Wagner et al., Nature Biotechnol. 1
4: 840-844, 1996). Most preferably, the antisense oligonucleotide is 2
It contains a complementary sequence of 0 to 30 bases. Oligonucleotides that are antisense to any region of the gene or mRNA transcript may be selected, but in a preferred embodiment, the antisense oligonucleotide is at the N-terminus or 5'upstream site, such as translation initiation, transcription. Corresponds to the start or promoter site. Furthermore, 3 '
-The untranslated region may be targeted. Targeting to mRNA splicing sites has also been used in the art, but may be less preferred if alternative mRNA splicing occurs. Further, the antisense is preferably mR
NA secondary structure is unexpected (see, eg, Sainio et al., Cell Mol. Neurobiol. 14
(5): 439-457, 1994), and is targeted to sites where the protein is not expected to bind. Finally, although the sequences listed are cDNA sequences, one of skill in the art may readily obtain genomic DNA corresponding to the cancer-associated antigen cDNA. Therefore, the present invention provides genomic DNA corresponding to nucleic acids encoding cancer-associated antigens.
Also provided are antisense oligonucleotides that are complementary to. Similarly, antisense to allelic or homologous cDNA and genomic DNA is given without undue experimentation.

In one set of embodiments, the antisense oligonucleotides of the invention are "natural".
It can be composed of deoxyribonucleotides, ribonucleotides or any combination thereof.

That is, as in the case of natural systems, the 5'end of one native nucleotide and the 3'end of another native nucleotide can be covalently linked by a phosphodiester internucleoside linkage. These oligonucleotides can be prepared by art-recognized methods that can be performed manually or by automated synthesizers. The oligonucleotide can also be produced recombinantly by a vector.

However, in a preferred embodiment, the antisense oligonucleotides of the invention may also include "modified" oligonucleotides. That is, the oligonucleotides are in a number of ways that prevent them from hybridizing to their targets, but enhance their stability or targeting, or otherwise enhance their therapeutic efficacy. Can be modified.

The term “modified oligonucleotide” as used herein (
1) At least two of its nucleotides are covalently linked by a synthetic internucleoside linkage (ie, a linkage other than a phosphodiester bond between the 5'end of one nucleotide and the 3'end of another nucleotide), and / or (2 3.) Describe an oligonucleotide in which a chemical group not normally associated with the nucleic acid is covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also includes oligonucleotides having covalently modified bases and / or sugars. For example, the modified oligonucleotide is 3 '
Includes oligonucleotides having a backbone sugar covalently linked to low molecular weight organic groups other than the hydroxyl group at the position and other than the phosphate group at the 5'position. Thus modified oligonucleotides may include a 2'-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. Accordingly, the present invention provides modified antisense molecules that are complementary to and hybridizable under physiological conditions with nucleic acids encoding breast cancer and / or T cell leukemia-associated antigen polypeptides. Intended to be contained together with an acceptable carrier.

Antisense oligonucleotides can be administered as part of a pharmaceutical composition. Such pharmaceutical compositions may include the antisense oligonucleotide in combination with any standard physiologically and / or pharmaceutically acceptable carrier known in the art. The composition should be sterile and contain a therapeutically effective amount of the antisense oligonucleotide in a weight or volume unit suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a biological system,
For example, it refers to non-toxic substances that are compatible with cells, cell cultures, tissues or organisms. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art as further described below. .

As used herein, a “vector” is one in which the desired sequence can be inserted by restriction and ligation for transfer between different genetic environments or for expression in a host cell. It can be any of a number of nucleic acids. Vectors are typically composed of DNA, although RNA vectors are also available. As a vector,
Examples include, but are not limited to, plasmids, phagemids and viral genomes. Is the cloning vector autonomously replicable in the host cell,
Or capable of integrating into the genome and cleaving the novel recombinant vector in a determinable manner so that it retains its ability to replicate in host cells, and joins the desired DNA sequences. Are further characterized by one or more endonuclease restriction sites that are capable. In the case of plasmids, replication of the desired sequence occurs multiple times if the plasmid is copy number-increasing in the host bacterium, or 1 per host before the host is propagated by mitosis.
It can happen only once. In the case of phage, replication can occur actively during the lytic phase and passively during the lysogenic phase. The expression vector is one in which the desired DNA sequence can be inserted by restriction and ligation so that it can be operably linked to regulatory sequences and expressed as an RNA transcript. The vector may further contain one or more marker sequences suitable for use in identifying whether cells have been transformed or transfected with the vector or not. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, enzymes whose activities are detectable by standard assays known in the art (e.g., p-galactosidase, Luciferase or alkaline phosphatase), as well as genes that visually affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (eg, green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene product present in the DNA segment to which they are operably linked.

As used herein, coding and regulatory sequences are "operably linked" when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. (Operably) "are said to be connected. When it is desired that the coding sequence be translated into a functional protein, induction of the promoter in the 5'regulatory sequence results in transcription of the coding sequence and the nature of the linkage between the two DNA sequences is (1
A) does not result in the introduction of frameshift mutations, (2) does not interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. For example, two DNA sequences are said to be operably linked. Thus, a promoter region is operably linked to a coding sequence if the promoter region is capable of effecting transcription of that DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide. Will be linked.

The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but in general, where necessary, TATA boxes, capping sequences, CA.
It includes 5'untranscribed and 5'untranslated sequences involved in initiation of transcription and translation, respectively, such as AT sequences and the like.

[0125] In particular, such 5'non-transcriptional regulatory sequences include a promoter region that includes a promoter sequence for transcriptional control of an operably linked gene.

Regulatory sequences may also include enhancer sequences or upstream activator sequences, if desired. The vector of the present invention may optionally include a 5'leader or signal sequence.

Selection and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art (eg Sambrook et al., Molecular Cloning: A Laborator.
y Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). The cell is genetically engineered by introducing into the cell a heterologous DNA (RNA) encoding a cancer-associated antigenic polypeptide or a fragment or variant thereof. The heterologous DNA (RNA) is placed under operable control of transcriptional elements to allow expression of the heterologous DNA in a host cell.

Preferred systems for mRNA expression in mammalian cells include genes conferring G418 resistance (which facilitate selection of stably transfected cell lines), and human cytomegalovirus (CMV) enhancer-promoter sequences. 3.1 and pRc / CMV containing various selectable markers (Invitrogen, Carlsb
(available from ad, CA).

Further suitable for expression in primate or canine cell lines is the pCEP4 vector, which contains the Epstein-Barr virus (EBV) origin of replication and promotes retention of the plasmid as a multicopy extrachromosomal element ( Invitrogen). Another expression vector is the pEF BOS plasmid containing the promoter of polypeptide extender 1a, which efficiently stimulates transcription in vitro. This plasmid is Mishiz
Uma and Nagata (Nuc. Acids Res. 18: 5322, 1990) and their use in transfection experiments is described, for example, in Demoulin (Mol. Cell. Bio.
16: 4710-4716, 1996). Yet another preferred expression vector is the adenovirus described by Stratford-Perricaudet, which lacks the E1 and E3 proteins (J. Clin. Invest. 90: 626-630, 199).
2). The use of adenovirus as an Adeno.P1A recombinant for the expression of antigens has been shown by Warnie in intradermal injection in mice for immunization against P1A.
r. et al. (Int. J Cancer, 67: 303-310, 1996). Additional vectors for delivery of nucleic acids are presented below.

The present invention also includes so-called expression kits that allow one skilled in the art to prepare the desired expression vector or expression vectors. Such expression kits include at least a separate portion of the vector and one or more of the cancer-associated antigen nucleic acid molecules described above. Other components can be added as desired as long as the above-mentioned nucleic acid molecule is contained. The invention also includes a kit for amplification of a cancer-associated antigen nucleic acid that includes at least a pair of amplification primers that hybridize to the cancer-associated antigen nucleic acid.
The primers are preferably 12-32 nucleotides in length and are non-overlapping to prevent the formation of "primer dimers". One of the primers hybridizes to one strand of the cancer-associated antigen nucleic acid and the second primer hybridizes to a complementary strand of the cancer-associated antigen nucleic acid with a sequence that allows amplification of the cancer-associated antigen nucleic acid. Selection of appropriate primer pairs is standard in the art. For example, the selection, optionally with the help of computer programs designed for such purposes, then
This can be done by examining the primers for amplification specificity and efficiency.

The present invention also allows the construction and transgenic overexpression of cancer-associated antigen gene “knockouts” in cells and animals, providing agents for testing cancer and certain aspects of the immune system response to cancer. To do.

The present invention also provides isolated polypeptides (including whole proteins and partial proteins) encoded by the cancer-associated antigenic nucleic acids described above. Such polypeptides are useful, for example, alone or as fusion proteins to produce antibodies, as components of immunoassays or diagnostic assays, or as therapeutic agents. The cancer-associated antigen polypeptide is isolated from a biological sample including tissue or cell homogenate, constructs an expression vector suitable for the expression system, introduces the expression vector into the expression system, and expresses the recombinantly expressed protein. Can be recombinantly expressed in a variety of prokaryotic and eukaryotic expression systems. Short polypeptides, including antigenic peptides (as presented by MHC molecules on the surface of cells for immune recognition) can also be chemically synthesized using well established methods of peptide synthesis. .

Unique fragments of cancer-associated antigenic polypeptides generally have the unique fragment characteristics and properties described above in connection with nucleic acids. As will be appreciated by those in the art, the size of the unique fragment will depend on the agent, such as whether the fragment forms part of a conserved protein domain. Therefore, some regions of cancer-associated antigens
Requires longer segments to be unique, while others typically range from 5
12 amino acids (eg 5, 6, 7, 8, 9, 10, including each integer up to the full length,
Only short segments (11 or 12 or more amino acids) are required.

Unique fragments of a polypeptide are preferably fragments that retain distinct functional capacity of the polypeptide. The functional capacity that can be retained in a unique fragment of a polypeptide includes interaction with antibodies, interaction with other polypeptides or fragments thereof, selective binding of nucleic acids or proteins, and enzymatic activity. One activity of interest is the ability to act as a feature for identifying polypeptides. Another is the ability to complex with HLA and elicit an immune response in humans. Those of skill in the art are typically familiar with methods for selecting unique amino acid sequences based on the ability of the unique fragment to selectively distinguish that sequence from non-family members. Typically, a comparison of the fragment's sequence with a sequence on a known database is all that is needed.

The present invention includes variants of the above cancer-associated antigenic polypeptides. As used herein, a "variant" of a cancer-associated antigen polypeptide is a polypeptide that contains one or more modifications to the primary amino acid sequence of the cancer-associated antigen polypeptide. Modifications that create cancer-associated antigenic variants are made to cancer-associated antigenic polypeptides to 1) reduce or eliminate the activity of cancer-associated antigenic polypeptides, and 2) properties of cancer-associated antigenic polypeptides, such as in expression systems Enhance protein stability or stability of protein-protein binding, 3) provide novel activity or property for cancer-associated antigenic polypeptides, eg addition of antigenic epitopes or addition of detectable moieties, or 4) HLA molecules To provide equivalent or better binding to.

Modifications to cancer-associated antigen polypeptides are typically made to nucleic acids that encode cancer-associated antigen polypeptides and include deletions, point mutations, truncations, amino acid substitutions and amino acid or non-amino acid moieties. It may include additions. Alternatively, the modification can be made directly to the polypeptide by, for example, cleavage, addition of linker molecules, addition of detectable moieties such as biotin, addition of fatty acids, substitution of L-amino acids with D-amino acids, and the like. .

