TADG-12 and CA125 are two proteins expressed with high specificity in ovarian cancer tumors. They thus would be potential antigens for immunotherapy in ovarian cancer. The invention is based on the discovery of peptides in TADG-12 and CA125 that can be used to induce an autologous T cell response that lyses ovarian cancer cells expressing TADG-12 or CA125. The peptides are contacted with dendritic cells in vitro to generate peptide-loaded dendritic cells. The peptide-loaded dendritic cells are contacted with T cells in vitro to amplify CD8+ T cells that recognize the peptide. At least one CA125 peptide and at least one TADG-12 peptide were found that amplified CD8+ T cells, even from cancer patients, that lysed autologous CA125-expressing or TADG-12-expressing tumor cells. The peptide-loaded dendritic cells can be administered to a cancer patient to amplify CD8+ T cells in vivo that attack the cancer cells. Alternatively, autologous CD8+ T cells can be amplified ex vivo and then infused into the cancer patient.

Description of the Invention

BACKGROUND OF THE INVENTION

Despite advances in post-surgical chemotherapy for ovarian cancer, nearly 90% of advanced cases will develop progressive disease that is refractory to salvage chemotherapy regimens. In response to the need for alternative treatments that prevent disease recurrence or progression, tumor-specific immunologic intervention has received some attention.

TADG-12 is a serine protease highly expressed in ovarian cancer, but with limited expression in normal human tissues (1). CA125/MUC16 is the best known ovarian tumor-associated antigen and its secreted form has long been recognized as the gold standard for monitoring patients with ovarian carcinoma.

Role of Dendritic Cells in T Cell Immunity

Dendritic cells (DC) are rare but highly potent antigen presenting cells of bone marrow origin that can stimulate both primary and secondary T and B cell responses (19-24). The combination of two cytokines (i.e., GM-CSF and IL-4) has been shown to generate large numbers of myeloid monocyte-derived DC (9-24). However, after 6-8 days of culture in vitro these DC are still immature. Although they may effectively capture antigens, these immature DC lack full T cell-stimulatory activity and are sensitive to the immunosuppressive effects of several immunoregulatory cytokines that can be produced by tumors (25). In contrast, when maturation is induced by appropriate stimuli, such as monocyte-conditioned medium, LPS, or a cocktail of inflammatory cytokines (e.g., TNF.alpha., IL1.beta., PGE2a) (26), DC demonstrate a reduced ability to phagocytose antigens, but show a significantly higher production of key cytokines (e.g., IL-12), increased resistance to the immunosuppressive effects of IL-10, increased expression of T cell adhesion and costimulatory molecules, and increased expression of chemokine receptors that guide DC migration into lymphoid organs for priming of antigen-specific T cells (24, 25).

DC and Human Tumor Immunotherapy

Monocyte-derived mature DC-based vaccinations have recently been shown to induce the rapid generation of broad T cell immunity in healthy subjects vaccinated with less than 3.times.10.sup.6 antigen-pulsed autologous DC (27, 28). In contrast, the administration of immature DC has been reported to result in inhibition of pre-existing effector T cell function (29). These recently published studies represent the first indisputable evidence of the efficacy of DC vaccination as novel and powerful tools for human immunization. However, at this time, the extent to which general conclusions can be drawn from these observations for the active immunization of cancer patients remain only partially established.

In this regard, only a few clinical trials of DC vaccination have been reported in cancer patients. These studies have sometimes documented the induction of an anti-tumor immune responses and therapeutic benefit. In a study of patients with low grade, chemotherapy-resistant non-Hodgkin's lymphoma, four patients were given a series of subcutaneous injections of DC cultured with tumor-derived idiotype protein (30). All four patients developed lymphoproliferative responses to their own idiotype protein. Clinical responses were also seen, with one patient with pericardial and periaortic masses experiencing complete remission (durable for 42 months at the time of publication), and a second patient becoming PCR-negative (using idiotype-specific primers) and remaining in complete remission for 36 months. The remaining two patients showed stabilization of disease.

In children, vaccination of patients with solid tumors with tumor lysate-pulsed DC has been shown to expand tumor specific T cells and mediate cancer regression (35). Indeed, significant regression of multiple metastatic sites were seen in 1 patient. Five patients showed stable disease, including 3 who had minimal residual disease at the time of vaccine therapy and remain free of tumor with 16-30 months follow-up. Only patients who had failed standard therapies and therefore had been heavily pretreated with chemotherapy were considered eligible for this study. Importantly, all pediatric patients were treated in an outpatient setting without any observable toxicity resulting by DC administration.

