Title:

In vivo activation of tumor-specific cytotoxic T cells

Document Type and Number:

United States Patent 7402314

Abstract:

The present invention relates to methods and compositions of activating cytotoxic T lymphocytes (CTLs) in vivo with specificity for particular antigenic peptides, and to methods and compositions of using activated CTLs in vivo for the treatment of a variety of disease conditions. In some preferred embodiments, the invention provides methods of employing a polypeptide of the amino acid sequence VMAGVGSPYV to specifically activating CTLs in subjects having a breast cancer overexpressing a Her-2/Neu protein, and methods of using the polypeptide to treat such subjects.

Inventors:

Sherman, Linda A. (La Jolla, CA, US)

Plaque It!

STATEMENT OF GOVERNMENT SUPPORT

This invention was made in part by government support by the National Institutes of Health Grant No. CA25803. The U.S. Government therefore has certain rights in the invention.

TECHNICAL FIELD

The present invention relates to methods, compositions, and peptides useful in activating CTLs in vivo with specificity for particular antigenic peptides. The invention also discloses the use of activated CTLs in vivo for the treatment of a variety of disease conditions, and compositions appropriate for these uses. Diagnostic kits, components, and methods are also described herein.

BACKGROUND

The efficiency with which the immune system cures or protects individuals from infectious disease has always been intriguing to scientists, as it has been believed that it might be possible to activate the immune system to combat other types of diseases. Such diseases include cancer, AIDS, hepatitis and infectious disease in immunosuppressed patients. While various procedures involving the use of antibodies have been applied in those types of diseases, few if any successful attempts using cytotoxic T lymphocytes have been recorded. Theoretically, cytotoxic T lymphocytes would be the preferable means of treating the types of disease noted above. However, no useful in vivo procedures have been available to specifically activate cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs), which are also called cytotoxic T cells or CD8 cells, represent the main line of defense against viral infections. CTLs specifically recognize and kill cells which are infected by a virus. Thus, the cost of eliminating a viral infection is the accompanying loss of the infected cells. The T cell receptors on the surface of CTLs cannot recognize foreign antigens directly. In contrast to antibodies, antigen must first be presented to the receptors.

The presentation of antigen to T cells is accomplished by major histocompatibility complex (MHC) molecules of the Class I type. The major histocompatibility complex (MHC) refers to a large genetic locus encoding an extensive family of glycoproteins which play an important role in the immune response. The MHC genes, which are also referred to as the HLA (human leucocyte antigen) complex, are located on chromosome 6 in humans. The molecules encoded by MHC genes are present on cell surfaces and are largely responsible for recognition of tissue transplants as “non-self”. Thus, membrane-bound MHC molecules are intimately involved in recognition of antigens by T cells.

MHC products are grouped into three major classes, referred to as I, II, and III. T cells that serve mainly as helper cells express CD4 and are primarily restricted by Class II molecules, whereas CTL-(CD8−) expressing cells, which mostly represent cytotoxic effector cells, interact with Class I molecules.

Class I molecules are membrane glycoproteins with the ability to bind peptides derived primarily from intracellular degradation of endogenous proteins. Complexes of MHC molecules with peptides derived from viral, bacterial and other foreign proteins comprise the ligand that triggers the antigen responsiveness of T cells. In contrast, complexes of MHC molecules with peptides derived from normal cellular products play a role in “teaching” the T cells to tolerate self peptides, in the thymus. Class I molecules do not present entire, intact antigens; rather, they present peptide fragments thereof, “loaded” onto their “peptide binding groove”.

For many years, immunologists have hoped to raise specific cytotoxic cells targeting viruses, retroviruses and cancer cells. While targeting against viral diseases in general may be accomplished in vivo by vaccination with live or attenuated vaccines, no similar success has been achieved with retroviruses or with cancer cells. Moreover, the vaccine approach has not had the desired efficacy in immunosuppressed patients. One way around this difficulty would be to immunize a healthy individual, isolate the CTLs from this individual, and inject these CTLs into the disease-afflicted person.

However, this experimental protocol is not always useful, as it is neither practical (nor ethical) in many circumstances to endeavor to immunize healthy individuals with tumor cells. Furthermore, it is problematic, at best, to endeavor to activate CTLs to recognize abnormal cells expressing abnormally high levels of peptides that are expressed on normal cells in lower quantities in normal, healthy individuals.

The use of mouse strains (including transgenic strains) to generate activated CTLs has not always been practical, particularly if the murine strain is unable to raise an immunologic response to the immunogen. Failure to raise an immunologic response may be due either to failure of the murine immune system to recognize the antigen, or its failure to generate activated cells that are compatible with the intended recipient of activated CTLs for therapeutic purposes.

For example, it has been observed that peptides are unique for a given MHC; in other words, certain antigenic peptides bind preferentially to particular MHC species and do not bind well to others, even in the absence of the “preferred” MHC molecule. Furthermore, MHC molecules are highly polymorphic, which fact generates at least two problems. First, the CTLs of an individual can only interact with peptides bound to precisely those three to six Class I molecules present in that individual. Second, CTLs react violently with all Class I molecules which are different from those expressed in the individual from whom the CTLs are obtained, regardless of what peptides the Class I molecules contain. This reactivity has been observed for some time and is termed allo-reactivity. It is the underlying cause of the immune rejection of transplanted organs.

Thus, apart from the rather heroic experimental protocol in which one individual is used as the donor of activated CTLs to another individual, it is difficult to find two unrelated persons with the exact same setup of Class I molecules. For this reason, at least one researcher has taken the rather non-specific approach of “boosting” existing CTLs by incubating them in vitro with IL-2, a growth factor for T cells. However, this protocol (known as LAK cell therapy) will only allow the expansion of those CTLs which are already activated. As the immune system is always active for one reason or another, most of the IL-2 stimulated cells will be irrelevant for the purpose of combating the disease. In fact, it has not been documented that this type of therapy activates any cells with the desired specificity. Thus, the benefits of LAK cell therapy are controversial at best, and the side effects are typically so severe that many studies have been discontinued.

Class I molecules bind peptides in a specific manner. All peptides have to be about 8-11 amino acids in length and their sequences must fit the peptide-binding pocket of the Class I molecules. In this respect, Class I molecules display some resemblance to antibodies. However, while a given antibody tends to bind only one antigen, a given Class I molecule can bind many hundred different peptides. As the number of viruses and other pathogens is quite large, it is apparent that our immune defense would be poor if we had only a single Class I molecule, even if it is capable of binding and altering many different peptides. For this reason, all humans have between three and six different Class I molecules, which can each bind many different types of peptides. Accordingly, the CTLs can recognize many thousands of peptides bound to one or another Class I molecule.

As selection seems to be the dominant force in evolution, pathogens emerge which cannot be recognized efficiently by the immune system. Thus, for example, a viral sequence, which gives rise to peptides that bind efficiently to a variety of Class I molecules, may mutate such that it is not recognized by any of the three to six Class I molecules present in an individual. This virus may therefore not be recognized by the immune system and may consequently cause the death of the affected individual. If all individuals had an identical set of Class I molecules, such a virus might conceivably eliminate an entire species.

However, individual variation is a safeguard against that possibility, as some 100 different forms of Class I molecules are present in the population.

If Class I molecules can bind a variety of peptides, including peptides derived from our own cellular proteins, one may wonder why the CTLs of the immune system do not recognize and destroy our own tissues. While the answer to this question is not entirely clear, two distinct mechanisms are presently believed to be operating. First, CTLs that can react with self peptides are eliminated in the thymus. Second, CTLs become non-responsive (anergic) to self peptides in the peripheral organs of the immune system. Since every possible type or epitope of cellular proteins is not synthesized by the cells in the thymus, the second mechanism would appear to be the more likely explanation. This mechanism appears to be operational for the level of self peptides normally encountered. If this level is increased by some means, it can be shown that individuals do indeed have CTLs that can recognize and destroy cells expressing self peptides. This latter observation is significant with regard to the concept of using the immune system to eliminate tumor cells.

Recently, it has become apparent that mutant and wild-type peptides derived from cellular oncogene proteins can be recognized by CTLs. This suggests that self peptides encoded by non-mutant genes, in addition to the peptides encoded by mutant genes, can be potential targets for T cell responses against tumor cells. (See, e.g., Melief and Kast, Curr. Op. Immunol . 5: 709-713 (1993); Boon, Adv. Cancer Res . 58: 177-210 (1992); Van der Bruggen, et al., Curr. Op. Immunol . 4: 608-612 (1992).)

Irrespective of the mode of activity, it is evident that the CTL response with respect to various tumor antigens is deficient in many cases. It would be desirable to stimulate the immune response in these individuals to respond to appropriate tumor antigens and thereby eliminate the cells and tissues so affected. Further, as there is no currently available vaccine for malignancies such as breast cancers, it is desirable to establish such a vaccine, preferably based on a range of antigenic determinants.

Accordingly, it is an object of the present invention to provide agents that strengthen or boost the ability of the cellular immune system to fight tumors and other malignancies. It is a further object to provide pharmaceutical compositions that strengthen or boost the cellular immune system for fighting tumor-related disease processes, both with reference to therapeutic and prophylactic uses.

These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides agents that strengthen or boost the cellular immune system to fight or prevent tumor growth or proliferation, or the growth or proliferation of other malignancies. In various embodiments of the present invention, the condition to be treated may comprise cancer, tumors, neoplasia, viral or retroviral infection, autoimmune or autoimmune-type conditions.

For example, the present invention is directed to a polypeptide having substantial homology with a CTL epitope selected from the group consisting of LLPENNVLSPL (SEQ ID NO 1); RMPEAAPPV (SEQ ID NO 2); STPPPGTRV (SEQ ID NO 3); LLGRNSFEV (SEQ ID NO 4); KIFGSLAFL (SEQ ID NO 10); TLQGLGISWL (SEQ ID NO 11); VMAGVGSPYV (SEQ ID NO 12); VLQGLPREYV (SEQ ID NO 13); and ILLVVVLGV (SEQ ID NO 14), or to a molecule that includes such a polypeptide or an analog or sequential subset thereof.

In addition, the present invention provides methods of provoking an immune response to p53 or Her-2/Neu antigens, comprising contacting a suitable cytotoxic T lymphocyte with an immune-response-provoking, effective amount of a molecule comprising a peptide selected from the group of epitopes listed above. The present invention further provides pharmaceutical compositions comprising at least one of the CTL-specific epitopes recited herein.