Modifications also include fusion proteins that include all or part of a cancer-associated antigen amino acid sequence. Those of skill in the art are familiar with methods of predicting the effects of changes in protein sequence on protein conformation, and thus may “design” variant cancer associated antigen polypeptides by known methods. An example of such a method is described by Dahiyat and Mayo in Sc
ience 278: 82-87, 1997, which allows proteins to be de novo designed. The method can be applied to known proteins to alter only a portion of the polypeptide sequence. By applying the computational method of Dahiyat and Mayo, specific variants of cancer-associated antigenic polypeptides are proposed and can be tested to determine if the variant retains the desired conformation. . Other computational and computer modeling methods for designing polypeptide mimetics that retain the activity of the polypeptides described herein, and selection methods such as phage display of peptide libraries are known in the art. Is.

Variants generally include cancer-associated antigenic polypeptides that are specifically modified to alter characteristics of the polypeptide that are not associated with its desired physiological activity.

For example, cysteine residues may be substituted or deleted to prevent unwanted disulfide bonds. Similarly, certain amino acids can be modified to enhance expression of cancer-associated antigenic polypeptides by eliminating proteolysis by proteases in the expression system (eg, in yeast expression systems where KEX2 protease activity is present). Dibasic amino acid residues).

Mutations in the nucleic acid encoding the cancer-associated antigenic polypeptide preferably preserve the amino acid reading frame of the coding sequence and are preferably hairpins or loops that may hybridize and be detrimental to the expression of the variant polypeptide. It does not create regions in nucleic acids that would form secondary structures such as.

Mutations can be made by selecting amino acid substitutions or by random mutagenesis of selected sites in the nucleic acid encoding the polypeptide. The variant polypeptide is then expressed and tested for one or more activities to determine which mutation (s) provide the variant polypeptide with the desired properties. Yet another mutation that is silent about the amino acid sequence of the polypeptide, but which provides preferred codons for translation in a particular host, is directed against the variant (or non-variant cancer-associated antigenic polypeptide). You can do it. Preferred codons for translation of nucleic acids in, for example, E. coli are well known to those of skill in the art. Still other mutations may be made to the non-coding sequences of the cancer-associated antigen gene or cDNA clone to enhance expression of the polypeptide. The activity of a variant of a cancer-associated antigen polypeptide is determined by cloning the gene encoding the variant cancer-associated antigen polypeptide into a bacterial or mammalian expression vector and introducing this vector into a suitable host cell. It can be tested by expressing the polypeptide and testing for the functional capacity of the cancer-associated antigenic polypeptide as disclosed herein above.

For example, variant cancer-associated antigen polypeptides can be tested for reactivity with autologous and allogeneic sera as disclosed in the examples. Preparation of other variant polypeptides may be convenient for testing other activities, as will be appreciated by those in the art.

Those skilled in the art will appreciate that conservative amino acid substitutions may be made in the cancer-associated antigen polypeptide to provide functionally equivalent variants of the above polypeptides, ie, where the variant is a cancer-associated antigenic polypeptide. It will also be recognized that the functional capacity of the peptide is retained. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants are found in references summarizing methods for altering polypeptide sequences known to those of skill in the art, for example, Molecular Cloning: A Laboratory Manual, J. Sambro.
ok, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, C
old Spring Harbor, New York, 1989 or Current Protocols in Molecular Bi
ology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Examples of functionally equivalent variants of cancer-associated antigenic polypeptides include conservative amino acid substitutions in the amino acid sequences of the proteins disclosed herein. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F
, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

For example, in determining that a peptide from a cancer-associated antigen polypeptide is presented by MHC molecules and recognized by CTL (eg, as described in the examples),
Conservative amino acid substitutions may be made at the residues that are not considered to be particularly direct contacts with the MHC molecule for the amino acid sequence of the peptide. For example, methods for identifying functional variants of HLA class II binding peptides are described in Strominger and Wucherpfennig, published P.
It is provided in the CT application (PCT / US96 / 03182). Peptides with one or more amino acid substitutions are also available, eg D'Amaro and Drijfhout (D'Amar
o et al., Human Immunol. 43: 13-18, 1995; Drijfhout et al., Human Immunol.
l. 43: 1-12, 1995) and can be tested for identity with known HLA / MHC motifs prior to synthesis using a computer program.
The substituted peptides can then be tested for binding to MHC molecules as well as recognition by CTL if bound to MHC. These variants have been tested for improved stability and are particularly useful in vaccine compositions.

Conservative amino acid substitutions in the amino acid sequence of a cancer-associated antigen polypeptide to produce a functionally equivalent variant of the cancer-associated antigen polypeptide typically refer to nucleic acid encoding the cancer-associated antigen polypeptide. Made by change. Such substitutions can be made by various methods known to those skilled in the art. For example, amino acid substitutions are Kunk
P by the method of el (Kunkel, Proc. Nat. Acad. Sci. USA 82: 488-492, 1985).
This can be done by CR-directed mutagenesis, site-directed mutagenesis, or by chemical synthesis of the gene encoding the cancer-associated antigenic polypeptide. Amino acid substitutions are made to small unique fragments of cancer-associated antigenic polypeptides such as antigenic epitopes recognized by autologous or allogeneic serum or cytolytic T lymphocytes (eg, SEQ ID NOS: 11-15). In the case of substitution, the substitution can be done by direct synthesis of the peptide. The activity of the functionally equivalent fragment of the cancer-associated antigen polypeptide is determined by cloning the gene encoding the modified cancer-associated antigen polypeptide into a bacterial or mammalian expression vector, introducing the vector into a suitable host cell, and It can be tested by expressing the relevant antigenic polypeptide and testing for the functional capacity of the cancer-associated antigenic polypeptide as disclosed herein. Chemically synthesized peptides can be tested directly for function, eg, binding to antisera that recognize related antigens.

The present invention has a number of uses as described herein, some of which are described elsewhere herein. First, the invention allows the isolation of cancer-associated antigenic protein molecules. Various methods well known to those of skill in the art can be utilized to obtain truncated cancer-associated antigenic molecules. The polypeptide can be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition. Alternatively, the expression vector can be introduced into a cell resulting in the production of the polypeptide. Alternatively, mRNA transcripts may be microinjected, or other methods introduced into the cell, resulting in production of the encoded polypeptide. Translation of mRNA in cell-free extracts, such as the reticulocyte lysate system, can also be used to produce the polypeptide. One of ordinary skill in the art can readily follow known methods for isolating cancer-associated antigenic polypeptides. Examples of these include, but are not limited to, immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography and immunoaffinity chromatography.

Isolation and identification of cancer-associated antigen genes also allows one of skill in the art to diagnose disorders characterized by cancer-associated antigen expression. These methods include determining the expression of one or more cancer-associated antigen nucleic acids and / or encoded cancer-associated antigen polypeptides and / or peptides derived therefrom. In the former case, such a determination can be carried out by any standard nucleic acid determination assay, including the polymerase chain reaction, or an assay using a labeled hybridization probe. In the latter case, such a determination can be performed by screening patient antisera for recognition of the polypeptide.

The present invention also allows isolation of proteins that bind to cancer-associated antigens as disclosed herein, including antibodies and cell-binding partners of cancer-associated antigens. Other uses for this are also described herein.

The invention also provides, in certain embodiments, "dominant negative" polypeptides derived from cancer-associated antigen polypeptides. A dominant negative polypeptide is an inactive variant of a protein that interacts with the cellular machinery to replace the active protein from that interaction with the cellular machinery or to compete with the active protein, thereby reducing the action of the active protein. Is. For example, a dominant negative receptor that binds the ligand but does not signal in response to the binding of the ligand may reduce the biological effects of ligand expression.

Similarly, a dominant negative catalytically inactive kinase that normally interacts with the target protein but does not phosphorylate the target protein may reduce phosphorylation of the target protein in response to cellular signals. Similarly, a dominant negative transcription agent that binds to a promoter site in the control region of a gene but does not increase gene transcription may reduce the action of a normal transcription agent by occupying the promoter binding site without increased transcription. .

The end result of expression of the dominant negative polypeptide in the cell is a reduction in the function of the active protein. One of ordinary skill in the art can assess the ability of a protein for dominant negative mutants using standard mutagenesis techniques to generate one or more dominant negative mutant polypeptides. For example, given the teachings contained herein for breast cancer, and T cell leukemia-related antigens, particularly those similar to known proteins with known activity, one of ordinary skill in the art would be familiar with site-directed mutagenesis. ,
The sequences of cancer-associated antigens may be modified by scanning mutagenesis, partial gene deletion or truncation, etc. (eg, US Pat. No. 5,580,723 and Sambrook et al., Molecular.
Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laborato
See ry Press, 1989). One of skill in the art can then test the population of mutated polypeptides for diminished selected activity and / or retention of such activity. Other similar methods for making and testing dominant negative variants of proteins will be apparent to those of skill in the art.

The present invention also includes agents such as polypeptides that bind to cancer-associated antigenic polypeptides. Such binding agents can be used, for example, in screening assays to detect the presence or absence of cancer-associated antigen polypeptides and complexes of cancer-associated antigen polypeptides with their binding partners, as well as isolated cancer-associated antigens. It can be used in purification protocols for the conjugation of polypeptides and cancer-associated antigenic polypeptides with their binding partners. Such agents can also be used to suppress the native activity of cancer-associated antigenic polypeptides, eg, by binding to such polypeptides.

Accordingly, the present invention includes peptide binding agents, which may be, for example, antibodies or fragments of antibodies that have the ability to selectively bind to a cancer-associated antigenic polypeptide. Antibodies include polyclonal and monoclonal antibodies prepared by conventional methods.

Significantly, as is well known in the art, only a small portion of the antibody molecule, the paratope, is involved in binding the antibody to its epitope (generally Clark, WR (1986) The.
Experimental Foundations of Modem Immunology Wiley & Sons, Inc., New Yo
rk;. Roitt, 1. (1991 ) Essential Immunologv 7 th Ed, Blackwell Scientific
See Publications, Oxford). For example, pFc 'and Fc regions are effectors of the complement cascade but are not involved in antigen binding. F (ab ') 2, in which the pFc' region was enzymatically cleaved or produced without the pFc 'region
Antibodies, called fragments, retain both the antigen binding sites of intact antibodies. Similarly, F
the c region was enzymatically cleaved or produced without the Fc region,
Antibodies called Fab fragments retain one of the antigen binding sites of an intact antibody molecule.
Continuing further, the Fab fragment consists of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.

The Fd fragment is the major determinant of antibody specificity (a single Fd fragment can associate with up to 10 different light chains without alteration of antibody specificity) and the Fd fragment is a single Retains the epitope binding ability in the separated state.

As is well known in the art, complementarity determining regions ((
CDR), which interacts directly with the epitope of the antigen and the framework regions (FR) that retain the tertiary structure of the paratope (generally Clark, 1986; Roi
See tt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1-FR4) separated by three complementarity determining regions (CDR1-CDR3).

CDRs, especially the CDR3 regions, and more particularly the heavy chain CDR3s, contribute significantly to antibody specificity.

The non-CDR regions of mammalian antibodies retain the epitope specificity of the original antibody,
It is well established in the art that similar regions of homologous or heterologous antibodies can be substituted. This is most clearly demonstrated in the development and use of "humanized" antibodies in which non-human CDRs are covalently linked to human FR and / or Fc / pFc 'regions to produce a functional antibody (eg, US Pat. , 816, 5
67, 5,225,539, 5,585,089, 5,693,7
62 and 5,859,205).

Thus, for example, PCT International Publication No. WO 92/04381 describes murine FR
It teaches the production and use of humanized murine RSV antibodies in which at least a portion of the region has been replaced by a FR region of human origin. Such antibodies, including fragments of intact antibodies that have the ability to bind antigen, are often referred to as "chimeric" antibodies.

Therefore, as will be apparent to one of skill in the art, the present invention provides F (ab ′) 2, Fab
, Fv and Fd fragments; Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions replaced by homologous human or non-human sequences; FR and / or CDR1 and / or CDR2 and Chimeric F (ab ′) 2 fragment antibody in which the light chain CDR3 region is replaced by a homologous human or non-human sequence; FR and / or CDR1 and / or CDR2 and / or light chain CDR3 region is a homologous human or non-human sequence Chimeric Fab fragment antibody replaced by; and FR and / or C
Chimeric Fd fragment antibodies in which the DR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences are also provided. The present invention also includes so-called single chain antibodies.