Treatments to prevent disease recurrence or progeression in ovarian and other cancers are needed.

SUMMARY

Both TADG-12 and CA125 have tightly limited tissue expressions. Their expression is much higher in ovarian cancer tissue than normal ovary. Obtaining sufficient amounts of tumor antigen from a patient for the development of DC-based immunotherapy against the patient's own tumor will be not possible in many cases. It would be preferable to predetermine immunogenic peptides of antigens commonly present on tumors that could be prepared synthetically in quantity for use in immunotherapy.

The invention is based on the discovery of peptides in TADG-12 and CA 125 that can be used to induce an autologous T cell response that lyses ovarian cancer cells expressing TADG-12 or CA125. A computer algorithm was used to select 9-mer and 10-mer peptides from CA125 and TADG-12 that were predicted to bind to the antigen-presenting groove of HLA class I protein A2. HLA A2 is the most common and the most well characterized HLA class I cell surface protein. It is present in approximately 50% of the population. Several 9-mer and 10-mer peptides from both TADG-12 and CA125 predicted to bind to HLA A2 were loaded onto dendritic cells having the HLA A2 antigen, and the peptide-loaded dendritic cells were used to amplify autologous CD8+ T cells ex vivo. Most of the peptides tested amplified CD8+ T cells that recognized and lysed autologous cells pulsed with the peptide. At least one CA125 peptide and at least one TADG-12 peptide consistently produced amplified CD8+ T cells, even from cancer patients, that lysed autologous CA 125-expressing or TADG-12-expressing tumor cells.

Accordingly, one embodiment of the invention provides a method of treating cancer in a patient whose cancer cells express CA125 involving: (a) contacting antigen-presenting cells with a purified peptide comprising an HLA-binding CA125 peptide of 7-12 amino acid residues to generate peptide-loaded antigen-presenting cells; (b) contacting the peptide-loaded antigen-presenting cells with T cells of the cancer patient to amplify CD8+ T cells that recognize the CA125 peptide; and (c) contacting the amplified CD8+ T cells with CA125-bearing cancer cells in the patient to lyse the CA125-bearing cancer cells. The CA125 peptide binds to a human class I HLA protein. When the CA125 peptide is bound to the HLA protein on the surface of antigen-presenting cells to generate peptide-loaded antigen-presenting cells, and the peptide-loaded antigen-presenting cells are contacted with T cells, the peptide-loaded antigen-presenting cells amplify CD8+ T cells that lyse autologous cells expressing CA125 in vivo or in vitro. Preferably the antigen-presenting cells are dendritic cells.

Another embodiment of the invention provides a method of treating cancer in a patient whose cancer cells express TADG-12 involving: (a) contacting antigen-presenting cells with a purified peptide comprising an HLA-binding TADG-12 peptide of 7-12 amino acid residues to generate peptide-loaded antigen-presenting cells; (b) contacting the peptide-loaded antigen-presenting cells with T cells of the cancer patient to amplify CD8+ T cells that recognize the TADG-12 peptide; and (c) contacting the amplified CD8+ T cells with TADG-12-bearing cancer cells in the patient to lyse the TADG-12-bearing cancer cells. The TADG-12 peptide binds to a human class I HLA protein. When the TADG-12 peptide is bound to the HLA protein on the surface of antigen-presenting cells to generate peptide-loaded antigen-presenting cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing TADG-12 in vivo or in vitro. Preferably, the antigen-presenting cells are dendritic cells.

Another embodiment of the invention provides a purified CA125 peptide of 7-50 amino acid residues, wherein the peptide binds to a human class I HLA protein, wherein when the peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing CA125 in vivo or in vitro.

Another embodiment of the invention provides a purified CA125 peptide of 7-50 amino acid residues, wherein the peptide binds to a human class I HLA protein, wherein when the peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse in vitro autologous lymphoblastoid cell line (LCL) cells pulsed with the peptide.

Another embodiment of the invention provides a purified TADG-12 peptide of 7-50 amino acid residues, wherein the peptide binds to a human class I HLA protein, wherein when the peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing TADG-12 in vivo or in vitro.

Another embodiment of the invention provides a purified CA125 peptide of 7-50 amino acid residues, wherein the peptide binds to a human class I HLA protein, wherein when the peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse in vitro autologous lymphoblastoid cell line (LCL) cells pulsed with the peptide.

Another embodiment of the invention provides a pharmaceutical composition comprising: dendritic cells loaded ex vivo with a purified peptide comprising an HLA-binding CA125 peptide of 7 to 12 amino acid residues; wherein the HLA-binding CA125 peptide binds to a human class I HLA protein on the surface of the dendritic cells, and wherein when the CA125 peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing CA125 in vivo or in vitro. Optionally, the dendritic cells could be replaced with other antigen-presenting cells.