Thus, in one embodiment, the present invention contemplates a polypeptide capable of specifically activating cytotoxic T lymphocytes in vivo, wherein the cytotoxic T lymphocytes (CTLs) specifically target malignant cells. In one variation, the polypeptide is derived from human p53 protein. Various p53 polypeptides are useful in this regard, including those with amino acid residue sequences such as STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), LLPENNVLSPL (SEQ ID NO 1), RMPEAAPPV (SEQ ID NO 2), and sequential subsets thereof.

In another variation, the polypeptide is derived from human Her-2/Neu protein. Various Her-2/Neu polypeptides are useful in this regard, including those with amino acid residue sequences such as KIFGSLAFL(SEQ ID NO 10), VMAGVGSPYV(SEQ ID NO 12), TLQGLGISWL (SEQ ID NO 11), VLQGLPREYV (SEQ ID NO 13), ILLVVVLGV (SEQ ID NO 14) and sequential subsets thereof.

Polypeptides having substantial homology with CTL epitopes are also disclosed herein. CTL epitopes identified with tumor-associated antigens are particularly preferred. Preferred CTL epitopes of the present invention include p53 and Her-2/Neu epitopes. Exemplary epitopes include STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), LLPENNVLSPL (SEQ ID NO 1), RMPEAAPPV (SEQ ID NO 2), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), TLQGLGISWL (SEQ ID NO 11), VLQGLPREYV (SEQ ID NO 13), and ILLVVVLGV (SEQ ID NO 14). The following CTL epitopes are somewhat more preferred: STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), and homologs, analogs and sequential subsets thereof.

The present invention also discloses a variety of pharmaceutical compositions. One embodiment of such a composition comprises a polypeptide having substantial homology with a CTL epitope; exemplary and preferred epitopes are noted above. A composition of the present invention may further comprise a pharmaceutically acceptable carrier.

Populations of specific cytotoxic T cells capable of lysing tumor cells displaying a specific peptide are also encompassed by the present invention. In one embodiment, the peptide is displayed exogenously. In another, the peptide is displayed endogenously.

In one embodiment of the disclosed populations, the CTLs are generated via in vivo immunization. In one variation, the specific peptide is derived from p53; in another, the specific peptide is derived from Her-2/Neu. Exemplary peptides useful according to the invention have already been identified hereinabove.

The present invention further contemplates a variety of useful anti-tumor vaccines. In one embodiment, a vaccine comprises an immunogenically effective amount of a cytotoxic T-lymphocyte-stimulating peptide. In alternative embodiments, the peptide may be derived from endogenously or exogenously displayed or processed proteins, analogs or portions thereof; preferably, such proteins, analogs, and portions thereof are tumor-associated. For example, p53 and Her-2/Neu proteins, analogs, and portions (or sequential subsets) thereof are preferred according to the present invention.

In various embodiments, the peptide for use in (or as) a vaccine is selected from the following group: STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), LLPENNVLSPL (SEQ ID NO 1), RMPEAAPPV (SEQ ID NO 2), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), TLQGLGISWL (SEQ ID NO 11), VLQGLPREYV (SEQ ID NO 13), and ILLVVVLGV (SEQ ID NO 14). In alternative variations, the peptide may be linked to a carrier. It may also be introduced into a mammal as a homopolymer, or as a heteropolymer.

The invention also discloses methods of generating activated CTL cells in vivo. In one embodiment, the method comprises contacting, in vivo, CTL cells with antigen-loaded Class I molecules surface-expressed on eucaryotic cells—e.g. mammalian cells, and more preferably murine cells—for a time period sufficient to activate, in an antigen-specific manner, the CTL cells. In one variation, the Class I molecules are human Class I MHC molecules. In another variation, the Class I molecules are chimeric human-mouse Class I MHC molecules. Appropriate antigens may be selected from the proteins, polypeptides, analogs and sequential subsets thereof which have already been described above.

The method may further comprise separating the activated CTL cells from the antigen-loaded Class I MHC molecules; suspending the activated CTL cells in an acceptable carrier or excipient; and administering the suspension to an individual in need of treatment.

The invention further contemplates methods of specifically killing target cells in a patient. In one embodiment, such a method comprises the steps of administering an immunogenic polypeptide specific to the target cells to an animal, thereby generating a population of antigen-loaded Class I molecules displaying the polypeptides on their cell surfaces; contacting, in vivo, a population of CTL cells with the population of antigen-loaded Class I molecules for a time period sufficient to activate, in an antigen-specific manner, the CTL cells; harvesting the activated CTL cells from the animal; and administering the activated CTL cells to the patient.

As noted previously, a variety of proteins, polypeptides, portions and sequential subsets thereof are available for use in this regard. For example, useful peptides include the following sequences: LLPENNVLSPL (SEQ ID NO 1), RMPEAAPPV (SEQ ID NO 2), STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), and sequential subsets thereof. In various embodiments, the Class I molecules are human Class I MHC molecules. In others, the Class I molecules are chimeric human-mouse Class I MHC molecules.

As noted previously, various methods of specifically killing target cells are contemplated herein. Another exemplary method uses specific, activated CTLs, prepared according to the following steps: obtaining a fluid sample containing T cells from an individual in need of treatment; loading empty Class I MHC molecules with at least one species of antigenic peptide, wherein the peptide is substantially homologous to at least a portion of a peptide derived from the target cell; admixing the T cells with an amount of peptide-loaded Class I MHC molecules sufficient to produce activated CTLs; harvesting the activated CTLs; and administering the activated CTLs to the individual. Useful antigenic molecules have already been disclosed hereinabove.

Also contemplated by the present invention are methods of provoking an immune response to a tumor-associated antigen. In one method, a cytotoxic T lymphocyte is contacted with an immune response-provoking amount of a molecule comprising a peptide derived from a tumor-associated protein. Exemplary proteins, polypeptides, analogs, homologs, and sequential subsets thereof are listed above and may be used in various embodiments of this method. For example, some peptides useful according to the present method include the amino acid residue sequences STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), or sequential subsets thereof.

In one variation of the foregoing method, the contacting step occurs in vivo—preferably, in a mammal. In another embodiment, the contacting occurs in vitro. In another variation, the method further comprises returning the contacted cytotoxic T cells to the host. Another embodiment discloses that a polypeptide is co-administered with a second polypeptide that induces a T helper response. In one variation, the polypeptide and the T helper-inducing polypeptide are conjugated to one another.

Also disclosed herein are methods of identifying specific cytotoxic T cells (CTLs) responsive to a specific T cell epitope. One such method includes the following steps: obtaining a test sample of lymphocytes from an individual, wherein the test sample is to be assayed for the presence of the specific CTLs; contacting target cells with a molecule comprising a peptide selected from the group consisting of STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), and sequential subsets thereof, wherein the target cells are of the same HLA class as the lymphocytes to be tested for the specific CTLs; contacting the test sample with a molecule according to step b, under conditions sufficient to restimulate the specific CTLs to respond to appropriate target cells; and determining whether the test sample of lymphocytes exerts a cytotoxic effect on the target cells, thereby confirming the presence of the specific CTLs.

Methods of detecting specific cytotoxic T cells (CTLs) having receptors capable of binding a specific T cell epitope in a tissue sample are also disclosed herein. One such method comprises the following steps: obtaining a test sample of lymphocytes from an individual, wherein the test sample is to be assayed for the presence of the specific CTLS; contacting the test sample with a molecule comprising a label and a tumor-associated peptide, to form an admixture; maintaining the admixture under suitable assay conditions for a predetermined period of time, sufficient to restimulate any specific CTLs in the test sample to respond to appropriate target cells; harvesting such contacted cells and washing with medium in the absence of the labeled molecule sufficient to remove any unbound labeled molecule; and measuring the bound labeled molecule using suitable measuring means.

Tumor-associated proteins and polypeptides for use according to the disclosed methods are described in detail herein. The invention also contemplates various alternative procedures for use according to the above-noted method. For example, the cells may be lysed using a hypotonic solution with or without unlabeled molecule—or via other means known in the art—and preparing a membrane fraction that is free of unbound labeled molecule.

The present invention also discloses methods of detecting anti-p53 antibodies in an individual. One such method comprises the following steps: obtaining a fluid sample from an individual to be tested; adding a predetermined amount of p53 polypeptide to the sample, to form an admixture; maintaining the admixture under biological assay conditions for a period of time sufficient to allow the p53 polypeptide to immunoreact with any anti-p53 antibodies present in the sample; and assaying for the presence of an immunoreaction product, thereby confirming the presence of anti-p53 antibodies. As before, useful p53 proteins, polypeptides, analogs, homologs, and sequential subsets thereof are described herein. Exemplary p53 polypeptides may include the following amino acid residue sequences (or sequential subsets thereof): STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12). It is also contemplated that the p53 polypeptide comprises two or more different polypeptides, e.g., polypeptides including sequences selected from the group consisting of STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), and sequential subsets thereof.

The present invention also contemplates various assay systems, including diagnostic assay systems. One exemplary assay system in kit form comprises a package containing, in an amount sufficient to perform at least one assay, at least one species of polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence derived from a tumor-associated protein. For example, in one embodiment, the tumor-associated protein is p53, and useful polypeptides may thus include one or more of the following amino acid residue sequences, or sequential subsets thereof: LLPENNVLSPL (SEQ ID NO 1), RMPEAAPPV (SEQ ID NO 2), STPPPGTRV (SEQ ID NO 3), or LLGRNSFEV (SEQ ID NO 4). Polypeptides substantially homologous thereto are also useful as described.

In various embodiments, the polypeptide may be affixed to a solid matrix. In another variation, the polypeptide comprises more than one species of polypeptide and wherein the species are present as an admixture. An assay system may further include, in a separate package, a labeled specific binding agent for signaling the presence of a polypeptide-containing immunoreaction product.

Another assay system of the present invention comprises an assay system in kit form comprising a package containing, in an amount sufficient to perform at least one assay, an antibody combining site-containing molecule capable of immunoreacting with a tumor-associated antigen. As noted previously, a wide variety of useful antigens are disclosed herein.