Accordingly, the present invention includes a number of sizes and types of polypeptides that specifically bind to cancer-associated antigenic polypeptides, and complexes of cancer-associated antigenic polypeptides with their binding partners. These polypeptides are also obtained from sources other than antibody technology. For example, such polypeptide binding agents can be provided in solution, in immobilized form, or by a degenerate peptide library that can be readily prepared as a phage display library. Combinatorial libraries of peptides containing one or more amino acids can also be synthesized. Libraries of peptoids and non-peptide synthetic moieties can be further synthesized.

Phage display may be particularly effective in identifying binding peptides useful according to the invention. Briefly, conventional techniques are used to prepare a phage library displaying inserts of 4 to about 80 amino acid residues (using, for example, m13, fd or lambda phage). The insert may represent, for example, a fully degenerate or biased array.

Next, a phage-carrying insert that binds to the cancer-associated antigen polypeptide can be selected.
This step can be repeated by several cycles of reselection of phage that bind the cancer-associated antigen polypeptide. The number of repeats results in the enrichment of phage-carrying specific sequences. DNA sequence analysis can be performed to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that binds to the cancer-associated antigen polypeptide can be determined. The procedure can be repeated using inserts containing some or all of the minimal linear portion plus a biased library containing one or more additional degenerate residues upstream or downstream thereof. .

Yeast two-hybrid screening methods can also be used to identify polypeptides that bind to cancer-associated antigenic polypeptides. Accordingly, the cancer-associated antigen polypeptides of the invention or fragments thereof are used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the cancer-associated antigen polypeptides of the invention. You can Such molecules can be used in screening assays, in purification protocols, in direct interference with the function of cancer-associated antigens, and for other purposes as will be apparent to those of skill in the art, as described above.

As detailed herein, the above antibodies and other binding molecules include
For example, it can be used to identify tissues that express the protein or to purify the protein. Antibodies can be further coupled by standard coupling techniques to specific diagnostic labeling agents for imaging cells and tissues expressing cancer-associated antigens, or therapeutically useful agents. Examples of the diagnostic agent include barium sulfate, iosetamic acid, iopanic acid, ipodate calcium, diatrizoate sodium, diatrizoate meglumine, metrizamide, sodium tyropanoate, and radiological diagnostic agents such as positron emitters such as fluorine-18 And carbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131 and indium-111, and nuclides for nuclear magnetic resonance such as fluorine and gadolinium. Other diagnostic agents useful in the present invention will be apparent to those of skill in the art.

As used herein, a “therapeutically useful agent” is desirably selectively labeled on cells expressing one of the cancer antigens disclosed herein. It includes any therapeutic molecule that may be mentioned, examples of which include antitumor agents, radioiodinated compounds, toxins, other cytostatics or cytolytic agents, and the like. Anti-tumor therapeutic agents are well known and examples include: aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, Dactinomycin, daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon-a, lomustine,
Mercaptopurine, methotrexate, mitotan, procarbazine HCI, thioguanine, vinblastine sulfate and vincristine sulfate. Further anti-tumor agents include Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce
A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gi
lman's “The Pharmacological Basis of Therapeutics,” Eighth Edition, 1
990, McGraw-Hill, Inc. (Health Professions Division). The toxin can be, for example, a protein such as pokeweed antiviral protein, cholera disinfectant, pertussis toxin, lysine, gelonin, abrin, diphtheria exotoxin, or Judemonas exotoxin. The toxin moiety can also be a high energy emitting radionuclide such as cobalt-60.

In the above methods and compositions, the antibodies prepared according to the present invention are also preferably specific for the cancer associated antigen / MHC complexes described herein.

As used herein, the term “disorder” refers to any pathological condition in which a cancer-associated antigen is expressed. One example of such a disorder is cancer, particular examples including breast cancer, kidney cancer and leukemia.

Tissue and / or cell samples for use in the various methods described herein include tissue biopsies including punch biopsy and cell scraping, as well as blood or other by aspiration or other methods. Can be collected by standard methods such as collection of bodily fluids.

In certain embodiments of the invention, immunoreactive cell samples are removed from a subject. By “immunoreactive cell” is meant a cell that can mature in an immune cell (eg, B cell, helper T cell or cytolytic T cell) upon appropriate stimulation. Thus, immunoreactive cells include CD34 + hematopoietic stem cells, immature T cells and immature B cells. When it is desired to produce cytolytic T cells that recognize the cancer-associated antigen, the immunoreactive cells express the cancer-associated antigen under conditions that favor the production, differentiation and / or selection of cytolytic T cells. Contacted with cells. Differentiation of T cell precursors to cytolytic T cells upon exposure to antigen is similar to clonal selection of the immune system.

Some therapeutic approaches based on this disclosure rely on a response by the subject's immune system to present one or more cancer-associated antigens, such as breast cancer, T cell leukemia cells, or other antigens. This results in lysis of the presentation cells. One such approach is the administration of autologous CTL specific for the cancer-associated antigen / MHC complex to a subject having abnormal cells of the phenotype in question. Developing such CTLs in vitro is within the ability of one of ordinary skill in the art. An example of a method for T cell differentiation is described in International Application PCT /
It is shown in US 96/05607. Generally, a sample of cells, such as blood cells, taken from a subject is contacted with cells that present the complex and are capable of inducing CTL to proliferate.

Target cells can be transfectants such as COS cells. These transfectants display the desired complex on their surface and when combined with the CTL of interest stimulate their proliferation. COS cells, like other suitable host cells, are widely available. Specific production of CTL clones is well known in the art. The clonally expanding autologous CTL is then administered to the subject.

CTL proliferation can be achieved by increasing tryptophan levels in T cell cultures, by inhibiting tryptophan catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO), or in T cell cultures. It can be increased by adding tryptophan therein. T cell proliferation is enhanced by increasing the rate of proliferation and / or prolonging the number of T cell divisions in culture. Furthermore, the increase in tryptophan in T cell culture also enhances the lytic activity of T cells grown in culture.

Another method for selecting antigen-specific CTL clones has recently been described (
Altman et al., Science 274: 94-96, 1996; Dunbar et al., Curr. Biol. 8: 413.
-416, 1998), in which the fluorogenic tetramer of the MHC class I molecule / peptide complex is used to detect specific CTL clones. In summary, soluble MHC class I molecules fold in vitro in the presence of 2-microglobulin and a peptide antigen that binds class I molecules. After purification, the MHC / peptide complex is purified and labeled with biotin. Tetramers are formed by mixing the biotinylated peptide-MHC complex with labeled avidin (eg phycoerythrin) in a 4: 1 molar ratio. The tetramer then receives CTL, such as peripheral blood or lymph nodes.
Contacted with a source of. The tetramer binds CTLs that recognize the peptide antigen / MHC class I complex. Cells bound by the tetramer can be sorted by fluorescence activated cell sorting to isolate reactive CTL. The isolated CTL can then be expanded in vitro for use as described herein.

Adoptive transfer (Greenberg, J. Immunol. 136 (5): 1917, 1986; Riddel et al., S
cience 257: 238, 1992; Lynch et al., Eur. J. Immunol. 21: 1403-1410, 1991;
Kast et al., Cell 59: 603-614, 1989), cells presenting the desired complex (eg, dendritic cells) have been combined with CTL to specifically target them, in order to detail the therapeutic methodology. Results in proliferation of CTLs. The expanded CTL is then administered to a subject who has a cellular abnormality characterized by certain abnormal cells that display the particular complex. The CTL then lyses the abnormal cells, thereby achieving the desired therapeutic purpose.

The above treatments assume that at least some of the subject's abnormal cells exhibit associated HLA / cancer associated antigen complexes. This is a method for identifying cells displaying a particular HLA molecule, as well as related sequences, in this case D of the cancer-associated antigen sequence.
This is very easy to determine as the art is familiar with how to identify cells that express NA. If cells displaying the relevant complex are identified by the screening methodologies described above, the cells can be combined with a sample from the patient, in which case the sample contains CTL. If the complex-presenting cells are lysed by the mixed CTL sample, then the cancer-associated antigen is present and that the subject is a suitable candidate for the set of therapeutic approaches above. Can be assumed.

Adoptive transfer is not the only form of treatment available according to the invention. CTL can also be initiated in vivo using a number of approaches. One approach is the use of non-proliferating cells that express the complex. The cells used in this approach include cells that normally express the complex, such as irradiated tumor cells, or cells that have been transfected with one or both of the genes required for the presentation of the complex (ie, antigenic peptide and presenting HLA. Molecule). Chen et al. (Proc
Natl. Acad. Sci. USA 88: 110-114, 1991) exemplify this approach and show the use of transfected cells expressing HPV-E7 peptide in a therapeutic regimen. Various cell types can be used.

Similarly, a vector carrying one or both of the genes of interest can be used. Viral or bacterial vectors are particularly preferred. For example, a nucleic acid encoding a cancer-associated antigenic polypeptide or peptide can be operably linked to promoters and enhancer sequences that direct the expression of the cancer-associated antigenic polypeptide or peptide in certain tissues or cell types. The nucleic acid can be incorporated into an expression vector.

Expression vectors are viral vectors constructed or modified to allow the insertion of exogenous nucleic acids, such as nucleic acids encoding cancer-associated antigens, as described elsewhere herein. It can be genomic, plasmid, or unmodified extracellular chromosomal nucleic acid. Nucleic acids encoding one or more cancer-associated antigens can also be inserted into the retroviral genome, thereby facilitating integration of the nucleic acid into the target tissue or cell type genome. In these systems, the gene is carried by microorganisms such as vaccinia virus, poxvirus, herpes simplex virus, retroviruses or adenoviruses, as well as substances that effectively "infect" the host cell. The resulting cells exhibit the complex and are recognized by autologous CTL, after which the cells proliferate.

A similar effect is obtained by combining the cancer-associated antigen or stimulatory fragment thereof with an adjuvant in v
This can be achieved by promoting incorporation into the antigen presenting cells in ivo.
Cancer-associated antigenic polypeptides are processed to produce peptide partners of the HLA molecule, whereas cancer-associated antigenic peptides can be presented without the need for further processing. Generally, a subject can receive an intradermal injection of an effective amount of a cancer-associated antigen. An initial dose followed by a booster dose can be administered according to immunization protocol standards in the art. Preferred cancer-associated antigens include those known to react with allogeneic cancer antisera shown in the examples below.

The present invention includes the use of various agents disclosed herein to “immunize” a subject or as a “vaccine”. As used herein, "immunization" or "vaccination" means increasing or activating an immune response to an antigen. It does not require elimination or eradication of symptoms, but rather a clinically favorable enhancement of the immune response to the antigen. Generally accepted animal models can be used for testing immunization against cancer with cancer-associated antigenic nucleic acids. For example, human cancer cells can be introduced into mice to create tumors and deliver one or more cancer-associated antigenic nucleic acids by the methods described herein. The effect on cancer cells (eg reduction in tumor size) can be assessed as a measure of the effectiveness of cancer-associated antigen nucleic acid immunization. Of course, testing of the above animal models using more conventional methods for immunization will include testing of one or more cancer-associated antigenic polypeptides or peptides derived therefrom to enhance the immune response. Administration may be included, optionally in combination with one or more adjuvants and / or cytokines. Methods for immunization, including formulation of vaccine compositions and selection of doses, routes of administration and dosing regimens (eg, initial and one or more booster doses) are well known in the art. The test can also be performed in humans, where the endpoint is to test for the presence of increased levels of circulating CTL on cells bearing the antigen, to test for levels of circulating antibodies to the antigen, on cells expressing the antigen. Test for the presence of.