Another embodiment of the invention provides a pharmaceutical composition comprising: dendritic cells loaded ex vivo with a purified peptide comprising an HLA-binding TADG12 peptide of 7 to 12 amino acid residues; wherein the TADG12 peptide binds to a human class I HLA protein on the surface of the dendritic cells, and wherein when the TADG 12 peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing TADG12 in vivo or in vitro. Optionally, the dendritic cells could be replaced with other antigen-presenting cells.

Another embodiment of the invention provides a pharmaceutical composition comprising: amplified CD8+ T cells that lyse autologous cells expressing CA125 in vivo or in vitro. The amplified CD8+ T cells are amplified by a process comprising: contacting T cells ex vivo with dendritic cells loaded ex vivo with a CA125 peptide of 7 to 12 amino acid residues; wherein the peptide binds to a human class I HLA protein on the surface of the dendritic cells. The peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing CA125 in vivo or in vitro. The T cells and dendritic cells share the same HLA class I protein. Optionally, the dendritic cells could be replaced with other antigen-presenting cells.

Another embodiment of the invention provides a pharmaceutical composition comprising: amplified CD8+ T cells that lyse autologous cells expressing TADG-12 in vivo or in vitro. The amplified CD8+ T cells are amplified by a process comprising: contacting T cells ex vivo with dendritic cells loaded ex vivo with a TADG-12 peptide of 7 to 12 amino acid residues; wherein the peptide binds to a human class I HLA protein on the surface of the dendritic cells. The peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing TADG-12 in vivo or in vitro. The T cells and dendritic cells share the same HLA class I protein. Optionally, the dendritic cells could be replaced with other antigen-presenting cells.

Another embodiment of the invention provides a method of identifying a CA125 peptide suitable for cancer immunotherapy comprising: (a) contacting one or more candidate peptides comprising an HLA-binding CA125 peptide of 7 to 12 amino acid residues with dendritic cells expressing an HLA class I protein to generate peptide-loaded dendritic cells; (b) contacting the peptide-loaded dendritic cells with HLA class I-matched T cells to generate amplified T cells that recognize the CA125 peptide; and (c) contacting the amplified T cells with target cells expressing CA125 to determine whether the amplified T cells lyse the target cells. Optionally, the dendritic cells could be replaced with other antigen-presenting cells.

Another embodiment of the invention provides a method of identifying a TADG12 peptide suitable for cancer immunotherapy comprising: (a) contacting one or more candidate peptides comprising an HLA-binding TADG12 peptide of 7 to 12 amino acid residues with dendritic cells expressing an HLA class I protein to generate peptide-loaded dendritic cells; (b) contacting the peptide-loaded dendritic cells with HLA class I-matched T cells to generate amplified T cells that recognize the TADG12 peptide; and (c) contacting the amplified T cells with target cells expressing TADG12 to determine whether the amplified T cells lyse the target cells. Optionally, the dendritic cells could be replaced with other antigen-presenting cells.

DETAILED DESCRIPTION

Dendritic cells are effective antigen-presenting cells. They are particularly adept at stimulating naive T cells. Dendritic cell function is reviewed in references 25 and 31. The invention involves stimulating CD8+ T cells, also known as cytotoxic T lymphocytes (CTL) that recognize particular peptides derived from the tumor antigens CA125 and TADG-12. That stimulation is preferably done with dendritic cells. It may be possible to do it with other antigen-presenting cells or by some other method developed in the future.

One embodiment of the invention provides a method of treating cancer in a patient whose cancer cells express CA125 involving: (a) contacting dendritic cells with a purified peptide comprising an HLA-binding CA125 peptide of 7-12 amino acid residues to generate peptide-loaded dendritic cells; (b) contacting the peptide-loaded dendritic cells with T cells of the cancer patient to amplify CD8+ T cells that recognize the CA125 peptide; and (c) contacting the amplified CD8+ T cells with CA125-bearing cancer cells in the patient to lyse the CA125-bearing cancer cells. The CA125 peptide binds to a human class I HLA protein.

The step of contacting dendritic cells with a purified peptide is preferably done ex vivo. It may be possible alternatively to perform the step in vivo in the patient.

The step of contacting dendritic cells with a purified peptide may be done by any suitable method, including mixing the dendritic cells with the purified peptide directly, mixing dendritic cells with purified peptide in liposomes, and expressing the purified peptide from a recombinant nucleic acid in the dendritic cells.