In one embodiment, the antibody combining site-containing molecule is affixed to a solid matrix. In another variation, the molecule is labeled.

Antibody combining site-containing molecules according to the present invention include antibody molecules or immunologically active portions thereof, including intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab′, F(ab′) ₂and F(v). In an exemplary embodiment, an antibody molecule of the present invention is able to immunoreact with a polypeptide as disclosed hereinabove. In one embodiment, the antibody molecule is monoclonal; in another, the antibody molecule is polyclonal. The invention further contemplates compositions comprising one or more antibody molecules as disclosed herein. In addition, the invention discloses hybridomas capable of secreting molecules containing antibody combining sites.

The invention further contemplates a molecule comprising a polypeptide having substantial homology with a CTL epitope. Various CTL epitopes are disclosed above. In an exemplary embodiment, CTL epitopes are selected from the group consisting of STPPPGTRV (SEQ ID NO 3), LLGRNSFEV (SEQ ID NO 4), KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), and sequential subsets thereof.

In one variation, the molecule comprises at least about eight amino acids and fewer than about 50 amino acids. In another, the molecule comprises at least about eight amino acids and fewer than about thirteen amino acids. In yet another embodiment, the polypeptide has an amino acid residue sequence substantially homologous to that of any of the CTL epitopes.

Another variation provides that the polypeptide is conjugated to a substance, wherein the substance is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a solid matrix, a carrier, and a second CTL epitope. In one embodiment, the substance is a second CTL epitope; in another, the second epitope is a T helper epitope. It is further contemplated that the carrier may comprise an immunogenic lipid or protein. Moreover, the polypeptide may be conjugated to the substance indirectly by a linker.

It is expressly to be understood that various embodiments as disclosed above and hereinbelow may be combined appropriately to describe further alternative embodiments of the within-described invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the ability of test peptides to bind A2.1 on the surface of target cells in binding inhibition assay as described in Example 1A2e. The percent specific lysis (% specific lysis of EA2/K ^btargets) is given on the X-axis and the test peptide is given on the Y-axis. The results of peptides M1 (1), FLU NP 365-373 (2), VSV-N-52-59 (3), HIV-Pol 510-518 (4), p53 264-272 (5), p53 149-157 (6), p53 65-73 (7), p53 25-35 (8), and no peptide (9) are given.

FIG. 2 illustrates CTL-mediated lysis of target cells which have p53-specific peptides bound to the cell surface as described in Example 1B1. The percent specific lysis (%-SL) is given on the Y-axis and the ratio of effector to target cells (E:T) is given on the X-axis.

FIG. 2A illustrates lysis of target cells with CTL that were generated from transgenic mice immunized with the p53.25-35 peptide (CTL A2K ^b25). The results of p53 peptides p53.25-35 and p53.149-157 peptide bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b+p53.25-35 (open circle) and EA2K ^b+p53.149-157 (open triangle), respectively), EA2K ^bcells without peptide (EA2K ^b; closed circle and EL-4 cells incubated in the presence of p53.25-35 cells (EL-4+p53.25-35; closed triangle) are given.

FIG. 2B illustrates lysis of target cells with CTL that were generated from transgenic mice immunized with the p53.65-73 peptide (CTL A2K ^b65). The results of p53 peptides p53.65-73 peptide bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b+p53.65-73; open circle), EA2K ^b+p53.149-157; open triangle, EA2K ^b; closed circle, and EL-4 cells incubated in the presence of p53.65-73 cells (EL-4+p53.65-73; closed triangle) are given.

FIG. 2C illustrates lysis of target cells with CTL that were generated from transgenic mice immunized with the p53.149-157 peptide (CTL A2K ^b149). The results of EA2K ^b+p53.149-157; open circle, p53.264-272 peptide bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b+p53.264-272; open triangle), EA2K ^b; closed circle, and EL-4 cells incubated in the presence of p53.149-157 cells (EL-4+p53.149-157; closed triangle) are given.

FIG. 2D illustrates lysis of target cells with CTL that were generated from transgenic mice immunized with the p53.264-272 peptide (CTL A2K ^b264). The results EA2K ^b+p53.264-272 (open circle), EA2K ^b+p53.149-157 (open triangle), EA2K ^b(closed circle), and EL-4 cells incubated in the presence of p53.264-272 cells (EL-4+p53.264-272; closed triangle) are given.

FIG. 3 illustrates CTL-mediated lysis of target cells which express endogenous p53 specific peptides bound to A2 on the cell surface as described in Example 1B2b. The percent specific lysis (%-SL) is given on the Y-axis and the ratio of effector to target cells (E:T) is given on the X-axis.

FIG. 3A illustrates lysis of target cells with CTL A2K ^b25. The results of EA2K ^b(open circles) and EA2K ^bwith A2/K ^b-bound endogenous p53 peptides expressed from a human p53 gene with a mutation at amino acid residue 273 (EA2K ^b.1 p53 (273); closed circle) are given.

FIG. 3B illustrates lysis of target cells with CTL A2K ^b65. The results of EA2K ^b(open circle) and EA2K ^b.1 p53 (273) (closed circle) are given.

FIG. 3C illustrates lysis of target cells with CTL A2K ^b149. The results of EA2K ^b(open circle), EA2K ^b.1 p53 (273) (closed circle), p53 peptide p53.149-157 peptide bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b+p53.149-157; open triangle), and EA2K ^b.1 p53 (273) with p53 peptide p53.149-157 peptide bound to A2.1/K ^bon the surface of the EA2K ^b.1p53 (273) (EA2K ^b.1 p53 (273)+p53.149-157; closed triangle) cells are given.

FIG. 3D illustrates lysis of target cells with CTL A2K ^b264. The results of EA2K ^b(open circle), EA2K ^b.1 p53 (273) (closed circle), p53 peptide p53.264-272 peptide bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b+p53.264-272; open triangle), and EA2K ^b.1 p53 (273) p53.264-272 peptide bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b.1 p53 (273)+p53.264-272; closed triangle) are given.

FIG. 3E illustrates lysis of target cells with CTL that were generated from transgenic mice immunized with the HIV pol 510-518 peptide (CTL CD8×A2K ^bHIV-pol). The results of EA2K ^b(open circle) and EA2K ^b.1 p53 (273) (closed circle) are given.

FIG. 4 illustrates CTL-mediated lysis of Saos-2 target cells as described in Example 1B2b. The percent specific lysis (%-SL) is given on the Y-axis and the ratio of effector to target cells (E:T) is given on the X-axis.

FIG. 4A illustrates lysis of target cells with CTL A2K ^b25. The results using Saos-2 target cells alone (Saos-2; open circle) and Saos-2 target cells which express a human mutant p53 gene with a mutation at amino acid residue 175 (Saos-2/175; closed circle) are shown. In FIG. 4B, lysis of target cells with CTL A2K ^b65 is illustrated. The results using Saos-2 (open circle) and Saos-2/175 (closed circle) target cells are given.

FIG. 4C illustrates lysis of target cells with CTL A2K ^b149. The results obtained with Saos-2 (open triangle) and Saos-2/175 target cells with the p53.149-157 peptide bound to A2 on their cell surface (Saos-2/175+p53.149-157; closed triangle) are shown. FIG. 4D illustrates lysis of target cells with CTL A2K ^b149. The results of Saos-2 (open circle) and Saos-2/175 (closed circle) target cells are given.

FIG. 4E illustrates lysis of target cells with CTL A2K ^b264. The results of Saos-2 (open circle) and Saos-2/175 (closed circle) target cells are illustrated. FIG. 4F illustrates lysis of target cells with CTL CD8×A2K ^bHIV-pol. The results of Saos-2 (open circle) and Saos-2/175 (closed circle) target cells are shown.

FIG. 5 illustrates CTL-mediated lysis of target cells which have specific peptides derived from Her-2/Neu bound to the cell surface as described in Example 2A3. The percent specific lysis (% ⁵¹Cr Release) is given on the Y-axis and the ratio of effector to target cells (Clone 12 (E/T)) is given on the X-axis. The CTL (Clone 12) was generated from transgenic mice immunized with the influenza G-matrix peptide (SEQ ID NO 8) as described in Example 1A2c. The G-matrix and M1(58-66) peptide have the same amino acid residue sequence. The results of the G-matrix peptide (G-MATRIX; open circle), Her-2/Neu peptides Her-3 (closed circle), Her-6 (open box), Her-7 (closed box), Her-8 (open triangle), and Her-9 (closed triangle) bound to A2.1/K ^bon the surface of the EA2K ^btarget cells are given.

FIG. 6 illustrates CTL-mediated lysis of target cells which have specific peptides derived from Her-2/Neu bound to the cell surface as described in Example 2A3. The percent specific lysis (% ⁵¹Cr release) is given on the Y-axis and the ratio of effector to target cells (E/T) is given on the X-axis. The CTL-mediated lysis of target cells with CTL that were generated from transgenic mice (A2K ^b×CD8) immunized with the either the Her-3 or Her-7 peptide (H3-pop and H7-pop, respectively) is illustrated. The results Her-3 bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b+Her-3-pep (closed circle) and EA2K ^b+Her-7-pep (open circle), respectively), and EA2K ^bcells without peptide (EA2K ^b; open triangle) are given. The results of Her-7 bound to A2.1/K ^bon the surface of the EA2K ^bcells (EA2K ^b+Her-3-pep (closed square) and EA2K ^b+Her-7-pep (open square), respectively), and EA2K ^bcells without peptide (EA2K ^b; closed triangle) are given.

FIG. 7 illustrates CTL-mediated lysis of target cells which express endogenous Her-2/Neu specific peptides bound A2.1/K ^bon the cell surface (EL4-A2K ^bTransfected With the Her-2/Neu Gene) as described in Example 2B2. The percent specific lysis (% ⁵¹Cr Release) is given on the Y-axis and the ratio of effector to target cells (E/T) is given on the X-axis. CTL were generated from transgenic mice (A2K ^b×CD8) immunized with the an HIV-derived peptide (SEQ ID NO 5) (HIV-pop). The results of the Her3 (H3-pop) CTL-mediated lysis of target cells EA2K ^b(EA2K ^b; open circle) and EA2K ^bwith A2/K ^b-bound endogenous Her-2/Neu peptides expressed from a Her-2/Neu gene (EA2K ^b-Her-2; closed circle), Her7 (H7-pop) CTL-mediated lysis of target cells EA2K ^b(EA2K ^b; open square) and EA2K ^bwith A2/K ^b-bound endogenous Her-2/Neu peptides (EA2K ^b-Her-2; closed square), HIVpol (HIV-pop) CTL-mediated lysis of target cells EA2K ^b(EA2K ^b; open triangle) and EA2K ^bwith A2/K ^b-bound endogenous Her-2/Neu peptides (EA2K ^b-Her-2; closed triangle), are given.