As part of an immunization composition, one or more cancer-associated antigens or stimulatory fragments thereof are administered with one or more adjuvants to induce or induce an immune response. Increase. Adjuvants are incorporated into or administered with the antigen, which enhances the immune response. Adjuvants enhance the immunological response by providing a reservoir of antigens (extracellular or in macrophages), activating macrophages and stimulating a specific set of lymphocytes.

Many types of adjuvants are well known in the art. Specific examples of adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham), a homologue obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 lipopolysaccharide of the genus Salmonella; saponins such as QS21 (SmithKline Beecham), Quillja. sapo
Pure QA-21 saponins purified from naria extract; PCT application WO 96/33
739 (SmithKline Beechm), DQS21; QS-7, QS-17
, QS-18 and QS-L1 (So et al., Mol. Tells 7: 178-186, 1997);
Freund's incomplete adjuvant; Freund's complete adjuvant; Montanide; Alum; CpG oligonucleotides (eg Kreig et al., Nature 374
: 546-9, 1995); and various water-in-oil emulsions prepared from biodegradable oils such as squalene and / or tocopherol. Preferably, the peptide is administered in admixture with the DQS21 / MPL combination. DQS
The ratio of 21 to MPL is typically about 1:10 to 10: 1, preferably about 1: 5.
5: 1, more preferably about 1: 1. Typically for human administration,
DQS21 and MPL are present in the vaccine formulation in the range of about 1 ug to about 100 Rg. Other adjuvants are known in the art and can be used in the present invention (eg, Goding, Monoclonal vlntibodies: Principles and Practice,
See 2nd Ed., 1986).

Methods of making mixtures or emulsions of peptides and adjuvants are well known to those of ordinary skill in the art of vaccination.

Other agents that stimulate a subject's immune response can also be administered to the subject. For example, other cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties. Many other cytokines useful for such purposes are known to those of skill in the art and have been shown, for example, to enhance the protective effects of vaccines (eg, Science 268: 1432-1434, 1995) interleukin-12 (IL-12), GM-CSF and IL-18.
Thus, cytokines can be co-administered with an antigen and an adjuvant to increase the immune response to the antigen.

There are a number of immune response-promoting compounds that can be used in vaccination protocols. These include costimulatory molecules provided in protein or nucleic acid form. Such costimulatory molecules include molecules that are expressed in dendritic cells (DC) and that interact with the CD28 molecule expressed in T cells, B7-1 and B7-2 (each C
D80 and CD86) molecules. This interaction is antigen / MHC / T
CR stimulation (Signal 1) Provides costimulation (Signal 2) to T cells to increase T cell proliferation and effector function. B7 is a T cell, CTLA4 (CD152)
A test that also interacts with CTLA4 and B7 ligands interacts with B7-CTLA.
We show that 4 interactions can enhance anti-tumor immunity and CTL proliferation (Zh
eng P., et al. Proc. Natl. Acad. Sci. USA 95 (11): 6284-6289 (1998)).

B7 are typically not expressed on tumor cells, where they are not efficient antigen presenting cells (APC) for T cells. Introduction of B7 expression allows tumor cells to more efficiently stimulate CTL proliferation and effector function. B7 / I
The combination of L-6 / IL-12 costimulation results in IFN-γ and Th in the T cell population.
1 has been shown to induce a cytokine profile leading to further enhancement of T cell activity (Gajewski et al., J. Immunol. 154: 5637-5648 (1995)).
. Tumor cell transfection with B7 is described by Wang et al. (J. Immunol., 19: 1-8
(1986)) and discussed in connection with in vitro CTL extensions for adoptive transfer immunotherapy.

Another delivery mechanism for the B7 molecule is nucleic acid (naked DNA) immunization (Ki
m J., et al. Nat Biotechnol., 15: 7: 641-646 (1997)) and recombinant viruses such as adeno and pox (Wendtner et al., Gene Ther., 4: 7: 726-735 (
1997)) is included. These systems allow the construction of expression cassettes for co-expression of B7 with other molecules, such as the antigens or fragment (s) of antigens (including polytopes) or cytokines discussed herein, among others. And easy to apply all. These delivery systems have been developed for the introduction of appropriate molecules in vitro as well as in
It can be used for the case of in vivo vaccination. T cells in vitro and in vi
The use of anti-CD28 antibody for direct stimulation with vo can also be considered.

Similarly, the inducible costimulatory molecule ICOS that induces a T cell response to foreign antigens.
Can be adjusted, for example, by the use of anti-ICOS antibodies (Hutloff et al.
, Nature 397: 263-266, 1999).

Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCs and some tumor cells and interacts with CD2 expressed on T cells. This interaction induces T cell IL-2 and IFN-γ production and thus may complement but does not replace the B7 / CD28 costimulatory interaction (Parra et al., J. Immunol., 158: 637- 64
2 (1997), Fenton et al., J. Immunother., 21: 2: 95-108 (1998)).

Lymphocyte function associated antigen-1 (LFA-1) is expressed on leukocytes and interacts with ICAM-1 expressed on APCs and some tumor cells. This interaction induces T-cell IL-2 and IFN-γ production and thus may complement but does not replace the B7 / CD28 costimulatory interaction (Fenton et al., J. Immunother., 2
1: 2: 95-108 (1998)).

LFA-1 is thus a further example of a costimulatory molecule that can be provided in vaccination protocols in the various methods discussed above for B7.

Complete CTL activation and effector function are associated with Th cell CD40L (CD40
T) due to the interaction between the ligand) molecule and the CD40 molecule expressed by DC
Requires h-cell help (Ridge et al., Nature, 393: 474 (1998), Bennett et
al., Nature, 393: 478 (1998), Schoenberger et al., Nature, 393: 480 (1
998)). This mechanism of this costimulatory signal is due to DC (APC) B7
And related upregulation of IL-6 / IL-12 production. Thus, the CD40-CD40L interaction complements the signal 1 (antigen / MHC-TCR) and signal 2 (B7-CD28) interaction.

The use of anti-CD40 antibodies to directly stimulate DC cells is predicted to enhance responses to tumor antigens normally encountered outside the inflammatory context or presented by non-professional APCs (tumor cells). To be done. Th help and B7 costimulatory signals are not provided in these situations. This mechanism can be used in the context of antigen-pulsed DC-based therapy or in situations where Th epitopes are not restricted to known TRA precursors.

Cancer-associated antigenic polypeptides or fragments thereof can also be used to isolate their native binding partners. Isolation of such a binding partner can be performed by a well-known method. For example, the isolated cancer-associated antigen polypeptide is bound to a support, such as a chromatographic medium, such as polystyrene beads or filters, and then a solution suspected of containing a binding partner is applied to the support. it can. If a binding partner that is capable of interacting with the cancer-associated antigen polypeptide is present in solution, it will bind to the support-bound cancer-associated antigen polypeptide. The binding partner can then be isolated.

The present invention is recombinant in expression vectors and in prokaryotes (eg E. coli) or eukaryotes (eg dendritic cells, B cells, CHO cells, COS cells, yeast expression systems, and insect cells). It will also be appreciated that this includes the use of cancer-associated antigen cDNA sequences to transfect host cells and cell lines, if baculovirus expression).

Particularly useful are mammalian cells such as humans, mice, hamsters, pigs, goats, primates and the like.

They can be of a wide variety of tissue types, including primate cells and cell lines.

Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells and fetal stem cells. Expression vectors require that the relevant sequences, ie, the nucleic acids described above, are operably linked to a promoter.

The present invention also contemplates delivery of nucleic acids, polypeptides or peptides for vaccination. Delivery of polypeptides and peptides can be accomplished by standard vaccination protocols well known in the art. In another embodiment, delivery of the nucleic acid is by ex vivo methods, i.e., removing cells from a subject, genetically engineering the cells to contain a cancer-associated antigen, and reintroducing the engineered cells into the subject. Can be achieved. An example of such an approach is abbreviated in US Pat. No. 5,399,346, as well as the documents filed in the package of that patent (all of which are publicly available documents). ). In general, it involves the in vitro introduction of a functional copy of the gene into the cell (s) of interest, as well as the return of the genetically engineered cell (s) to the subject. A functional copy of a gene is under operable control of regulatory elements that allow expression of the gene in genetically engineered cell (s). Many transfection and transduction techniques, as well as suitable expression vectors, are well known to those of skill in the art, and some of them are in PCT application WO 95/00.
654. In vivo nucleic acid delivery using vectors such as viruses and targeted liposomes is also contemplated by the present invention.

In a preferred embodiment, the viral vector for delivering a nucleic acid encoding a cancer-associated antigen is adenovirus, adeno-associated virus, poxvirus such as vaccinia virus and attenuated poxvirus, Semliki Forest Fever virus, Venezuelan equine. It is selected from the group consisting of encephalomyelitis virus, retrovirus, Sindbis virus and Ty virus-like particle. Examples of viruses and virus-like particles that have been used to deliver exogenous nucleic acids include replication defective adenoviruses (e.g., Xiang et al., Virology 219: 220-227, 1996; Eloit et al.,
J. Virol. 7: 5375-5381, 1997; Chengalvala et al., Vaccine 15: 335-339,
1997), modified retrovirus (Townsend et al., J. Virol. 71: 3365-3374, 19).
97), a non-replicating retrovirus (Irwin et al., J. Virol. 68: 5036-5044, 1994.
), Replication-defective Semliki Forest Fever virus (Zhao et al., Proc. Natl. Acad. Sci.
USA 92: 3009-3013, 1995), Canarypox virus and highly attenuated vaccinia virus derivatives (Paoletti, Proc. Natl. Acad. Sci. USA 93: 11349-11353, 1996).
), Non-replicating vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA 93: 11341).
-11348, 1996), replication vaccinia virus (Moss, Dev. Biol. Stand. 82: 55-63).
, 1994), Venezuelan equine encephalomyelitis virus (Davis et al., J. Virol. 70: 37.
81-3787, 1996), Sindbis virus (Pugachev et al., Virology 212: 587-.
594, 1995) and Ty virus-like particles (Allsopp et al., Eur. J. Immunol 26:
1951-1959, 1996). In a preferred embodiment, the viral vector is an adenovirus.

Another preferred virus for certain applications is the adeno-associated virus, double stranded DNA virus. Adeno-associated virus can infect a wide range of cell types and cell types and primaries, and can be engineered to be replication defective. It further allows multiple sets of transductions with the advantages of heat and lipid solvent stability, high transduction frequency in cells of diverse lineages including hematopoietic cells, and lack of suppression of superinfection. Adeno-associated virus may integrate into human cell DNA in a site-specific manner, thereby minimizing the potential for insertional mutagenesis and variability in inserted gene expression. Furthermore, wild-type adeno-associated virus infections were passaged in tissue culture for more than 100 passages in the absence of selection pressure, indicating that adeno-associated virus genome integration was a relatively stable event. means. Adeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on noncytotoxic eukaryotic viruses in which non-essential genes have been replaced by those genes. Non-cytotoxic viruses include retroviruses whose life cycle involves reverse transcription of genomic viral RNA into DNA and subsequent proviral integration into host cell DNA.

Adenoviruses and retroviruses have been approved for human gene therapy trials. In general, retroviruses are replication-defective (ie, they can direct the synthesis of desired proteins, but are unable to produce infectious particles). Such genetically modified retroviral expression vectors have general utility for high efficiency transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses (steps of incorporating exogenous genetic material into a plasmid, steps of transfecting a packaging cell line with a plasmid, steps of producing a recombinant retrovirus by a packaging cell line). , Including the steps of collecting viral particles from the tissue medium, as well as infecting target cells with viral particles).
Kriegler, M., “Gene Transfer and Expression, A Laboratory Manual,” WH
Freeman Co., New York (1990) and Murry, EJ Ed. ”Methods in Molecul
ar Biology, ”vol. 7, Humana Press, Inc., Clifton, New Jersey (1991).