In one embodiment, step (a) is performed ex vivo, step (b) involves infusing the peptide-loaded dendritic cells into the patient to amplify the CD8+ T cell in vivo in the patient, and step (c) occurs in vivo in the patient.

In another embodiment, steps (a) and (b) are performed ex vivo, and step (c) involves infusing the amplified CD8+ T cells into the patient to contact the CA125-bearing cancer cells in vivo in the patient.

The purified peptide contacted with the dendritic cells may be a peptide of any appropriate length, e.g., 7 to 5000 amino acid residues. CA125 has a long N-terminal domain, a multiple repeat domain, and a transmembrane C-terminal domain (2, 32). Suitable peptide antigens may be found in any domain. The peptides tested herein are from the multiple repeat domain, which extends from residue 12070 to residues 21868 of the CA125 sequence in reference 32 (GenBank accession number AAL65133). The multiple repeat domain consists of 156-amino-acid repeat units that are homolgous to each other. The individual repeat units are homologous to each other. In some embodiments, the HLA-binding CA125 peptide of 7-12 amino acid residues is from the multiple repeat domain.

In a preferred embodiment, the purified peptide contacted with dendritic cells is a short peptide that does not need to be proteolytically processed to be presented by the dendritic cells. In particular embodiments, the purified peptide is 7-50, 7-30, 7-20, 7-12, or 8-10 amino acid residues in length. The purified peptide may comprise only CA125 sequence or may comprise other sequences. It may be a naturally-derived fragment of CA 125. Alternatively, it may be, for instance, a long peptide comprising a multiple repeat of a single CA125 8- to 10-mer peptide sequence.

The CA125 peptide used in the invention binds to a human class I HLA protein. There are several variants of HLA class I protein, including HLA A*0201. Other variants include HLA A1, A24, B14, and CwO301. The affinity of particular 8-10 mer peptides for one of these or other HLA class I cell surface proteins can be calculated with an algorithm on the NIH website bimas.dcrt.nih.gov/molbio/hla_bind. The algorithm is described in reference (3). Candidate peptides for screening can be identified by screening the CA125 protein sequence (GenBank accession number AAL65133 and disclosed in reference 32) for 8- to 10-mer peptide sequences with affinity for HLA class I proteins with the BIMAS program.

Whether a peptide binds to an HLA class I protein can be determined experimentally as described in Examples 3 and 4 below.

When the CA125 peptide is bound to the HLA class I protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing CA125 in vivo or in vitro. Dendritic cells can be loaded with the peptides and used to amplify CD8+ T cells as described in Examples 5 and 6 below. The ability of the amplified CD8+ T cells to lyse autologous cells expressing CA125 can be tested as described in Examples 9 and 10 below. Preferably the autologous cells are cancer cells expressing CA125.

The amplified T cells can also be tested for the ability to lyse autologous cells pulsed with the CA 125 peptide of 7-12 amino acid residues as described in Examples 7 and 8 below.

In the assay and in the method of treating cancer, the T cells are ordinarily autologous with the cancer cells or other cells expressing CA125. Preferably, the dendritic cells are also autologous to the T cells and the cells expressing CA125. However allogeneic dendritic cells sharing at least one HLA class I cell surface protein may also be used.

To treat a patient for cancer, dendritic cells may be prepared in vitro and loaded with the appropriate peptide. The peptide-loaded dendritic cells may then be infused into a patient. They will then amplify T cells in the patient that recognize the peptide and recognize and lyse cancer cells expressing CA125.

The dendritic cells may be infused intravenously into the patient as described in Example 12 below. They may also be administered by another route, such as subcutaneously. Preferably dendritic cells are administered multiple times to a patient, e.g., three times with two weeks between treatments.

As an alternative to administering peptide-loaded dendritic cells to the patient, the peptide-loaded dendritic cells can be used to amplify CD8+ T cells from the patient ex vivo, as described in Examples 5 and 6 below. The amplified T cells may be then infused into the patient. As many amplified T cells as can be obtained would typically be infused.

The dendritic cells to amplify T cells ex vivo, or for infusion into the patient, may be allogeneic or autologous. Whether allogeneic or autologous, they will have a short life span in the body, so they are not expected to induce a hazardous autoimmune response. The CD8+ T cells, if prepared ex vivo, are preferably autologous, but may be allogeneic. If they are allogeneic, they may produce a graft-versus-host disease.

In the method of treating cancer, the CA125-bearing cancer cells may be any type of cancer expressing CA125. Ovarian carcinoma is best known for expressing CA125, but other cancer types are also known to often express CA125, including lymphoma, and specifically non-hodgkin's lymphoma.