FIG. 8 illustrates Her-3, Her-7, and HIV CTL-mediated lysis of breast carcinoma cell lines as described in Example 3A2a. The percent specific lysis (% ⁵¹Cr Release) is given on the Y-axis and the ratio of effector to target cells (E/T) is given on the X-axis. The results of the Her-3 CTL-mediated lysis of target cells MCF-7, MDA 23.1, and MDA 435 (MCF-7-A2 ⁺ Neu ⁺ (open circle), MDA 23.1- ^A2+ Neu ⁺ (closed square), and MDA 435 A2 ⁺ Neu ⁺ (open square)) and Her-7 CTL-mediated lysis of target cells MCF-7 (closed circle), MDA 23.1 (open triangle), and MDA 435 (closed triangle) which express a Her-2/Neu gene (EA2K ^b-Her-2) are given.

FIG. 9 illustrates the effect of A2 concentration and anti-A2 monoclonal antibody on the ability of Her-7 and HIV CTL-mediated lysis of the breast carcinoma cell line MDA-23.1 as described in Example 3. The percent specific lysis (%-SL) is given on the Y-axis and the ratio of effector to target cells (E:T) is given on the X-axis. The results of the Her-7 CTL-mediated lysis of target cells MDA 23.1 in the absence and presence of anti-A2 (Her2-7 CTL and +anti-A2 (Her2-7 CTL) (open square and closed square, respectively) and HIVpol CTL-mediated lysis of target cells MDA 23.1 in the absence and presence of anti-A2 (HIVpol CTL and +anti-A2 (HIVpol CTL) (open circle and closed circle, respectively) are given.

FIGS. 10A-H illustrate A2.1-restricted recognition of endogenously synthesized p53 epitopes by p53-specific CTL from A2.1/K ^b-Tg and A2.1-Tg mice. Effector CTL were generated by peptide-priming of Tg mice. In FIGS. 10A and B, the CTL cell lines were A2K ^b149-primed; in FIGS. 10C and D, the CTLs were primed with A2K ^b264. In FIGS. 10E and F, the CTL cell lines were A2 149-primed; 10 G and H, the CTLs were primed with A2 264. In FIGS. 10A-H, effector:target (E:T) ratios were plotted against specific ⁵¹Cr release (%). CTL were assayed for cytotoxicity in a 5-hour ⁵¹Cr release assay against the indicated targets: FIGS. 10A and C: T2A2/K ^b(open circles, ∘) or T2A2/K ^b+p53.149-157 (closed circles, ●) or T2A2/K ^b+p53.264-272 (closed squares, ▪). FIGS. 10E and G: T2 (∘) or T2 pulsed with p53.149-157 (●) or p53.264-272 (▪). FIGS. 10B, D, F, H: Saos-2 (open triangles, Δ) or the same cells transfected with the human p53 gene, Saos-2/175 (closed triangles, ▴). (See, e.g., Dittmer, et al., Nature Genet . 4: 42-6 (1993); Masuda, et al., PNAS USA 84: 7716-9 (1987); Hinds, et al., Cell Growth Diff . 1: 571-580 (1990).) Both lines expressed similar levels of A2.1 as detected by flow cytometry. (See, e.g., Irwin, et al., J. Exp. Med . 170: 1091-1101 (1989).)

FIGS. 11A and B illustrate the efficiency of peptide recognition by p53-specific CTL lines. CTL lines specific for hu-wt-p53.149-157 and 264-272 were established from A2.1-Tg (CTL A2 149 and CTL A2 264) and A2.1/K ^b-Tg mice (CTL A2/K ^b149 and CTL A2/K ^b264) and assayed at an E:T ratio of 10:1 for lytic activity against nonpeptide and p53.149-157-pulsed T2 (FIG. 11A) or nonpeptide and p53.264-272-pulsed T2 targets (FIG. 118). Peptides were used at the indicated concentrations to pulse T2 targets after ⁵¹Cr labeling. Effector cells were CTL A2 149 (closed circles, ●), CTL A2/K ^b149 (open circles, ∘), CTL A2 264 (closed squares, ▪) and CTL A2/K ^b264 (open squares, □). The data represent the results of a 4-hour ⁵¹Cr release assay, whereby specific ⁵¹Cr release (%) is plotted against peptide concentration (M).

FIG. 12 illustrates the in vitro binding of peptides to A2.1/K ^b. The efficiency with which each Her-2/neu-specific peptide bound A2.1/K ^bwas determined in a competitive binding assay as described in Example 5 below. The binding of the test peptide to the target cells could be detected by the competitive inhibition of the binding of the influenza A-specific peptide as evidenced by a decrease in the ability of the influenza A-specific CTL to lyse the target cells. The competitor peptide is identified on the vertical axis; % inhibition of lysis is indicated on the horizontal axis. Data are given in percent inhibition of lysis by each of the peptides. No inhibition represented 71% lysis.

FIGS. 13A and B illustrate the efficiency of peptide recognition by Her-2/neu-specific CTL lines. The H7-and H3-specific CTLs established from A2.1-Tg or A2/K ^b-Tg mice were assayed for lytic activity against the H7 and H3 peptides, respectively. Peptides were used to pulse T2 labeled targets at the indicated concentrations. Percent specific lysis is plotted against peptide concentration (molar). In FIG. 13A, the open circles (∘) represent H7-A2.1/K ^b×CD8, while the closed circles ●) represent H7-A2.1. In FIG. 13B, open circles (∘) represent H3-A2.1/K ^b×CD8, while the closed circles (●) represent H3-A2.1. Data represent lysis at effector to target ratios (E:T) of 1:1 in a four-hour assay.

FIGS. 14A-D illustrate the inhibition of specific killing by anti-A2 antibody. An anti-A2 mAb (PA2.1) was used to determine if CTL lysis was A-2 restricted. Prior to the addition of the effector cells, tumor cells were incubated in the presence or absence of 0.5 mg/ml of PA2.1 mAb. Percent specific lysis is plotted against E:T ratio in each of FIGS. 14A-D. In FIG. 14A, closed circles (●) represent NCI-H1355, while closed squares (▪) represent NCI-H1355-PA2.1. In FIG. 14B, closed circles (●) represent MDA-231, while closed squares (▪) represent MDA-231-PA2.1. In FIG. 14C, closed circles (●) represent SAOS-175, while closed squares (▪) represent SAOS-175-PA2.1. In FIG. 14D, closed circles (●) represent T98G, while closed squares (▪) represent T98G-PA2.1.

FIGS. 15A-D show that H3 and H7 peptides are presented on the surface of tumor cells. Peptides from the MDA.MB.231 and MCF-7 tumor cell lines were extracted by acid elution and fractionated as described in Example 5, using a C18 analytical column. Following HPLC fractionation, the samples were lyophilized and resuspended in 100 μl of PBS. Fifty (50) μl of each fraction from MDA.MB.231 (FIGS. 15A and 15C) and MCF-7 (FIGS. 15B and 15D) were used to pulse T2-A2K ^btarget cells and assayed for recognition by the H3 (FIGS. 15A and 15B) and H7 (FIGS. 15C and 15 D) CTL populations. Data represents lysis at E:T 10.1 in a four-hour assay. In each of FIGS. 15A-D, % specific lysis is plotted against HPLC-Fractions.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Amino Acid Residue: An amino acid, e.g., one formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH ₂refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature (described in J. Biol. Chem . 243:3552-59 (1969) and adopted at 37 C.F.R. § 1.822(b)(2)), abbreviations for amino acid residues are shown in the following Table of Correspondence:


TABLE OF CORRESPONDENCE
SYMBOL
1-Letter	3-Letter	AMINO ACID

Y	Tyr	tyrosine
G	Gly	glycine
F	Phe	phenylalanine
M	Met	methionine
A	Ala	alanine
S	Ser	serine
I	Ile	isoleucine
L	Leu	leucine
T	Thr	threonine
V	Val	valine
P	Pro	proline
K	Lys	lysine
H	His	histidine
Q	Gln	glutamine
E	Glu	glutamic acid
Z	Glx	Glu and/or Gln
W	Trp	tryptophan
R	Arg	arginine
D	Asp	aspartic acid
N	Asn	asparagine
B	Asx	Asn and/or Asp
C	Cys	cysteine
X	Xaa	Unknown or other

It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxy-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those listed in 37 C.F.R. § 1.822(b)(4), and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH ₂or to a carboxy-terminal group such as COOH.

The term conservative substitution as used herein is meant to denote that one amino acid residue has been replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as Ile, Val, Leu or Met for another, or the substitution of one polar residue for another such as between Arg and Lys, between Glu and Asp or between Gln and Asn, and the like. The term “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that such a polypeptide also displays the requisite binding activity.

In some instances, the replacement of an ionic residue by an oppositely charged ionic residue such as Asp by Lys has been termed conservative in the art in that those ionic groups are thought to merely provide solubility assistance. In general, however, since the replacements discussed herein are on relatively short synthetic polypeptide antigens, as compared to a whole protein, replacement of an ionic residue by another ionic residue of opposite charge is considered herein to be a “radical replacement”, as are replacements between nonionic and ionic residues, and bulky residues such as Phe, Tyr or Trp and less bulky residues such as Gly, Ile and Val.

The term antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. Illustrative antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab′, F(ab′) ₂and F(v).

The term antibody combining site refers to that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) antigen.

The term correspond in its various grammatical forms is used herein and in the claims in relation to polypeptide sequences to mean the polypeptide sequence described plus or minus up to three amino acid residues at either or both of the amino-and carboxy-termini and containing only conservative substitutions in particular amino acid residues along the polypeptide sequence.

Polypeptide and Peptide are terms used interchangeably herein to designate a series of no more than about 50 amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

Protein: Protein is a term used herein to designate a series of greater than 50 amino acid residues connected one to the other as in a polypeptide.