Preferably, the nucleic acid delivery vector described above contains (1) exogenous genetic material capable of being transcribed and translated into mammalian cells, and capable of inducing an immune response in a host, and ( 2) The surface contains a ligand that selectively binds to a receptor on the surface of a target cell, eg, a mammalian cell, thereby acquiring entry into the target cell.

Various techniques for introducing the nucleic acids of the invention into cells can be used, depending on whether the nucleic acid was introduced into the host in vitro or in vivo.

Such techniques include transfection of nucleic acid-CaPO ₄ precipitate,
Examples include transfection of a nucleic acid associated with DEAE, transfection or infection with the above-mentioned virus containing the nucleic acid, liposome-mediated transfection, and the like. For certain applications it is preferable to target the nucleic acid to specific cells. In such cases, the vehicle used to deliver the nucleic acid of the invention into cells (eg, retroviruses or other viruses; liposomes) can have targeting molecules attached to it. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell, or a ligand for a receptor on the target cell can be attached to or incorporated into the nucleic acid delivery vehicle. Preferred antibodies include antibodies that selectively bind a cancer-associated antigen, either alone or in complex with MHC molecules. Monoclonal antibodies are particularly preferred. When liposomes are used to deliver the nucleic acids of the invention, proteins that bind surface membrane proteins associated with endocytosis are incorporated into liposome formulations for targeting and / or to facilitate uptake. obtain.

Such proteins include capsid proteins or fragments thereof that are tropic for specific cell types, antibodies to proteins that undergo internalization during the cycle, intracellular localization targets, and intracellular half-lives. Examples include proteins that enhance the. Macromolecular delivery systems have also been successfully used to deliver nucleic acids into cells, as is known to those of skill in the art. Such systems even allow oral delivery of nucleic acids.

The therapeutic agents of the invention can be administered by any conventional route, including injection, or by gradual infusion over time. Administration can be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal.

When the cancer-associated antigenic peptide is used for vaccination, a mode of administration that effectively delivers the cancer-associated antigen and adjuvant can be used so that the immune response against the antigen is increased. For administration of the cancer-associated antigenic peptide in an adjuvant, preferred methods include intradermal, intravenous, intramuscular and subcutaneous administration. Although these are preferred embodiments, the invention is not limited to the particular mode of administration disclosed herein.

Standard references in the art (eg, Remington's Pharmaceutical Sciences,
18 ^th edition, 1990) provide a mode of administration and formulations for the delivery of immunogens containing adjuvants or in non-adjuvant carriers. When the antibody is used therapeutically, the preferred route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. In general, such systems should utilize components that do not significantly impair the biological properties of the antibody, eg, paratope binding capacity (eg, Sciarra and Cutie, “Aerosols,” in
^{Remington's Pharmaceutical Sciences, 18 th edition} , 1990, pp 1694-1712 reference, the contents of which are incorporated herein by reference). One of ordinary skill in the art can readily determine various parameters and conditions for producing antibody aerosols without resorting to undue experimentation. When using the antisense preparations of the present invention, slow intravenous administration is preferred.

The compositions of the invention are administered in an effective amount. An "effective amount" is an amount of a cancer-associated antigen composition that alone or in combination with additional doses produces a desired response, eg, enhances an immune response against a cancer-associated antigen. When treating a particular disease or condition characterized by the expression of one or more cancer-associated antigens, such as breast cancer, or T cell leukemia, the desired response is inhibition of disease progression. This may include only temporarily slowing the progression of the disease, but more preferably involves permanently arresting the progression of the disease. This can be monitored by routine methods, or by the diagnostic methods of the invention discussed herein. The desired response to treatment of a disease or condition is also to delay or even prevent the onset of the disease or condition.

Such an amount will, of course, depend on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size and weight, duration of treatment, nature of the combination treatment (if any). (If any), depending on the particular route of administration, and similar agents within the knowledge and expertise of health workers. These agents are well known to those of ordinary skill in the art and can be handled with no more than routine experimentation. It is generally preferred that the maximum dose of the individual components or combinations thereof be used, ie the maximum safe dose according to sound medical judgment. However, one of ordinary skill in the art will appreciate that a patient may claim a low or tolerable dose for medical reasons, psychological reasons, or for virtually any other reason.

The pharmaceutical compositions used in the above methods are preferably sterile and are in an effective amount to produce the desired response in the unit of weight or volume suitable for administration to a patient. Contains nucleic acid encoding the relevant antigen. The response is mediated by the cancer-associated antigens via the reporter system, for example by measuring downstream effects such as gene expression or by measuring the physiological effects of the cancer-associated antigen composition such as tumor regression or reduction of disease symptoms. It can be measured by determining the immune response after administration of the composition. Other assays are known to those of skill in the art and can be used to measure the level of response.

The dose of the cancer-associated antigenic composition (eg, polypeptide, peptide, antibody, cell or nucleic acid) administered to the subject should be selected according to different parameters, particularly according to the mode of administration used and the condition of the subject. You can Other agents include the desired duration of treatment. If the response in the subject is insufficient at the initial dose applied, higher doses (or effective high doses via different, more localized delivery routes) can be used to the extent that the patient's tolerability permits. .

In general, for treatment to elicit or increase an immune response, a dose of a cancer-associated antigen is prescribed and doses of 1 ng to 1 mg, preferably 10 ng to 100 ug, according to any standard technique in the art. Is administered.

If a nucleic acid encoding a cancer-associated antigen or variant thereof is used, a dose from 1 ng to 0.1 mg will generally be prescribed and administered according to standard procedures.
Other protocols for administration of cancer-associated antigen compositions are known to those of skill in the art,
In that case, the dose, the injection plan, the injection site, the administration method (for example, in the tumor) and the like are different from the above. Administration of the cancer-associated antigenic composition to a non-human mammal, eg, for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above.

When administered, the pharmaceutical compositions of the invention are applied in pharmaceutically acceptable amounts as well as pharmaceutically acceptable preparations. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffers, preservatives, compatible carriers and optionally other therapeutic agents. When used in a medicament, the salt should be pharmaceutically acceptable, but the non-pharmaceutically acceptable salt may be conveniently used to prepare its pharmaceutically acceptable salts Is not excluded from the range. Such pharmacologically and pharmaceutically acceptable salts include the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid,
Examples thereof include, but are not limited to, those prepared from succinic acid and the like.

Furthermore, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.

The cancer-associated antigen composition may be optionally combined with a pharmaceutically acceptable carrier.
As used herein, the term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to humans. The term "carrier" means an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical composition are in a manner such that there are no interactions that substantially impair the desired pharmaceutical efficacy,
It can be mixed with the molecules of the invention and with one another.

The pharmaceutical compositions may contain suitable buffering agents, for example salt acetic acid, salt citric acid, salt boric acid and salt phosphoric acid.

The pharmaceutical compositions may also optionally contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens and thimerosal.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with the carrier which constitutes one or more accessory ingredients. generally,
The compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of active compound. Other compositions include suspensions in aqueous or non-aqueous liquids, such as syrups, elixirs or emulsions.

Compositions suitable for parenteral administration are preferably isotonic with the blood of the recipient,
It is convenient to include sterile aqueous or non-aqueous preparations of cancer-associated antigenic polypeptides or nucleic acids. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations are for example 1,3
It can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. Moreover, sterile fixed oils are conveniently used as a solvent or suspending medium. For this purpose any bland fixed oil can be used including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectable drugs. Suitable carrier formulations for oral, subcutaneous, intravenous, intramuscular administration, etc. are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Eas
Can be found in ton, PA.

Examples Example 1: SEREX Screening of Breast Cancer Cells The cDNA library preparation and screening procedure was standard. c
ZAP Express using DNA library kit (Stratagene, La Jolla, CA)
A breast cancer library was constructed in a vector from mRNA isolated from breast tumors of a 59 year old female patient. The T cell leukemia library was made from mRNA isolated from adult T cell leukemia cells. Sahin et al., Proc. Natl. Acad. Sci. USA 92:
As described by 11810-11813, 1995, using autologous patient sera to obtain cDNA.
The A library was screened.

The following modifications were made to the standard procedure. E. coli lysate and E. coli phage lysate bound to Sepharose 4B (5 prime-3 prime, Boulder Co, Cat #
5307-050901 and # 5307-110901) were used to preabsorb serum. Preabsorbed serum was diluted 1: 200 and incubated overnight at + 4 ° C with nitrocellulose membranes containing phage plaques. Peroxidase-conjugated goat anti-human IgG (Jackson Immunoresearch Labs. Inc. Cat. # 10) specific for Fc-γ fragment
9-035-008) and after visualization with 3,3′-diaminobenzidine, clones coding for human immunoglobulin sequences were identified. After washing, the membranes were incubated with alkaline phosphatase-conjugated goat anti-human IgG (Jackson Immunoresearch Labs. Inc. Cat. # 109-055-008), which is Fc-γ fragment specific, and 5-bromo-4-chloro-3. Reactive phage plaques were visualized by incubation with indolyl-phosphate and nitroblue tetrazolium.

Clones were purified and analyzed for nucleic acid sequences. Sequence comparisons showed that these clones represent cDNA from two distinct genes.

The first gene contains the four nucleotide sequences shown herein. Clone HOM-TALL1-5 was isolated from an adult T cell leukemia library. H
The OM-TALL1-5 sequence is HOM-TALL1-5 (5 ′) (SEQ ID NO: 1).
And HOM-TALL1-5 (3 ′) (SEQ ID NO: 3). Clone MO-
BC-203 was isolated from a breast cancer library. The MO-BC-203 sequence is M
O-BC-203 (5 ') (SEQ ID NO: 4) and MO-BC-203 (3') (
SEQ ID NO: 6) is included.

The polypeptides encoded by the long open reading frames of these sequences are SEQ ID NO: 2 (HOM-TALL1-5) and SEQ ID NO: 5 (MO-B).
C-203).

[0232] T cell and breast cancer clones are not identical and therefore may represent allelic variants of the same gene from different individuals. The overlap in the clone is shown below: nucleotides 469-1 of HOM-TALL1-5 (5 ') (SEQ ID NO: 1).
178 is nucleotides 1 to 71 of MO-BC-203 (5 ′) (SEQ ID NO: 4).
2 and the nucleotides 1 to 524 of HOM-TALL1-5 (3 ′) (SEQ ID NO: 3) match nucleotides 44 to 567 of MO-BC-203 (3 ′) (SEQ ID NO: 6). To do.

The clones MO-BC-203 and HOM-TALL1-5 were 80-90%.
Of the TRF1-interacting ankyrin-related ADP-ribose polymerase and is partially homologous to human tankyrase. The base full-length RNA in tissue blot analysis is approximately 7 kb. Full-length RNA is expressed in skeletal muscle and placenta, and to a lesser extent in other tissues.

National Cancer Institute Cancer Genome Analysis Project (NCI CGAP: National Institu
te Cancer Genome Anatomy Project) database HOM-TALL1
Sequences similar to -5 and MO-BC-203 have been shown for kidney cancer, adrenal adenomas, colon adenocarcinomas,
From pancreatic and uterine adenocarcinomas, some of which are listed in Table 4.

The protein encoded by clone MO-BC-203 was tested 11
React positively with four of the breast cancer sera of.

From the breast cancer library, the second gene, MO-BC-416 (SEQ ID NO: 7)
) Was isolated. The long open reading frame polypeptide encoded by the MO-BC-416 nucleotide sequence is set forth as SEQ ID NO: 8.

Clone MO-BC-416 represents a novel alternative transcript of the known gene Cxorf5, which does not contain exon 19 of Cxorf5. Cxorf
Two alternative transcripts of 5 have been previously described (deConciliis et al., Genomi
cs 51 (2): 243-250 (1998)). The previously described alternative transcripts are exon 10 (transcript Cxorf5-1, emb! Y15164) HS717AX) or exon 10a (transcript Cxorf5-2, emb1Y163551HS71).
7AXAS).