The preferred HLA-binding CA125 peptide of 7-12 amino acids is YTLDRDSLYV (SEQ ID NO: 10). This peptide is shown below to amplify CD8+ T cells that consistently lyse autologous ovarian tumor cells expressing CA125. Several other CA125 peptides were shown to bind to HLA A*0201, to amplify CD8+ T cells that lyse autologous cells pulsed with the peptide. And some of these appeared to lyse tumor cells expressing CA 125 at least inconsistently, but only SEQ ID NO: 10 was found to amplify T cells that lysed the tumor cells consistently. Given that one CA125 peptide can amplify CD8+ T cells that lyse tumor cells, others could be identified with further screening.

In particular embodiments, the CA125 peptide comprises SEQ ID NO:10. In other embodiments, it comprises at least 7 amino acid residues of SEQ ID NO: 10 in the same order and with the spacing as in SEQ ID NO: 10 (i.e., where 3 of the 10 residues of SEQ ID NO: 10 are replaced with other residues or are absent on the ends of the peptide). In other embodiments, it comprises at least 8, or at least 9 amino acid residues of SEQ ID NO: 10 in the same order and with the spacing as in SEQ ID NO: 10. In particular embodiments, the peptide comprises at least 7, 8, or 9 contiguous residues of SEQ ID NO: 10.

In the detailed description above, the term "CA125" can be replaced with "TADG-12" to describe the analogous method of treating cancer in a patient whose cancer cells express TADG-12.

The affinity of particular 8-10-mer peptides of TADG-12 and TADG-12V for HLA class I cell surface proteins can be calculated with the BIMAS algorithm on the NIH website bimas.dcrt.nih.gov/molbio/hla_bind. The algorithm is described in reference (3). Candidate peptides for screening can be identified by screening the TADG-12 and TADG-12V protein sequences (SEQ ID NO:21 and 22, Tables 1 and 2 (see Original Patent)) for 8-10-mer peptide sequences with affinity for HLA class I proteins using the BIMAS algorithm on the NIH website bimas.dcrt.nih.gov/molbio/hla_bind. TADG-12 and TADG-12V are identical through the first 256 amino acid residues.

For use in the method of treating cancer in a patient whose cancer cells express TADG-12, the preferred TADG-12 peptide is YLPKSWTIQV (SEQ ID NO: 17). This peptide is shown below to amplify CD8+ T cells that consistently lyse autologous ovarian tumor cells expressing TADG-12. Several other TADG-12 peptides were shown to bind to HLA A*0201, to amplify CD8+ T cells that lyse autologous cells pulsed with the peptide. And some of these appeared to lyse tumor cells expressing TADG-12 at least inconsistently, but only SEQ ID NO: 17 was found to amplify T cells that lysed the tumor cells consistently. Given that one TADG-12 peptide can amplify CD8+ T cells that lyse tumor cells, others most likely could be identified with further screening.

In particular embodiments, the TADG-12 peptide comprises SEQ ID NO:17. In other embodiments, it comprises at least 7 amino acid residues of SEQ ID NO: 17 in the same order and with the spacing as in SEQ ID NO: 17 (i.e., where 3 of the 10 residues of SEQ ID NO: 17 are replaced with other residues or are absent on the ends of the peptide). In other embodiments, it comprises at least 8, or at least 9 amino acid residues of SEQ ID NO: 17 in the same order and with the spacing as in SEQ ID NO: 17. In particular embodiments, the peptide comprises at least 7, 8, or 9 contiguous residues of SEQ ID NO:17.

Claim 1 of 17 Claims

1. A method of treating cancer in a patient whose cancer cells express CA125 comprising: (a) contacting dendritic cells with a purified peptide comprising an HLA-binding CA125 peptide of 7-12 amino acid residues to generate peptide-loaded dendritic cells; (b) contacting the peptide-loaded dendritic cells with T cells of the cancer patient to amplify CD8+ T cells that recognize the CA125 peptide; and (c) contacting the amplified CD8+ T cells with CA125-bearing cancer cells in the patient to lyse the CA125-bearing cancer cells; wherein the CA125 peptide binds to a human class I HLA protein, wherein when the CA125 peptide is bound to the HLA protein on the surface of dendritic cells to generate peptide-loaded dendritic cells, and the peptide-loaded dendritic cells are contacted with T cells, the peptide-loaded dendritic cells amplify CD8+ T cells that lyse autologous cells expressing CA125 in vivo or in vitro; wherein the purified peptide comprises SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; wherein the purified peptide is 7 to 50 amino acid residues in length.

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