Receptor and receptor protein are terms used herein to indicate a biologically active proteinaceous molecule that specifically binds to (or with) other molecules.

Substantially homologous means that a particular subject sequence or molecule, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences. For purposes of the present invention, amino acid sequences having greater than 90 percent similarity, equivalent biological activity, and equivalent expression characteristics are considered substantially homologous and are included within the scope of proteins defined by the terms “p53” and “Her-2/Neu”. Amino acid sequences having greater than 40 percent similarity are considered substantially similar. For purposes of determining homology or similarity, truncation or internal deletions of the reference sequence should be disregarded, as should subsequent modifications of the molecule, e.g., glycosylation. Sequences having lesser degrees of homology and comparable bioactivity are considered equivalents.

Transfection as the term is used herein means the acquisition of new genetic markers by incorporation of added DNA in eucaryotic cells, whereas transformation refers to the acquisition of new genetic markers by incorporation of added DNA in procaryotic cells.

As used herein, the term vector refers to a DNA molecule capable of autonomous replication and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.

Vectors capable of directing the expression of DNA segments (genes) encoding one or more proteins are referred to herein as “expression vectors”. Also included are vectors which allow the cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.

B. Detailed Description

1. Enhancing Tumor Immunogenicity Using Tumor-Specific Antigens

a. The p53 Protein

Normal p53 protein acts as a regulator of the cell cycle. In response to DNA damaging influences, such as UV light, normal p53 protein accumulates in cell nuclei, causing cell cycle arrest at the G ₁phase, thus allowing cells to repair the DNA damage. This function of p53 is lost in tumor cells in which p53 is inactivated by mutation of the gene or by binding of the proteins encoded by viral or cellular oncogenes to p53. As a result, genetic alterations accumulate at a rapid rate in affected cells, leading to malignant transformation. (See, e.g., Lane, Nature 358: 15-16 (1992); Ullrich, et al., J. Biol. Chem . 267: 15259-15262 (1992); Hartwell, Cell 71: 543-546 (1992).)

It is not known to what extent the overexpression of p53—the expression of which is seen as a normal response to DNA damage—leads to an immune response to the p53 protein. In any event, mutation of the p53 gene (p53) is the most frequent genetic change associated with human cancer. Moreover, in many tumor cells carrying mutations in p53, the p53 protein is also overexpressed due to decreased breakdown.

In addition, this overexpression is often associated with the formation of anti-p53 antibodies. For example, in one recent study, all small cell lung cancer patients with demonstrable serum antibodies against the p53 protein had mis-sense mutations in p53 and overexpressed p53 antigen in their tumor cell lines. One study reported that anti-p53 antibodies were not detected in sera from patients with other types of p53 mutation (Winter, et al., Cancer Res . 52: 4168-74 (1992)).

It has also been reported that the antibody response to p53 in breast cancer patients is directed against immunodominant epitopes unrelated to the mutational “hot spot” (Schlichtholz, et al., Cancer Res . 52: 6380-4 (1992)). The antibodies were reactive with two immunodominant regions located at the carboxy-and amino-termini of the protein, outside the mutational “hot spot” region (Id.).

The detection of antibodies directed against immunodominant epitopes suggests that such antibodies are actually autoantibodies, as they are directed against normal p53 sequences. In turn, this finding implies that the low level of p53 in normal cells is “ignored” by the immune system, which means that immunotherapies directed against p53 would likely cause little or no damage to normal cells.

The concept of autoimmunity to p53 as a possible therapeutic principle is also supported by the in vitro arousal of CTL responses against a wild type p53 peptide presented by the HLA A2.1 MHC class I molecule. In the relevant study, CTLs against a mutant p53 peptide presented by HLA A2.1 were also obtained.

Responses against both peptides were obtained with responding T lymphocytes from healthy donors. The extent to which these CTLs can recognize HLA-matched tumor cells with p53 overexpression mutants was not tested, however. Interestingly, no CTLs were obtained by stimulation with a p53 self-peptide that binds HLA A2.1 with even higher affinity, suggesting that this peptide may have induced immunological tolerance. (See Melief and Kast, Curr. Op. Immunol . 5: 709-13 (1993)).

b. Her-2/Neu

Her-2/Neu, which is also known as c-erbB-2, is a proto-oncogene that encodes a 185 kDa transmembrane receptor glycoprotein with tyrosine-specific kinase activity. Expression of this protein is enhanced in a number of breast and ovarian tumors and correlates with tumor aggressiveness, suggesting that it may play an important role in tumor growth. (See, e.g., Ioannides, et al., Cellular Immunol . 151: 225-234 (1993).) The Her-2/Neu protein has also been described as a growth factor receptor-like protein. (See, e.g., DiFiore, et al., Science 237: 178-182 (1987); Bargmann, et al., Nature 319: 226-230 (1986); Yamamoto, et al., Nature 319: 230-234 (1986).)

Her-2/Neu is similar in structure and sequence to the epidermal growth factor receptor (Coussens, et al., Science 230: 1132 (1985)). The Her-2/neu oncogene (also referred to as erbB-2) is amplified and overexpressed in approximately 30% of human breast and ovarian tumors, and the overexpression of the Her-2/Neu protein correlates with a poor prognosis in these diseases (Slamon, et al., Science 244: 707 (1989)).

Recent in vitro experiments submit that at least three antigenic epitopes are recognized on ovarian cancer cells by tumor-specific CTL (Ioannides, et al., J. Immunol . 146: 1700 (1991)). Another study has proposed that the sensitivity of ovarian epithelial tumor cells to CTL-mediated lysis is associated with the level of expression of Her-2/Neu, intimating that this oncogene product may serve as a source of tumor-associated antigens or as an inducer of such peptides (Yoshino, et al., J. Immunol . 152: 2393 (1994)). The identity or source of these tumor-associated antigens (TAA) is unknown, but the oncogene products seem to be logical candidates. The potential relationship between Her-2/Neu expression and the immune response to ovarian cancer is unclear, however, but it has been proposed that Her-2/Neu expression may be inversely related to lymphokine-activated killer cell-mediated killing (Lichtenstein, et al., Cancer Res , 50: 7364 (1990)).

Another study proposes that CTL expanded from tumor-associated lymphocytes with HLA-A2 ⁺ and Her-2/Neu ⁺ tumors can specifically recognize synthetic peptides corresponding to amino acids 971-980 of Her-2/Neu protein (Ioannides, et al., Cellular Immunol . 151: 225-234 (1993)).

2. Polypeptides

A polypeptide or peptide of the present invention is preferably derived from a protein expressed by a “target” cell or tissue—e.g., tumor cells or other malignant cells or tissues. In one embodiment, such a protein from which useful peptides may be derived is unique to target cells or tissues. Alternatively, an exemplary peptide may be derived from a protein which is expressed in “normal” cells, but is overexpressed in “abnormal” cells such as tumor cells.

For example, a polypeptide of the present invention may be derived from p53 protein, Her-2/Neu protein, or from other candidate (i.e., tumor-associated) proteins. The terms “polypeptide” and “peptide” may be used interchangeably herein.

Thus, an exemplary polypeptide of the present preferably invention corresponds in amino acid residue sequence to one or more amino acid residue sequences of a normal p53 protein, a mutated form of p53 protein, a p53 protein analogue, or a derivative of any of the foregoing. For example, a p53-derived polypeptide may have an amino acid residue sequence corresponding to the formula STPPPGTRV (SEQ ID NO 3), or a sequential subset thereof.

Another exemplary polypeptide of the present invention corresponds in amino acid residue sequence to one or more amino acid residue sequences of normal Her-2/Neu protein, a mutated form of Her-2/Neu protein, a Her-2/Neu protein analogue, or a derivative of any of the foregoing. For example, a Her-2/Neu-derived polypeptide may have an amino acid residue sequence corresponding to the formula KIFGSLAFL (SEQ ID NO 10), VMAGVGSPYV (SEQ ID NO 12), or any sequential subsets thereof.

A polypeptide of the present invention also can exhibit homology in sequence to a polypeptide portion of a protein expressed or abnormally expressed in a target cell or tissue. Preferably, a polypeptide of the present invention corresponds to a sequential subset of p53 protein or Her-2/Neu protein, wherein “sequential subset” refers to the fact that a polypeptide has an amino acid residue sequence corresponding to that of a subset of the amino acid residue sequence of a larger protein or polypeptide. For example, if “ABCDEFGH” represented an amino acid residue sequence of a polypeptide, exemplary sequential subsets thereof would include “ABC”, “BCDE”, “DEFGH”, “ABCDEFG”, and so forth.

The present invention provides certain polypeptides that stimulate HLA class I restricted cytotoxic T lymphocyte (“CTL”) responses against certain tumor antigens, particularly when such antigens are expressed in a host cell that is capable of expressing such antigens. Such polypeptides are useful in compositions and methods for the treatment, prevention, and diagnosis of tumors and malignancies—e.g., carcinoma of the breast. For example, stimulated CTLs of the present invention are able to specifically target and kill specific antigen-expressing cells, thereby preventing, impeding, or reversing the course of the relevant disease process. Novel combinations of epitopes are contemplated within the context of the present invention, such that the CTL response described in brief above, and in greater detail below, is combined with a T-helper response or multiple CTL response directed at different antigens, for example.

The polypeptides of interest are derived from various regions of tumor-related proteins or nucleotide sequences encoding same. For example, p53 peptides having the following amino acid residue sequences (or sequential subsets thereof) are contemplated herein: p53.25-35, LLPENNVLSPL (SEQ ID NO 1); p53.65-73, RMPEAAPPV (SEQ ID NO 2); p53.149-157, STPPPGTRV (SEQ ID NO 3); p53.264-272, LLGRNSFEV (SEQ ID NO 4). In addition, Her-2/Neu peptides having the following amino acid residue sequences (or sequential subsets thereof) are also contemplated herein: HER-3, KIFGSLAFL (SEQ ID NO 10); HER-6, TLQGLGISWL (SEQ ID NO 11); HER-7, VMAGVGSPYV (SEQ ID NO 12); HER-8, VLQGLPREYV (SEQ ID NO 13); and HER-9, ILLVVVLGV (SEQ ID NO 14).