Exon 10 of clone MO-BC-416 was clone Cxorf5-1 (e
mb1Y151641HS717AX). In addition to the lack of exon 19, clone MO-BC-416 showed 10 compared to Cxorf5-1.
It is truncated by 42 bp at its 5'end and its 3'end is shorter than in Cxorf5-1 due to the alternative polyadenylation signal.

The two previously known transcripts of the gene Cxorf5 have different distributions in human tissues. For example, the Cxorf5-1 transcript is ubiquitously expressed,
On the other hand, the Cxorf5-2 transcript is not expressed in skeletal muscle, liver and heart.

Sequences similar to MO-BC-416 in the National Cancer Society Cancer Genome Analysis Project (NCI CGAP) database are from germ cell tumors, colon adenocarcinoma, ovarian cancer, Wilms tumor and breast tumors, of which Some are listed in Table 4.

The protein encoded by clone MO-BC-416 does not react with 24 normal sera, but with one of 11 breast cancer sera and with 3 kidney cancer sera (not with Wilms tumor). Reacts with one of them. Renal cell carcinoma and Wilms tumor (nephroblastoma) are not the same. Wilms tumor is a fairly rare form of renal cancer that occurs predominantly in children under the age of 4 years and contains a chromosome 11 mutation.

Example 2 Preparation of Recombinant Cancer-Associated Antigens Recombinant proteins are prepared according to standard procedures to facilitate the screening of patient sera for antigens reactive with cancer-associated antigens, eg, by ELISA. In one method of production, a clone encoding a cancer-associated antigen is subcloned in a baculovirus expression vector and the recombinant expression vector is introduced into a suitable insect cell. The baculovirus / insect cloning system is preferred because the translational modifications are performed in insect cells. Another preferred eukaryotic system is Invitrog
Drosophila expression system from En. A clone expressing a large amount of recombinant protein is selected and used to produce the recombinant protein. Any of the patients used to isolate the clone, either with the serum from the patient used to isolate the particular clone or, in the case of a cancer-associated antigen recognized by allogeneic serum, Recombinant proteins are tested for antibody recognition with either sera or sera that recognize the cloned gene product.

Alternatively, the cancer-associated antigen clone is inserted into a prokaryotic expression vector for production of recombinant protein in bacteria. Other systems may also be used, including yeast expression systems and mammalian cell culture systems.

Example 3 Preparation of Antibodies Against Cancer-Associated Antigens Recombinant cancer-associated antigens produced as in Example 2 above are used to generate polyclonal antisera and monoclonal antibodies according to standard procedures.

The so-produced antisera and antibodies can be used to treat cancers by using antisera / antibodies in assays of cell extracts of patients known to express particular cancer-associated antigens (eg, ELISA assay). Test for proper recognition of related antigens.
These antibodies are used for experimental purposes (eg, localization of cancer-associated antigens, immunoprecipitation, Western blot, etc.) and diagnostic purposes (eg, examination of tissue biopsy extracts, tests for the presence of cancer-associated antigens). obtain.

Example 4: Breast Cancer and T Cell Leukemia-Associated Antigen Expression in Cancers of Similar and Different Origins Expression of one or more breast cancer and T cell leukemia associated antigens was tested in a series of tumor samples. , Other methods, if any, are diagnosed and / or treated by the methods described herein. Tumor cell lines and tumor samples are tested for cancer-associated antigen expression, preferably by RT-PCR according to standard procedures. Northern blots are also used to test for expression of cancer-associated antigens. Antibody-based assays such as ELISA and Western blot can also be used to determine protein expression. The preferred method of testing for expression of cancer-associated antigens (in other cancers, as well as in patients with yet another same-type cancer) is allogeneic serotyping using a modified SEREX protocol (as above).

In all of the above, extracts from tumors of patients who provided serum for the initial isolation of cancer-associated antigens are used as positive controls. Cells containing the recombinant expression vector described in the above example can also be used as a positive control.

The results resulting from the above experiments are diagnostic (eg to determine the presence of cancer, to determine the prognosis of a treated patient etc.) and therapeutic (eg vaccine composition etc.)
A panel of multiple cancer-associated nucleic acids and / or polypeptides for use in.

Example 5: HLA Typing of Patients Positive for Cancer-Associated Antigens To determine whether HLA molecules represent peptides derived from cancer-associated antigens, express breast cancer and / or T-cell leukemia-associated antigens. Patient cells are HLA typed. Peripheral blood lymphocytes are taken from the patient and typed for HLA class I or class II as well as for specific subtypes of class I or class II. Tumor biopsy samples may also be used for typing. By any of the standard methods in the field of clinical immunology, for example by recognition with specific monoclonal antibodies,
Alternatively, HLA typing can be performed by HLA allele-specific PCR (eg, as described in WO 97/31126).

Example 6 Characterization of Cancer-Associated Antigen Peptides Presented by MHC Class I and Class II Molecules Antigens that elicit an antibody response in a subject can also elicit a cell-mediated immune response.
The cells have cell surface MHC class I or class I for immune surveillance.
The protein is processed into peptides for display on the I molecule. Some kind of M
The peptides presented by the HC / HLA molecule generally match the motif.
These motifs are known in some cases and can be used to screen breast cancer, and / or T cell leukemia-associated antigens for the presence of potential class I and / or class II peptides. A summary of class I and class II motifs has been published (eg Rammensee et al., Immunogenetics 41: 178-
228, 1995). Based on the above experimental results, individual breast cancer and / or T
The HLA type, which represents a cell leukemia-associated antigen, is known. Therefore, these HLA
Preference is given to the motif of the peptide represented by the molecule.

Computer algorithms can also be used to examine class I and class II motifs. For example, computer programs have been described for predicting potential CTL epitopes based on known class I motifs (eg Parker et al., J Immunol. 152: 163, 1994; D'Amaro et al., Human Immuno
l. 43: 13-18, 1995; Drijfhout et al., Human Immunol. 43: 1-12, 1995).
. The HLA binding prediction is available at the URL http://bimas.dcrt.nih.gov. At National Institu
This can be conveniently done using the algorithms available on the tes of Health World Wide website over the Internet.

Methods for determining HLA class II peptides and making substitutions therefor are also known (eg, Stringer and Wucherpfen.
nig (International application PCT / US96 / 03182)).

Example 7: Identification of Portions of Cancer-Associated Polypeptides Encoding Antigens To determine whether cancer-associated antigens isolated as described above can elicit a cytolytic T lymphocyte response, The following method is implemented. Localizing an antigenic peptide within a cancer-associated antigen clone by matching the consensus for one of the clones encoding the cancer-associated antigen polypeptide, or corresponding to a putative protein, and the appropriate HLA class I molecule (as above) CTL clones are generated by stimulating peripheral blood lymphocytes (PBLs) of a patient with autologous normal cells transfected with irradiated PBLs loaded with activating synthetic peptides (eg, Knuth et al., Proc. Natl
Acad. Sci. USA 81: 3511-3515, 1984; van der Bruggen et al., Eur. J. Im
munol. 24: 3038-3043, 1994). Brichard et al. (Eur. J. Immunol. 26: 224-2
30, 1996), these CTL clones are screened for specificity against cancer-associated antigen clones and COS cells transfected with autologous HLA alleles. By measuring the release of TNF from cytolytic T lymphocytes or by the 5'Cr release assay (Herin et al.,
Int. J. Cancer 39: 390-396, 1987), which determines CTL recognition of cancer-associated antigens. C
If the TL clone specifically recognizes the transfected COS cells, a shorter fragment of the cancer-associated antigen clone transfected in the COS cells is tested to identify the region of the gene encoding the peptide. To do. Fragments of cancer-associated antigen clones are prepared by exonuclease III digestion or other standard molecular biology methods. Synthetic peptides are prepared to confirm the exact sequence of the antigen.

[0254] Optionally, shorter fragments of the cancer-associated antigen cDNA are generated by PCR. Shorter fragments are used to trigger TNF release or 5'Cr release as described above.

Synthetic peptides are prepared that correspond to some of the shortest fragments of cancer-associated antigen clones that elicit TNF release. The progressively shorter peptides are synthesized to determine the optimal cancer-associated antigen tumor rejection antigen peptide for a given HLA molecule.

A similar method is performed to determine if the cancer-associated antigen contains one or more HLA class II peptides recognized by T cells. The sequences of cancer-associated antigenic polypeptides can be examined for HLA class II motifs as described above.
In contrast to class I peptides, class II peptides are displayed by a limited number of cell types. Therefore, for these experiments, preferably HLA class I
Dendritic cells or B cell clones expressing the I molecule are used.

Example 8: Determination of full length MO-BC-203 nucleic acid molecule The MO-BC-203 clone identified in Example 1 was extended by standard techniques to obtain full length nucleotides and the amino acid sequence of this gene product. .

The nucleotide sequence is shown as SEQ ID NO: 9 and the amino acid sequence is shown as SEQ ID NO: 10.

Example 9: Expression of MO-BC-203 Antigen in Cancer Patient Sera MO-B with respect to reactivity with sera from various normal cancer patients listed below.
The C-203 antigen was tested.

[Table 1]

Example 10: Determination of peptide epitopes of MO-BC-203 To identify possible epitopes of MO-BC-203, MO-BC-2.
We synthesized overlapping peptides derived from 03 protein and
Tested for recognition by 8'T lymphocytes. According to the manufacturer's instructions using a commercially available enzyme-linked immunosorbent spot (ELISPOT) assay for ^IFN-∧ y, it was determined CD8'T lymphocyte responses. Five MO-as recognized by CD8 + T lymphocytes of HLA-A2 positive breast cancer patient (NW1100).
A peptide derived from BC-203 was identified: DLADPSAKAV (SEQ ID NO: 11).
, LLSYGADPTL (SEQ ID NO: 12), LLAHGADAPTL (SEQ ID NO: 1)
3), GMFGAGIYFA (SEQ ID NO: 14), ATADALFQV (SEQ ID NO: 15).

The specificity of recognition of these five peptides was confirmed by a proliferation assay. HL
A-A2 + T2 target cells were labeled with 5'Cr as described above, then MO-BC.
Incubated for 30 minutes in 96 well microplates in the presence of -203 peptide.

Next, an HLA-A2 positive breast cancer patient (N
CTLs from W1100) were added in equal volume of medium at various effector: target ratios (see Table 2). Chromium-51 release was measured after 4 hours. The percentage of specific lysis was calculated as follows:

[Equation 1] The peptide GMFGAGIYFA (SEQ ID NO: 14) was positive in these cytotoxicity assays, as shown in Table 2 below.

[Table 2]

Example 11: SEREX Screening of Ovarian Cancer Cells A cDNA library was prepared from mRNA isolated from ovarian cancer cells. As described above, Sahin et al., Proc. Natl. Acad. Sci. USA 92: 11810-11813, 199.
A cDNA library was screened using autologous patient sera from 5. Isolated sequence MOVA-91 (nucleotide sequence: SEQ ID NO: 16, polypeptide sequence:
SEQ ID NO: 17) appears to be derived from the Homo sapiens histone deacetylase 3 gene (HDAC3, Yang et al., J. Biol. Chem. 272 (44): 28001-2800.
7, 1997; Genbank deposit number NM003883.1, U66914, AF0397
03, U75697, U75696, AF005482).

Known transcripts of the HDAC3 gene are ubiquitously expressed in many cell types (Yang e
al., 1997), expression in brain tissue is modest, and expression in tumor cell lines is unchanged.

GenBank database (National Cancer Society Cancer Genome Analysis Project (NCI CG
Similar sequences to MOVA-91 in AP) and other sources) are found in squamous cell carcinoma, glioblastoma (pooled), anaplastic oligodendroglioma, B cell chronic lymphocytic leukemia, larynx, uterus. And from germinal center B cell tumors. Other sequences with limited identity were found in colon cancer (HCC) and T84 cancer cell lines.

[0266]   Some of the above are listed in Table 4.