In certain embodiments of the present invention, the polypeptides of interest will have the sequences just recited as well as others listed below, or will have sequences that are substantially homologous thereto. Two polypeptides are said to be substantially homologous if at least 50% of the amino acid (“aa”) residues are the same in the same or analogous position. By analogous position, it is intended the relative position of the polypeptide of interest itself, regardless of any flanking polypeptide or other chemical elements that may be attached to the polypeptide of interest.

Preferred peptides employed in the subject invention, accordingly, need not be identical, but are at least substantially homologous, to the following peptides: LLPENNVLSPL (SEQ ID NO 1); RMPEAAPPV (SEQ ID NO 2); STPPPGTRV (SEQ ID NO 3); LLGRNSFEV (SEQ ID NO 4); KIFGSLAFL (SEQ ID NO 10); TLQGLGISWL (SEQ ID NO 11); VMAGVGSPYV (SEQ ID NO 12); VLQGLPREYV (SEQ ID NO 13); and ILLVVVLGV (SEQ ID NO 14).

The present invention relates to a polypeptide having substantial homology with a CTL epitope selected from the same group of polypeptides identified above. Preferred polypeptides include LLPENNVLSPL (SEQ ID NO 1); RMPEAAPPV (SEQ ID NO 2); STPPPGTRV (SEQ ID NO 3); LLGRNSFEV (SEQ ID NO 4); KIFGSLAFL (SEQ ID NO 10); TLQGLGISWL (SEQ ID NO 11); VMAGVGSPYV (SEQ ID NO 12); VLQGLPREYV (SEQ ID NO 13); and ILLVVVLGV (SEQ ID NO 14).

In particular, the present invention relates to a suitable molecule comprising a polypeptide having substantial homology with one of the CTL epitopes recited above. The molecule of the present invention comprises at least about five amino acids and as many as about 50 amino acids. A preferred range of amino acids for the molecule of the present invention is from about seven amino acids to fewer than about twenty-five amino acids. A more preferred range of amino acids is from about eight amino acids to fewer than about fifteen. An even more preferred range of amino acids is from about eight amino acids to fewer than about 13 amino acids.

It may be desirable to optimize peptides of the invention to a length of about eight to twelve amino acid residues, commensurate in size with endogenously processed peptides that are bound to major histocompatibility complex (“MHC”) class I molecules on the cell surface. See generally, Schumacher et al., Nature , 350, 703-706 (1991); Van Bleek et al., Nature , 348, 213-216 (1990); Rotzschke et al., Nature , 348, 252-254 (1990); and Falk et al., Nature , 351, 290-296 (1991). Methods of selecting and generating class I MHC molecules are also disclosed in U.S. Pat. No. 5,314,813, the disclosures of which are incorporated by reference herein.

As set forth in more detail below, usually the peptides will have at least a majority of amino acids that are homologous to a corresponding portion of contiguous residues of the p53 or Her-2/Neu sequences disclosed hereinabove, and contain a CTL-inducing epitope.

The peptides of the present invention can be prepared by any suitable means, such as synthetically using standard peptide synthesis chemistry or by using recombinant DNA technology. Although the peptide preferably will be substantially free of other naturally occurring p53 or Her-2/Neu proteins and fragments thereof, in some embodiments the peptides can be synthetically conjugated to native fragments or particles, or other compounds that are nonproteinaceous. The term peptide is used interchangeably with polypeptide or oligopeptide in the present specification to designate a series of amino acids connected one to the other by peptide bonds between the alpha-amino and alpha-carboxy groups of adjacent amino acids. The polypeptides or peptides can be any suitable length, either in their neutral (actually zwitterionic) forms or in forms that are salts, and either free of modifications, such as glycosylation, side chain oxidation, or phosphorylation, or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides, as herein described.

Desirably, the peptide will be as small as possible while still maintaining substantially all of the biological activity of the larger peptides first disclosed herein. By biological activity is meant the ability to bind an appropriate MHC molecule and induce a cytotoxic T lymphocyte response against p53 or Her-2/Neu antigen or antigen mimetic. By a cytotoxic T lymphocyte response is meant a CD8 ⁺ T lymphocyte response specific for an antigen of interest, wherein CD8+, MHC class I-restricted T lymphocytes are activated. The activated T lymphocytes secrete lymphokines (e.g., gamma interferon) and liberate other products (e.g., serine esterases) that inhibit viral replication in infected autologous cells or transfected cells, with or without cell killing.

Various modifications can be effected at noncritical amino acid positions within the polypeptide of interest without substantially disturbing its biological activity. Such modifications include, but are not limited to, substitutions, deletions and additions of other peptidyl residues, C ₁-C ₇alkyl or C ₁-C ₁₀aralkyl, as described herein and as appreciated in the art.

A polypeptide of the present invention may or may not be glycosylated, depending on the means of synthesis. For example, if a non-glycosylated polypeptide is preferred, it may be synthesized either directly by standard peptide synthesis techniques or by procaryotic host expression of a recombinant DNA molecule of the present invention. A eucaryotically produced polypeptide of the present invention is not typically glycosylated.

A polypeptide of the present invention can also incorporate a variety of changes, such as insertions, deletions, and substitutions of amino acid residues which are either conservative or nonconservative as long as the resulting polypeptide molecule exhibits the desired properties. The “desired properties” as referred to herein include that the polypeptide is immunogenic in a suitable host and able to generate antibodies to a desired protein, polypeptides derived therefrom, or proteins or polypeptides substantially homologous to the desired protein, whether it is present in the denatured state (as is found in an SDS-PAGE gel) or in its natural state, as expressed in or on cells. In various alternative embodiments, the desired protein may be p53, Her-2/Neu, or another protein associated with tumors or other malignancies.

A majority of the amino acids of the polypeptides of the present invention will be identical or substantially homologous to the amino acids of the corresponding portions of naturally occurring p53 or Her-2/Neu proteins or epitopes identified above, wherein the selected polypeptide can be flanked and/or modified at one or both termini as described herein.

Accordingly, a molecule of the present invention in one of its embodiments comprises a polypeptide as described hereinabove that has conjugated thereto a substance, wherein the substance is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a solid matrix, a carrier, and a second CTL epitope. The substance can be conjugated to the polypeptide at any suitable position, including the N and C termini and points in between, depending on the availability of appropriate reactive groups in the side chains of the constituent amino acids of the polypeptide of interest. Additionally, the substance can be conjugated directly to the polypeptide or indirectly by way of a linker. Preferred radiolabels include ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, and other suitable radiolabels for use in various radioimmunoassays and the like. Preferred fluorescent labels include fluorescein, rhodamine, and other suitable fluorescent labels for use in fluorescent assays and the like.

Preferred enzymes include alkaline phosphatase and other suitable enzymes useful for any suitable purpose, including as a marker in an assay procedure. Preferred solid matrices are glass, plastic, or other suitable surfaces, including various resins such as Sephadex® chromatography media and the like. Preferred carriers include immunogenic lipids, proteins, and other suitable compounds, such as a liposome or bovine serum albumin. Preferred second CTL epitopes include T-helper specific antigens, antigens that would foster a B cell response, and other suitable antigens that stimulate CTLs.

Additional amino acids can be added to the termini of a peptide of the present invention to provide for ease of linking peptides one to another, for coupling to a carrier, support or a larger peptide, for reasons discussed herein, or for modifying the physical or chemical properties of the peptide, and the like. Suitable amino acids, such as tyrosine, cysteine, lysine, glutamic or aspartic acid, and the like, can be introduced at the C-or N-terminus of the peptide. In addition, a peptide of the present invention can differ from the natural sequence by being modified by terminal-NH ₂acylation, e.g., acetylation, or thioglycolic acid amidation, terminal-carboxy amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.

It is understood that the p53 or Her-2/Neu peptides of the present invention or analogs or homologs thereof that have cytotoxic T lymphocyte stimulating activity may be modified as necessary to provide certain other desired attributes—e.g., improved pharmacological characteristics—while increasing or at least substantially retaining the biological activity of the unmodified peptide. For instance, the within-described peptides can be modified by extending, decreasing or substituting amino acids in the peptide sequence by, for example, the addition or deletion of suitable amino acids on either the amino terminal or carboxy terminal end, or both, of peptides derived from the sequences disclosed herein.

When a polypeptide of the present invention incorporates conservative substitutions of the sequences corresponding to the p53 or Her-2/Neu proteins or polypeptides depicted above, the substituted amino acid residues are replaced by another, biologically similar amino acid residue such that the resulting polypeptide has an amino acid residue sequence that is different from (other than) a sequence of p53 or Her-2/Neu. Some examples of conservative substitutions include substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue. Also, a polar residue such as arginine, glycine, glutamic acid, aspartic acid, glutamine, asparagine, and the like, can be conservatively substituted for another member of this group.

Still another aspect of a polypeptide incorporating conservative substitutions occurs when a substituted amino acid residue replaces an unsubstituted parent amino acid residue. Examples of substituted amino acids may be found at 37 C.F.R. § 1.822(b)(4), which species are incorporated herein by reference.

The peptides may be modified to enhance substantially the CTL inducing activity, such that the modified peptide analogs have CTL activity greater than a peptide of the wild-type sequence. For example, it may be desirable to increase the hydrophobicity of the N-terminus of a peptide, particularly where the second residue of the N-terminus is hydrophobic and is implicated in binding to the HLA restriction molecule. By increasing hydrophobicity at the N-terminus, the efficiency of the presentation to T cells may be increased. Peptides prepared from other disease associated antigens, particularly those containing CTL inducing epitopes for which a host may not have significant CTL activity, may be made CTL-inducing by substituting hydrophobic residues at the N-terminus of the peptide where the second residue is normally hydrophobic.

Therefore, peptides of the present invention may be subject to various modifications, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such modifications provide for certain advantages in their use. By “conservative substitution” is meant replacing an amino acid residue with another that is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Preferably, the portion of the sequence that is intended to mimic substantially a p53 or Her-2/Neu cytotoxic T lymphocyte-stimulating epitope will not differ by more than about 20% from the sequence of at least one portion or segment of p53 protein or Her-2/Neu protein, except where additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, for example, ease of linking or coupling, and the like.

Within the peptide sequences identified by the present invention, including the representative peptides listed above, there are residues (or those that are substantially functionally equivalent) that allow a particular peptide to retain its biological activity, i.e., the ability to stimulate a class I-restricted cytotoxic T-lymphocytic response against cells that express p53 or Her-2/Neu antigen. These residues can be identified by suitable single amino acid substitutions, deletions, or insertions, followed by suitable assays, such as testing for cytotoxic activity by so-stimulated CTLs.

In addition, the contributions made by the side chains of the residues can be probed via a systematic replacement of individual residues with a suitable amino acid, such as Gly or Ala. Systematic methods for determining which residues of a linear amino acid sequence are required for binding to a specific MHC protein, one of the characteristics of the peptides of the present invention, are known. See, for instance, Allen et al., Nature 327: 713-717; Sette et al., Nature 328: 395-399; Takahashi et al., J. Exp. Med . 170: 2023-2035 (1989); and Maryanski et al., Cell 60: 63-72 (1990).

Peptides that tolerate multiple amino acid substitutions generally incorporate small, relatively neutral molecules, e.g., Ala, Gly, Pro, or similar residues. The number and types of residues that can be substituted, added or subtracted will depend on the spacing necessary between the essential epitopic points and certain conformational and functional attributes that are sought. By types of residues, it is intended, e.g., to distinguish between hydrophobic and hydrophilic residues, among other attributes. If desired, increased binding affinity of peptide analogs to its MHC molecule for presentation to a cytotoxic T-lymphocyte can also be achieved by such alterations. Generally, any spacer substitutions, additions or deletions between epitopic and/or conformationally important residues will employ amino acids or moieties chosen to avoid stearic and charge interference that might disrupt binding.

Peptides that tolerate multiple substitutions while retaining the desired biological activity may also be synthesized as D-amino acid-containing peptides. Such peptides may be synthesized as “inverso” or “retro-inverso” forms, that is, by replacing L-amino acids of a sequence with D-amino acids, or by reversing the sequence of the amino acids and replacing the L-amino acids with D-amino acids. As the D-peptides are substantially more resistant to peptidases, and therefore are more stable in serum and tissues compared to their L-peptide counterparts, the stability of D-peptides under physiological conditions may more than compensate for a difference in affinity compared to the corresponding L-peptide. Further, L-amino acid-containing peptides with or without substitutions can be capped with a D-amino acid to inhibit exopeptidase destruction of the antigenic peptide.

In addition to the exemplary peptides described herein, the present invention provides methods for identifying other epitopic regions associated with said peptide regions capable of inducing MHC-restricted cytotoxic T lymphocyte responses against tumor cells or tissues. The methods comprise obtaining peripheral blood lymphocytes (PBL) from affected and/or unaffected individuals and exposing (i.e., stimulating) the PBL cells with synthetic peptide or polypeptide fragments derived from a peptide region (e.g., p53 derivatives such as p53.25-35, LLPENNVLSPL (SEQ ID NO 1); p53.65-73, RMPEAAPPV (SEQ ID NO 2); p53.149-157, STPPPGTRV (SEQ ID NO 3); and p53.264-272, LLGRNSFEV (SEQ ID NO 4)). Peptides derived from Her-2/Neu proteins are useful in this regard as well, and include exemplary peptides such as HER-3, KIFGSLAFL (SEQ ID NO 10); HER-6, TLQGLGISWL (SEQ ID NO 11); HER-7, VMAGVGSPYV (SEQ ID NO 12); HER-8, VLQGLPREYV (SEQ ID NO 13); and HER-9, ILLVVVLGV (SEQ ID NO 14).

Pools of overlapping synthetic peptides randomly selected from the p53 or Her-2/Neu protein's amino acid residue sequence, each typically about 8 to 20 residues long, preferably 8-12 residues, can be used to stimulate the cells. Alternatively, as exemplified hereinbelow, peptides fitting a binding motif for CTL-directed antigens of a particular HLA class I allele (Falk et al., Nature 351: 290-296 (1991)) were selected for testing. It is contemplated that peptides fitting the analogous binding motifs for other HLA class I alleles may be identified by following the methods disclosed herein, and accordingly are viewed as part of the present invention. (See, e.g., Guo et al., Nature 360: 364-366 (1992); Jardetzky et al., Nature 353: 326-329 (1991).)

Active peptides can be selected from pools that induce cytotoxic T lymphocyte activity. The ability of the peptides to induce specific cytotoxic activity is determined by incubating the stimulated PBL cells with autologous labeled (e.g., ⁵¹Cr) target cells (such as HLA matched macrophages, T cells, fibroblasts or B lymphoblastoid cells) expressing p53 or Her-2/Neu proteins, polypeptides, or derivatives thereof (or subgenomic fragments thereof), such that the targeted antigen is synthesized endogenously by the cell (or the cell is pulsed with the peptide of interest), and measuring specific release of label.

Once a peptide having an epitopic region that stimulates a cytotoxic T lymphocyte response is identified, the MHC restriction element of the response can be determined and/or confirmed. This involves incubating the stimulated PBL or short term lines thereof with a panel of (labeled) target cells or known HLA types that have been pulsed with the peptide of interest, or appropriate controls. The HLA allele(s) of cells in the panel that are lysed by the CTL are compared to cells not lysed, and the HLA restriction element(s) for the cytotoxic T lymphocyte response to the antigen of interest is identified.

Carbone et al. ( J. Expo. Med . 167: 1767 (1988)) have reported that stimulation with peptides may induce cytotoxic T lymphocytes with low affinity for corresponding endogenous protein, such that repetitive peptide stimulation may yield cytotoxic T lymphocytes that recognize peptide but not native antigen. As the inability of stimulated cytotoxic T lymphocytes to recognize native Her-2/Neu proteins, for example, would be undesirable in the development of anti-Her-2/Neu peptide therapeutics and vaccine compositions, methods to circumvent this potential limitation are preferably used. For example, a sequential restimulation of cytotoxic T cells may be employed according to the present invention to identify and select T cells with a higher affinity for naturally processed antigen than for a synthetic peptide. Short term cytotoxic T lymphocyte lines are established by restimulating activated PBL.

Cells stimulated with peptide are preferably restimulated with peptide and recombinant or native p53 or Her-2/Neu antigen, e.g., a Her-2/Neu-derived peptide. Cells having activity may also be stimulated with an appropriate T cell mitogen, e.g., phytohemagglutinin (PHA). The restimulated cells are provided with irradiated allogeneic PBLs as an antigen nonspecific source of T cell help, and the appropriate antigen.

To expand selectively the population of cytotoxic T lymphocytes that recognize, e.g., native Her-2/Neu antigen and to establish long term lines, a sample of PBL from a patient is first stimulated with peptide and recombinant or native tumor-related antigen, followed by restimulation with HLA-matched B lymphoblastoid cells that stably express the corresponding tumor-related antigen polypeptide. The cell lines are re-confirmed for the ability to recognize endogenously synthesized antigen using autologous and allogeneic B-lymphoblastoid or other cells transfected or infected so as to produce the appropriate antigen.

Having identified different peptides of the invention that contribute to inducing anti-tumor cytotoxic T lymphocyte responses in one or more patients or HLA types, in some instances it may be desirable to join two or more peptides in a composition, either by chemical linkage or as a physical mixture. The peptides in the composition can be identical or different, and together they should provide equivalent or greater biological activity than the parent peptide(s). For example, using the methods described herein, two or more peptides may define different or overlapping cytotoxic T lymphocyte epitopes from a particular region, e.g. p53-derived peptides STPPPGTRV (SEQ ID NO 3) and LLGRNSFEV (SEQ ID NO 4), which peptides can be combined in a “cocktail” to provide enhanced immunogenicity for cytotoxic T lymphocyte responses. Moreover, suitable peptides of one p53 or Her-2/Neu region can be combined with suitable peptides of other p53 or Her-2/Neu regions, respectively, from the same or different protein, particularly when a second or subsequent peptide has a MHC restriction element different from the first. The present disclosure thus includes exemplary proteins, polypeptides, and epitope sequences derived from various p53 or Her-2/Neu regions.

This composition of peptides can be used effectively to broaden the immunological coverage provided by therapeutic, prophylactic, or diagnostic methods and compositions of the present invention for the benefit of a diverse population. For example, the different frequencies of HLA alleles among prevalent ethnic groups (Caucasian, asian and african blacks) are shown in the following table. Therapeutic or vaccine compositions of the invention may be formulated to provide potential therapy or immunity to as high a percentage of a population as possible.


HLA ALLELE
FREQUENCIES AMONG PREVALENT ETHNIC GROUPS
	HLA Allele	EUC	NAC	AFR	JPN

	A2	45.3	46.6	27.3	43.2
	A29	7.4	8.1	12.3	0.4
	A31	5.4	6.2	4.4	15.3
	A32	8.8	7.1	3	0.1
	A33	3.3	3.4	9	13.1
	A28 ¹	7.7	9.9	16.6	1.1

	Abbreviations: EUC, European Caucasian; NAC, North American Caucasian; AFR, African blacks; JPN, Japanese.
	¹A28 represents the two alleles A268 and A269.

The peptides of the present invention may further be combined via linkage to form polymers (multimers), or can be formulated in a composition without linkage, as an admixture. Where the same peptide is linked to itself, thereby forming a homopolymer, a plurality of repeating epitopic units are presented. When the peptides differ, heteropolymers with repeating units are provided, forming a cocktail of, for example, epitopes specific to different tumor antigen segments, different epitopes to the same protein or gene region, different epitopes to different proteins or gene regions, different HLA restriction specificities, and/or a peptide that contains T helper epitopes. In addition to covalent linkages, noncovalent linkages capable of forming intermolecular and intrastructural bonds are included.

Linkages for homo- or hetero-polymers or for coupling to carriers can be provided in a variety of ways. For example, cysteine residues can be added at both the amino-and carboxy-termini, where the peptides are covalently bonded via controlled oxidation of the cysteine residues. Also useful are a large number of heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, including N-succinimidyl-3-(2-pyridyl-dithio) proprionate (SPDP). This latter reagent creates a disulfide linkage between itself and a cysteine residue in one protein and an amide linkage through the amino on a lysine or other free amino group in the other. A variety of such disulfide/amide forming agents are known. See, for example, Immun. Rev . 62: 185 (1982).

Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioether forming agents are commercially available (from, for example, Aldrich Chemical Company, Inc., Milwaukee, Wis.) and include reactive esters of 6-maleimidocaproic acid, 2 bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. A particularly preferred coupling agent is succinimidyl-4-(n-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). It will be understood that suitable linkage does not substantially interfere with either of the linked groups to function as described, e.g., as an anti-tumor cytotoxic T cell determinant/stimulant, peptide analogs, or T helper determinant/stimulant.

In another aspect of the present invention, the peptides of the invention can be combined or coupled with other suitable peptides that present anti-tumor T-helper cell epitopes, i.e., epitopes that stimulate T cells that cooperate in the induction of cytotoxic T cells to tumor antigens, such as those derived from p53 or Her-2/Neu protein. The T-helper cells can be either the T-helper 1 or T-helper 2 phenotype, for example.

The peptides of the present invention can be prepared using any suitable means. Because of their relatively short size (generally, fewer than 50 amino acids, and preferably fewer than 20), the peptides can be synthesized in solution or on a solid support in accordance with conventional peptide synthesis techniques. Various automatic synthesizers are commercially available (for example, from Applied Biosystems) and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis (2d. ed., Pierce Chemical Co., 1984); Tam et al., J. Am. Chem. Soc . 105: 6442 (1983); Merrifield, Science 232: 341-347 (1986); and Barany and Merrifield, The Peptides (Gross and Meienhofer, eds., Academic Press, New York, 1979), 1-284.

Alternatively, suitable recombinant DNA technology may be employed for the preparation of the peptides of the present invention, wherein a nucleotide sequence that encodes a peptide of interest is inserted into an expression vector, transformed or transfected into a suitable host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., Molecular Cloning, A Laboratory Manual (2d ed., Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989), and Current Protocols in Molecular Biology (Ausubel et al., eds., John Wiley and Sons, Inc., New York, 1991), and U.S. Pat. Nos. 4,237,224, 4,273,875, 4,431,739, 4,363,877 and 4,428,941, for example.

Thus, recombinant DNA-derived proteins or peptides, which comprise one or more peptide sequences of the invention, can be used to prepare the anti-tumor cytotoxic T cell epitopes identified herein or identified using the methods disclosed herein. For example, a recombinant p53-derived peptide of the present invention may be prepared in which the p53 amino acid sequence is altered so as to present more effectively epitopes of peptide regions described herein to stimulate a cytotoxic T lymphocyte response. By this means, a polypeptide is used that incorporates several T cell epitopes into a single polypeptide.

As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc ., 103, 3185 (1981), modifications can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available.

For expression of fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in a suitable cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts, yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.

It is also preferable that the polypeptide is antigenic when expressed on cells or in its denatured state so that antibodies immunoreactive with the desired protein molecule also immunoreact with a polypeptide of the present invention. Accordingly, a polypeptide of the present invention can also be used to generate a variety of useful antibodies by means described herein. A polypeptide of the present invention may also be used to specifically trigger an immune response—e.g., to generate specific cytotoxic T lymphocytes (CTLs). These and other utilities of the polypeptides will be apparent from the discussions provided hereinbelow.

A polypeptide of the present invention can be synthesized by any of the peptide synthetic techniques known to those skilled in the art. A summary of some of the techniques available can be found in J. M. Stuard and J. D. Young, “Solid Phase Peptide Synthesis”, W. H. Freeman, Co., San Francisco (1969), J. Meinhofer,“Hormonal Proteins and Peptides” Vol. 2, pp. 46, Academic Press (New York) 1983, and U.S. Pat. No. 4,631,211, which description is incorporated herein by reference. When a polypeptide desired for use in the present invention is relatively short (less than about 50 amino acid residues in length) direct peptide synthetic techniques are generally favored, usually by employing a solid phase technique such as that of Merrifield ( JACS 85: 2149 (1963)).

A polypeptide of the present invention can also be synthesized by recombinant DNA techniques. Such recombinant techniques are favored especially when the desired polypeptide is relatively long (greater than about 50 amino acids residues in length). When recombinant DNA techniques are employed to prepare a polypeptide of the present invention, a DNA segment coding for the desired polypeptide is incorporated into a preselected vector that is subsequently expressed in a suitable host. The expressed polypeptide, containing at least one of the amino acid residue sequences corresponding to p53 or Her-2/Neu proteins or polypeptides identified above, is preferably purified by a routine method such as gel electrophoresis, immunosorbent chromatography, and the like.

3. Hybridomas and Antibody Compositions

a. Hybridomas

Hybridomas of the present invention are those which are characterized as having the capacity to produce an antibody, including a monoclonal antibody, of the present invention. Methods for producing hybridomas producing (secreting) antibody molecules having a desired immunospecificity, i.e., having the ability to immunoreact with a particular protein, an identifiable epitope on a particular protein and/or a polypeptide, are generally well known in the art. For example, useful methods are described by Niman et al., PNAS USA 80: 4949-4953 (1983), and by Galfre et al., Meth. Enzymol . 73: 3-46 (1981). Other methods are described in U.S. Pat. Nos. 5,180,806, 5,114,842, 5,204,445, and RE 32,011, the disclosures of which are incorporated by reference herein.

A hybridoma cell is typically formed by fusing an antibody-producing cell and a myeloma or other self-perpetuating cell line. Such a procedure was described by Kohler and Milstein, Nature 256: 495-497 (1975).

Typically, hybridomas of the present invention are produced by using, in the above techniques as an immunogen, a substantially pure p53 or Her-2/Neu protein, polypeptide, homolog, or a sequential subset of a polypeptide of the present invention.

b. Inocula

In another embodiment, a protein or polypeptide of this invention, an antigenicaily related variant thereof, or a protein or polypeptide at least 75% homologous to at least a portion of a p53 or Her-2/Neu protein or polypeptide identified herein is used in a pharmaceutically acceptable aqueous diluent composition to form an inoculum that, when administered in an effective amount, is capable of inducing antibodies that immunoreact with a p53 or Her-2/Neu protein or polypeptide.

The word “inoculum” in its various grammatical forms is used herein to describe a composition containing a p53 or Her-2/Neu protein or polypeptide of this invention as an active ingredient used for the preparation of antibodies against a p53 or Her-2/Neu protein or polypeptide.

When a polypeptide is used to induce antibodies it is to be understood that the polypeptide can be used alone, or linked to a carrier as a conjugate, or as a polypeptide polymer, but for ease of expression, the various embodiments of the polypeptides of this invention are collectively referred to herein by the term “polypeptide”, and its various grammatical forms.

For a polypeptide that contains fewer than about 35 amino acid residues, it is preferable to use the peptide bound to a carrier for the purpose of inducing the production of antibodies as already noted.

As previously noted, one or more additional amino acid residues can be added to the amino-or carboxy-termini of the polypeptide to assist in binding the polypeptide to a carrier. Cysteine residues added at the amino-or carboxy-termini of the polypeptide have been found to be particularly useful for forming conjugates via disulfide bonds. However, other methods well known in the art for preparing conjugates can also be used. Exemplary additional linking procedures include the use of Michael addition reaction products, di-aldehydes such as glutaraldehyde, Klipstein et al., J. Infect. Dis . 147: 318-326 (1983) and the like, or the use of carbodiimide technology as in the use of a water-soluble carbodiimide to form amide links to the carrier. For a review of protein conjugation or coupling through activated functional groups, see Aurameas, et al., Scand. J. Immunol . 8 ( Suppl . 7): 7-23 (1978).

Useful carriers are well known in the art, and are generally proteins themselves. Exemplary of such carriers are keyhole limpet hemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine serum albumin (BSA) or human serum albumin (HSA), red blood cells such as sheep erythrocytes (SRBC), tetanus toxoid, and cholera toxoid, as well as polyamino acids such as poly (D-lysine: D-glutamic acid), and the like.

The choice of carrier is more dependent upon the ultimate use of the inoculum and is based upon various criteria. For example, a carrier that does not generate an untoward reaction in the particular animal to be inoculated should be selected.

The present inoculum contains an effective, immunogenic amount of a p53 or Her-2/Neu protein or polypeptide of this invention. As noted above, a smaller polypeptide may be used as a conjugate (i.e., linked to a carrier). The effective amount of polypeptide or protein per unit dose depends, among other things, on the species of animal inoculated, the body weight of the animal, and the chosen inoculation regimen as is well known in the art. Inocula typically contain polypeptide or protein concentrations of about 10 micrograms to about 500 milligrams per inoculation (dose), preferably about 50 micrograms to about 50 milligrams per dose.

The term “dose” or “unit dose” as it pertains to the inocula of the present invention refers to physically discrete units suitable as unitary dosages for animals, each unit containing a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent; i.e., carrier, or vehicle. The specifications for the novel unit dose of an inoculum of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular immunologic effect to be achieved, and (b) the limitations inherent in the art of compounding such active material for immunologic use in animals, as disclosed in detail herein, these being features of the present invention.

Inocula are typically prepared by dispersing a polypeptide, polypeptide-conjugate, or protein in a physiologically tolerable (acceptable) diluent or vehicle such as water, saline or phosphate-buffered saline to form an aqueous compos

4950598	Process for making T cell hybridomas	August, 1990	Engleman et al.	435/172.2
5112948	Methods and compositions for inducing monocyte cytotoxicity	May, 1992	Jones
5292642	Methods and compositions for the detection of monocyte cytotoxicity inducing factor	March, 1994	Jones
5434247	Peptides for inducing monocyte cytotoxicity in diagnostics	July, 1995	Jones
5679641	Peptides of human P53 protein for use in human T cell response inducing compositions, and human P53 protein-specific cytotoxic t-lymphocytes	October, 1997	Melief et al.
5726023	Immune reactivity to HER-2/neu protein for diagnosis and treatment of malignancies in which the HER-2/neu oncogene is associated	March, 1998	Cheever et al.	435/7.1
6419931	Compositions and methods for eliciting CTL immunity	July, 2002	Vitiello et al.	424/201.1

WO/1993/024525	December, 1993			PEPTIDES OF HUMAN p53 PROTEIN FOR USE IN HUMAN T CELL RESPONSE INDUCING COMPOSITIONS, AND HUMAN p53 PROTEIN-SPECIFIC CYTOTOXIC T-LYMPHOCYTES
WO/1994/020127	February, 1994			HLA-A2.1 BINDING PEPTIDES AND THEIR USES