The protein encoded by clone MOVA-91 reacted positively with cancer sera in serology tests as shown in Table 3.

[Table 3]

[0268]

[Table 4]

Equivalents With no more than routine experimentation, one of ordinary skill in the art will recognize or be able to ascertain a large number of equivalents to the particular embodiments of the invention described herein. Such equivalents are intended to be covered by the appended claims. All references disclosed herein are incorporated by reference in their entireties.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 39/39 A61K 39/395 D 4C085 39/395 E 4C086 L 4C087 N 4H045 T 45/00 48/00 45 / 00 A61P 35/00 48/00 43/00 105 A61P 35/00 C07K 14/82 43/00 105 16/32 C07K 14/82 C12N 1/15 16/32 1/19 C12N 1/15 1/21 1 / 19 C12Q 1/68 A 1/21 G01N 33/53 D 5/10 C12P 21/08 C12Q 1/68 C12N 15/00 ZNAA G01N 33/53 A61K 37/02 // C12P 21/08 C12N 5/00 A (72) Inventor Lagarkova, Marka Russian Federation Moscow, Moscow State University, Belozersky, Institute of Physio-Kemica Biology (72) Inventor Korolewa, Ekaterina Russia Moscow, Moscow State University, Belozersky, Institute of Physio-Chemical Biology (72) Inventor Tarskaya, Regina Russian Federation Moscow, Russian Academy of Sciences, Engelhard Institute of Molecular Biology (72) Inventor Udvichenko, Konstantin Russian Federation Moscow, Moscow State University, Belozelsky, Institute of Physio-Chemical Biology (72) Inventor Mesher Yakov, Russian Federation Moscow, Russian Acad Me of Medical Science, Oncological Research Center (72) Inventor Litchnitzer, Michael Lo Russian Federation Moscow, Russian Academy of Medical Sciences, Oncological Research Center (72) Inventor Kuplash, Dmitry Russia Federation Moscow, Academy of Sciences, Engelhard Institute of Molecular Biology (72) Inventor Ned Spasov, Sergey Russian Federation Moscow , Moscow State University, Belozersky, Institute of Physio-Chemical Biology (72) Inventor Tracy, Ozlem Germany Republic Day-66421 Hambruk / Saar, Inéle Medizin 1 (72) Inventor Zahin, Yuguar Federal Republic of Germany De 66421 Hamburk / Saar, Inélé Medizín 1 (72) Inventor Pfluends, Michael Russian Federation Moscow, Mo Mulberry-state University, Berozerusuki, Institute of Physiology - Ke Michal Biology (72) inventor Old, Lloyd Jay. United States New York 10158, New York, Third Avenue 605 (72) Inventor Knutus, Alex Germany Day 60488 Frank Furt am Main, Hall 2-28 (72) Inventor Jaguar, Elke Germany Day 60488 Frank Furt am Main, Hall 2-28 F-term (reference) 4B024 AA01 AA12 BA36 CA01 GA11 HA12 HA17 4B063 QA19 QQ02 QQ03 QQ42 QQ79 QQ96 QR08 QR32 QR42 QR55 QR62 QS25 QS33 QS34 QX01 4B064 CA08 CA4 CA04 CA04 CA04 CAB4 CA4 CA04 AA03 AA06 AA07 AA13 AA17 BA44 CA53 CA56 DA01 DA27 DA32 NA14 ZB211 ZB212 ZB261 ZB262 4C085 AA13 AA14 AA38 BB01 BB11 CC03 CC21 CC23 DD21 DD62 DD63 EE01 EE06 EA06 A02 A05 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 A01 A02 MA02 NA05 NA13 NA14 ZB21 ZB26 4H045 AA11 AA30 BA10 CA41 DA86 EA28 EA31 EA51 FA74

Claims (104)

[Claims] 1. A method of diagnosing a disorder characterized by the expression of a human cancer-associated antigen precursor encoded by a nucleic acid molecule, the method comprising the step of treating a biological sample isolated from a subject with a nucleic acid molecule, its expression product or Contacting an agent that specifically binds with a fragment of its expression product complexed with an HLA molecule,
Wherein the nucleic acid molecule is an NA Group 1 nucleic acid molecule, and determining the interaction between the agent and the nucleic acid molecule or expression product as determinant of the disorder. 2. The agent is, (A) a nucleic acid molecule comprising an NA group 1 nucleic acid molecule or a fragment thereof, (B) a nucleic acid molecule containing an NA group 3 nucleic acid molecule or a fragment thereof, (C) a nucleic acid molecule containing an NA group 5 nucleic acid molecule or a fragment thereof, (D) an antibody that binds to the expression product of the NA group 1 nucleic acid, (E) an antibody that binds to the expression product of the NA group 3 nucleic acid, (F) an antibody that binds to the expression product of the NA group 5 nucleic acid, (G) an agent that binds to a complex of an HLA molecule and a fragment of an expression product of an NA group 1 nucleic acid, (H) an agent that binds to a complex of a fragment of an expression product of an HLA molecule and an NA group 3 nucleic acid, and (I) An agent that binds to a complex of an HLA molecule and a fragment of an NA group 5 nucleic acid expression product The method of claim 1, selected from the group consisting of: 3. The disorder is characterized by the expression of multiple human cancer-associated antigen precursors, and the agents are multiple agents, each of which is specific for a different human cancer-associated antigen precursor, and The agent is at least 2, at least 3, at least 4, at least 4, at least 6, at least 7, or at least 8, at least 9
Or the method of claim 1, wherein there are at least 10 such agents. 4. The method according to any one of claims 1 to 3, wherein the agent is specific for a human cancer-associated antigen precursor which is a breast cancer, T-cell leukemia and / or ovarian cancer-associated antigen precursor. 5. A method for determining regression, progression or onset of a symptom characterized by the expression of an abnormal level of a protein encoded by a nucleic acid molecule which is a NA group 1 molecule, which has or is suspected of having the symptom. A sample from a patient specific for (i) protein, (ii) protein-derived peptide, (iii) protein or antibody that selectively binds the peptide, and (iv) protein-derived peptide and MHC molecule complex Monitoring a parameter selected from the group consisting of conventional cytolytic T cells as determinative of the regression, progression or onset of said condition. 6. The method of claim 5, wherein the sample is body fluid, body exudate or tissue. 7. The step of monitoring comprises: (a) an antibody that selectively binds the protein of (i) or the peptide of (ii); (b) a protein or peptide that binds to the antibody of (iii); And (c) (iv) contacting with a detectable agent selected from the group consisting of cells displaying a complex of peptide and MHC molecule. 8. The method of claim 7, wherein the antibody, protein, peptide or cell is labeled with a radioactive label or an enzyme. 9. The method of claim 5, comprising assaying the sample for the peptide.
The method described in. 10. The method according to claim 5, wherein the nucleic acid molecule is NA group 3 molecule. 11. The method according to claim 5, wherein the nucleic acid molecule is NA group 5 molecule. 12. The protein is a plurality of proteins, the parameter is a plurality of parameters, each of the plurality of parameters is specific to a different one of the plurality of proteins, at least one of which is defined by one molecule of NA group. The method according to claim 5, which is an encoded cancer-associated protein. 13. A pharmaceutical formulation for a human subject, which, when administered to a subject, selectively enriches for the presence of a complex of HLA molecule and human cancer associated antigen, and a pharmaceutically acceptable agent. A pharmaceutical preparation comprising the obtained carrier, wherein the human cancer-associated antigen is a fragment of human cancer-associated antigen precursor encoded by a nucleic acid molecule containing NA group 1 molecule. 14. An agent comprises a plurality of agents, each of which selectively enriches a complex of HLA molecules and different human cancer associated antigens in a subject, wherein at least one of the human cancer associated antigens is a NA group 1 molecule. Pharmaceutical formulation according to claim 13, encoded by: 15. The pharmaceutical formulation according to claim 14, wherein the plurality of agents are at least 2, at least 3, at least 4 or at least 5 different. 16. The pharmaceutical preparation according to claim 13, wherein the nucleic acid molecule is an NA group 3 nucleic acid molecule. 17. The agent comprises the following: (1) an isolated polypeptide containing a human cancer-associated antigen or a functional variant thereof, (2) a promoter for expressing the isolated polypeptide or a functional variant thereof. Nucleic acid operably linked to (3) a host cell expressing an isolated polypeptide or a functional variant thereof, and (4) an isolated complex of a polypeptide or a functional variant thereof and an HLA molecule. The pharmaceutical preparation according to claim 13, which is selected from the group consisting of: 18. The pharmaceutical preparation according to any one of claims 13 to 17, which further comprises an adjuvant. 19. The pharmaceutical preparation according to claim 13, wherein the agent is a cell expressing an isolated polypeptide containing a human cancer-associated antigen or a functional variant thereof, and the cell is nonproliferative. 20. The agent of claim 13, wherein the agent is a cell expressing an isolated polypeptide comprising a human cancer-associated antigen or a functional variant thereof, the cell expressing an HLA molecule that binds the polypeptide. Pharmaceutical formulation. 21. The human cancer wherein the agent is at least 2, at least 3, at least 4 or at least 5 different polypeptides, each encoding a different human cancer-associated antigen, or a functional variant thereof. 14. The pharmaceutical preparation according to claim 13, wherein at least one of the related antigens is encoded by NA group 1 molecule. 22. The pharmaceutical preparation according to claim 13, wherein the agent is a PP group 2 polypeptide. 23. The pharmaceutical preparation according to claim 13, wherein the agent is a PP group 3 polypeptide or a PP group 4 polypeptide. 24. The pharmaceutical formulation of claim 20, wherein the cells recombinantly express one or both of the polypeptide and HLA molecule. 25. The pharmaceutical preparation according to claim 20, wherein the cells are non-proliferative. 26. An isolated agent that selectively binds a PP group 1 polypeptide,
A composition comprising: 27. The agent of claim 2, wherein the agent selectively binds a PP group 2 polypeptide.
7. A composition of matter according to 6. 28. The agent of claim 2, wherein the agent selectively binds a PP group 3 polypeptide.
7. A composition of matter according to 6. 29. The agent of claim 2, wherein the agent selectively binds a PP group 4 polypeptide.
7. A composition of matter according to 6. 30. The agent of claim 2, wherein the agent selectively binds a PP group 5 polypeptide.
7. A composition of matter according to 6. 31. Any of claims 26-30, wherein the agent is a plurality of different agents that selectively bind at least 2, at least 3, at least 4 or at least 5 different such polypeptides. The composition according to. 32. The composition according to any of claims 26-30, wherein the agent is an antibody. 33. The composition of claim 31, wherein the agent is an antibody. 34. A composition of matter comprising a complex of the agent according to any one of claims 26 to 30 and a therapeutic or diagnostic agent. 35. A composition of matter comprising a complex of the agent of claim 31 and a therapeutic or diagnostic agent. 36. The composition of matter of claim 34, wherein the complex of agent and therapeutic or diagnostic agent is a toxin. 37. A pharmaceutical composition comprising an isolated nucleic acid molecule selected from the group consisting of NA group 1 molecule and NA group 2 molecule and a pharmaceutically acceptable carrier. 38. The pharmaceutical composition of claim 37, wherein the isolated nucleic acid molecule comprises NA group 3 or NA group 4 molecules. 39. The pharmaceutical composition of claim 37, wherein the isolated nucleic acid molecule comprises at least two isolated nucleic acid molecules encoding two different polypeptides, each polypeptide comprising a different human cancer-associated antigen. . 40. The pharmaceutical composition according to any of claims 37 to 39, further comprising an expression vector having a promoter operably linked to the isolated nucleic acid molecule. 41. The pharmaceutical composition of any of claims 37-39, further comprising a host cell that recombinantly expresses the isolated nucleic acid molecule. 42. A pharmaceutical composition comprising an isolated polypeptide comprising a PP group 1 or PP group 2 polypeptide and a pharmaceutically acceptable carrier. 43. The pharmaceutical composition of claim 42, wherein the isolated polypeptide comprises a PP group 3 or PP group 4 polypeptide. 44. The pharmaceutical composition of claim 42, wherein the isolated polypeptide comprises at least two different polypeptides, each comprising a different human cancer-associated antigen. 45. The pharmaceutical composition according to claim 42, wherein the isolated polypeptide is a PP group 11 polypeptide or an HLA-binding fragment thereof. 46. The pharmaceutical composition according to claim 42, wherein the isolated polypeptide is a PP group 12 polypeptide or an HLA-binding fragment thereof. 47. The pharmaceutical composition according to any of claims 42-46, further comprising an adjuvant. 48. An isolated nucleic acid molecule comprising NA group 3 molecules. 49. An isolated nucleic acid molecule comprising NA group 4 molecules. 50. The following: (a) SEQ ID NOs: 1, 3, 4, 6 identifying nucleic acids encoding human cancer-associated antigen precursors of sufficient length to exhibit sequence uniqueness within the human genome. , A fragment of a nucleic acid molecule having a nucleotide sequence selected from the group consisting of the nucleotide sequences already described as 7, 9 and 16, (b) an isolated nucleic acid molecule selected from the group consisting of the complement of (a) However, the fragment is as follows: (1) a sequence having the GenBank accession number of Table 4, (2) a complement of (1), and a sequence consisting of (3) (1) and (2) fragments An isolated nucleic acid molecule comprising a sequence of contiguous nucleotides that is not identical to any sequence selected from the group. 51. A sequence of consecutive nucleotides   (1) at least two consecutive nucleotides that are not identical to the sequence group,   (2) at least 3 contiguous nucleotides that are not identical to the sequence group,   (3) at least 4 contiguous nucleotides that are not identical to the sequence group,   (4) at least 5 contiguous nucleotides that are not identical to the sequence group,   (5) at least 6 contiguous nucleotides that are not identical to the sequence group,   (6) At least 7 consecutive nucleotides that are not identical to the sequence group 51. The isolated nucleic acid molecule of claim 50, selected from the group consisting of: 52. A fragment comprising at least 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, 24 nucleotides,
51. The isolated nucleic acid molecule of claim 50, having a size selected from the group consisting of 26 nucleotides, 28 nucleotides, 30 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, and 200 nucleotides. 53. The isolated nucleic acid molecule of claim 50, which encodes a polypeptide or fragment thereof that binds the human HLA receptor or human antibody. 54. An expression vector comprising the isolated nucleic acid molecule of any of claims 48-53 operably linked to a promoter. 55. An expression vector comprising a nucleic acid operably linked to a promoter, wherein the nucleic acid is NA group 2 molecule. 56. An expression vector comprising a nucleic acid encoding an NA group 1 or group 2 molecule and an HLA molecule. 57. A host cell transformed or transfected with the expression vector of claim 54. 58. A host cell transformed or transfected with the expression vector of claim 55 or 56. 59. A host cell transformed or transfected with an expression vector according to claim 54 and further comprising a nucleic acid encoding HLA. 60. A host cell transformed or transfected with an expression vector according to claim 55 and further comprising a nucleic acid encoding HLA. 61. An isolated polypeptide encoded by the isolated nucleic acid molecule of claim 48 or 49. 62. A fragment of the polypeptide of claim 61 which is immunogenic. 63. The fragment of claim 62, wherein the fragment or a portion of the fragment binds HLA or human antibody. 64. An isolated fragment of a human cancer-associated antigen precursor that binds HLA or human antibody, or a portion thereof, wherein the precursor is encoded by a nucleic acid molecule that is a NA group 1 molecule. 65. The fragment of claim 64, wherein the fragment is part of a complex with HLA. 66. The fragment of claim 65, wherein the fragment is 8-12 amino acids long. 67. An isolated poly containing fragments of the polypeptide of claim 61, which identifies a human cancer-associated antigen precursor polypeptide of sufficient length to exhibit sequence uniqueness within the human genome. peptide. 68. A kit for detecting the presence of expression of a human cancer-associated antigen precursor, which comprises a pair of isolated nucleic acid molecules, each of which comprises (a) a nucleotide sequence of any one of NA group 1 molecules. 12-32 nucleotide contiguous segment and (b) ("a")
Said kit comprising said pair of isolated nucleic acid molecules consisting essentially of a molecule selected from the group consisting of the complements of, said contiguous segments not overlapping. 69. The kit of claim 68, wherein pairs of isolated nucleic acid molecules are constructed and arranged to selectively amplify isolated nucleic acid molecules that are NA group 3 molecules. 70. A method of treating a subject having a disorder characterized by expression of a human cancer-associated antigen precursor, the method comprising the presence of a complex of HLA molecule and human cancer-associated antigen effective to ameliorate the disorder. Wherein the human cancer-associated antigen comprises the following: (a) a nucleic acid molecule comprising a NA group 1 nucleic acid molecule, and (b) a NA group. A nucleic acid molecule comprising 3 nucleic acid molecules, (c) a nucleic acid molecule comprising an NA group 5 nucleic acid molecule, a fragment of a human cancer-associated antigen precursor encoded by a nucleic acid molecule selected from the group consisting of: 71. The disorder is characterized by the expression of multiple human cancer-associated antigen precursors, the agent selectively enriching in a subject each for the presence of a complex of HLA molecules and a different human cancer-associated antigen. At least one of the human cancer-associated antigens
71. The method of claim 70, wherein one is encoded by the NA group 1 molecule. 72. The method of claim 71, wherein the plurality is at least 2, at least 3, at least 4 or at least 5 such agents. 73. The agent is PP group 1, PP group 2, PP group 3, PP group 4 or PP.
73. The method of any of claims 70-72, which is an isolated polypeptide selected from the group consisting of group 5. 74. The method of any of claims 70-72, wherein the disorder is cancer. 75. The method of claim 73, wherein the disorder is cancer. 76. A method of treating a subject having a condition characterized by expression of a human cancer-associated antigen precursor in cells of the subject, comprising: (i) removing a sample containing immunoreactive cells from the subject, (ii) ) Contacting the host cell with a sample containing immunoreactive cells under conditions that favor the production of cytolytic T cells against the human cancer-associated antigen that is a fragment of the precursor; (iii) cells expressing the human cancer-associated antigen Introducing into the subject a cytolytic T cell in an amount effective to lyse the host cell, the host cell being transformed with an expression vector comprising an isolated nucleic acid molecule operably linked to a promoter. Alternatively, the method as described above, wherein the isolated nucleic acid molecule is transfected and is selected from the group of nucleic acid molecules consisting of NA group 1, NA group 2, NA group 3, NA group 4 and NA group 5. 77. The method of claim 76, wherein the host cell recombinantly expresses an HLA molecule that binds a human cancer-associated antigen. 78. The method of claim 76, wherein the host cell endogenously expresses an HLA molecule that binds a human cancer-associated antigen. 79. A method of treating a subject having a condition characterized by expression of a human cancer-associated antigen precursor in a cell of the subject, the method comprising: (i) identifying a nucleic acid molecule expressed by the cell associated with the condition. Wherein the nucleic acid molecule is NA group 1 molecule, (ii) (a) the identified nucleic acid molecule, (b) a fragment of the identified nucleic acid containing a segment encoding a human cancer-associated antigen, c) transfecting a host cell with a nucleic acid selected from the group consisting of a deletion, substitution or addition to (a) or (b) and a degeneracy of (d) (a), (b) or (c). (Iii) culturing the transfected host cells to express the transfected nucleic acid molecule, (iv) an amount of the host effective to enhance an immune response to the cells of the subject associated with the condition. Introducing into a subject a vesicle or an extract thereof, including the method. 80. further comprising identifying an MHC molecule that exhibits a portion of the expression product of the nucleic acid molecule, wherein the host cell expresses the same MHC molecule as the one identified, and the host cell expresses the expression product of the nucleic acid molecule. 80. The method of claim 79, which presents a MHC binding moiety. 81. The immune response comprises a B cell response or a T cell response.
9. The method according to 9. 82. The response is a T cell response which comprises the generation of cytolytic T cells specific for a host cell displaying a portion of the expression product of a nucleic acid molecule or a cell of interest expressing a human cancer-associated antigen. 82. The method of claim 81. 83. The method of claim 79, wherein the nucleic acid molecule is NA group 3 molecule. 84. The method of claim 79 or 80, further comprising treating the host cells to render them non-proliferative. 85. A method for treating, diagnosing, or monitoring a subject having a condition characterized by the expression of an abnormal amount of a protein encoded by a nucleic acid molecule that is a NA group 1 molecule, the method comprising: Or an antibody that specifically binds to a peptide derived therefrom,
Administering to a subject an antibody conjugated to a therapeutically useful agent in an amount effective to treat the condition. 86. The method of claim 85, wherein the antibody is a monoclonal antibody. 87. The method of claim 86, wherein the monoclonal antibody is a chimeric antibody or a humanized antibody. 88. A method of treating a condition characterized by the expression in a subject of an abnormal amount of a protein encoded by a nucleic acid molecule that is a NA group 1 nucleic acid molecule, which prevents the condition in the subject and prevents its onset. 48. The method, comprising administering to the subject a pharmaceutical composition according to any of claims 13-25 and 37-47 in an amount effective to delay or suppress. 89. The method of claim 88, wherein the condition is cancer. 90. The method of claim 88, further comprising first identifying that the subject expresses an abnormal amount of protein in the tissue. 91. The method of claim 89, further comprising first identifying that the subject expresses an abnormal amount of protein in the tissue. 92. A method of treating a subject having a condition characterized by the expression of an abnormal amount of a protein encoded by a nucleic acid molecule that is a NA group 1 nucleic acid molecule, the method comprising: (i) expressing an abnormal amount of the protein. (Ii) isolating a sample of cells, (iii) culturing the cells, and (iv) introducing the cells into the subject in an amount effective to elicit an immune response against the cells. Comprising: 93. The method of claim 92, further comprising rendering the cells non-proliferative prior to introducing them into the subject. 94. A method of treating a pathological cellular condition characterized by aberrant expression of a protein encoded by a nucleic acid molecule that is a NA group 1 nucleic acid molecule, the method comprising the step of expressing the protein in a subject in need thereof. Administering an effective amount of an agent that inhibits activity. 95. The method of claim 94, wherein the agent is a suppressor antibody that selectively binds a protein and the antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody. 96. The method of claim 94, wherein the agent is an antisense nucleic acid molecule that selectively binds to a nucleic acid molecule that encodes a protein. 97. The method of claim 94, wherein the nucleic acid molecule is an NA Group 3 nucleic acid molecule. 98. A composition of substances useful for stimulating an immune response against a plurality of proteins encoded by a nucleic acid molecule that is a NA group 1 molecule, the composition comprising a plurality of peptides derived from the amino acid sequence of the protein. , A composition of matter, wherein the peptide binds to one or more MHC molecules displayed on the surface of cells which express an abnormal amount of protein. 99. The composition of matter of claim 98 wherein at least a portion of the plurality of peptides binds to an MHC molecule and elicits a cytolytic response thereto. 100. The composition of matter of claim 99 further comprising an adjuvant. 101. The composition of matter of claim 100 wherein the adjuvant is saponin, GM-CSF or interleukin. 102. Further comprising at least one peptide useful in stimulating an immune response against at least one protein not encoded by a nucleic acid molecule that is a NA group 1 molecule, wherein at least one peptide is one or more MHs.
99. The composition of matter of claim 98, which is associated with a C molecule. 103. The following: (i) a peptide derived from a protein encoded by a nucleic acid molecule that is a NA group 1 molecule, and (ii) a complex and selection of MHC molecules that form a complex by binding with the peptide. An isolated antibody that specifically binds, wherein (i) or (ii) alone does not bind. 104. The antibody of claim 103, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fragment thereof.