Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/80—Vaccine for a specifically defined cancer
  • A61K2039/852—Pancreas

Definitions

• the present invention relates to a novel combination therapy for pancreatic cancer that utilizes an antigenic peptide and chemotherapeutic agent.
• Pancreatic cancer has one of the highest mortality rates of any malignancy, and the 5-year-survival rate of patients is 4%. Approximately 28,000 patients are diagnosed with pancreatic cancer each year, and nearly all patients will die of their disease (Greenlee, R. T., et al., (2001) CA Cancer J Clin, 51: 15-36). The poor prognosis of this malignancy results from the difficulty of early diagnosis and the poor response to current therapeutic methods (Greenlee, R. T., et al. (2001) CA Cancer J Clin, 51: 15-36, Klinkenbijl, J. H., et al. (1999) Ann Surg, 230: 776-82; discussion 782-4.). In particular, there are currently no identified tumor markers that allow for reliable screening at an early, potentially curative stage of the disease.
• FTI farnesyltransferase inhibitor
• Tyrosine kinase inhibitor STI-571 is an inhibitor which selectively deactivates the bcr-abl fusion protein. This pharmaceutical agent was subsequently developed for the therapy of chronic myeloid leukemia, wherein the constant activation of bcr-abl tyrosine kinase plays a significant role in the transformation of white blood cells.
• Such pharmaceutical agents are designed to inhibit the carcinogenic activity of specific gene products (Molina MA, et al., (2000) Cancer Res, 16:4744-9).
• gene products with promoted expression generally serve as potential targets for the development of novel anti-tumor agents.
• nucleic acid synthesis inhibitors may also be used as anti-tumor agents.
• gemcitabine (Gemzar R ) is a first- line treatment of pancreatic cancer. The combination therapy of gemcitabine and paclitaxel has also been applied to treatment of pancreatic cancer.
• tumor angiogenesis is critically involved in the progression of tumors. It has previously been demonstrated that an effective vaccine against tumor angiogenesis could be developed according to an endothelial cell-based approach, targeting vascular endothelial growth factor receptors (VEGFRs) 1 and 2, as HLA class I molecules are not down-regulated on endothelial cells (Wada S et al., Cancer Res 2005 Jun 1, 65(11): 4939-46; Ishizaki H et al., Clin Cancer Res 2006 Oct 1, 12(19): 5841-9).
• VAGFRs vascular endothelial growth factor receptors
• CTLs cytotoxic T lymphocytes
• the present invention addresses the need in the art for an improved pancreatic cancer therapy by providing a novel combination therapy for pancreatic cancer that utilizes an antigenic peptide, particularly antigenic peptides and cancer vaccines that target VEGFR2, and a chemotherapeutic agent such as gemcitabine.
• VEGFR2 is strongly expressed in tumoral tissue endothelial cells and is thought be involved in the proliferation of endothelial cells on the VEGF signal. Accordingly, the present invention focused on possible cancer vaccine therapies that target VEGFR2 (KDR/flk-1; referred to below as KDR). It was subsequently discovered that the therapeutic effect of chemotherapeutic agents such as gemcitabine is potentiated by VEGFR2 (KDR/flk-1; referred to below as KDR) peptides that induce cytotoxic T- cells against cells expressing VEGFR2. Thus, it is an object of the present invention to provide:
• a method of treating cancer in a subject that includes the step of administering to the subject (i) and (ii);
• AMFFWLLLV SEQ ID NO: 7
• VIAMFFWLL SEQ ID NO: 8
• AVIAMFFWL SEQ ID NO: 9
• KLIEIGVQT SEQ ID NO: 10
• YMISYAGMV SEQ ID NO: 11
• IQSD VWSFGV SEQ ID NO: 12
• VLAMFFWLL SEQ ID NO: 13
• kits for treating cancer in a subject containing pharmaceutical compositions that include (i) and (ii) as active ingredient respectively and pharmaceutical acceptable carrier;
• kits of [4] wherein the subject is HLA-A24-positive or HLA-A02-positive.
• the kit of [4] wherein the cancer is pancreatic cancer.
• An anti-cancer agent for treating cancer in a subject that includes (i) in combination with (ii);
• chemotherapeutic agents selected from the group consisting of gem- citabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof.
• [l]-(i) for manufacturing a pharmaceutical composition that enhances the therapeutic effect of gemcitabine.
• Figure 1 presents the administration protocol for the antigenic peptide and chemotherapeutic agent utilized in the present examples.
• Figure 2 presents the results of flow cytometry analysis of naive, memory, and effector T cells among CD8-positive T cells, wherein the functional lymphocyte fractions were determined by perforin staining.
• Figure 3 presents the numerical change of regulatory T cells (e.g., CD25high and Foxp3-positive cells among CD4-positive T cells) before and after vaccine admin- istration as measured by flow cytometry after four color staining.
• Figure 4 presents the results of a PET scan on Case 3, particularly a lymph- adenopathy near the inoculation site after vaccination.
• Figure 6 presents the levels of specific CTL reaction arising in Case 3 before and after vaccination.
• Figure 7 presents a series of CT scans on Case 4, depicting the reducing effect of the treatment on pancreatic cancer primary focus.
• Figure 8 presents a series of CT scans on Case 4, depicting the tumor reducing effect on pancreatic cancer liver metastatic focus 1.
• Figure 9 presents a series of CT scans on Case 4, depicting the tumor reducing effect on pancreatic cancer liver metastatic focus 2.
• Figure 10 presents the changes in the tumor markers, CEA and CA 19-9, arising in Case 4 over the course of treatment.
• Figure 11 presents the specific CTL reaction arising in Case 4 before and after vaccination.
• Figure 12 presents a series of CT scans on Case 6, depicting the tumor reducing effect on pancreatic cancer primary focus.
• Figure 14 presents the specific CTL reaction arising in Case 6 before and after vaccination.
• Figure 16 presents the changes in the tumor marker CA 125 arising in Case 7 over the course of treatment.
• Figure 17 presents the specific CTL reaction arising in Case 7 before and after vaccination.
• FIG.19 Figure 19 presents the changes in the tumor marker CA 125 arising in Case 10 over the course of treatment. [0011] Detailed Description of the Invention
• the term "several" as applies to amino acid additions, deletions, and/or substitutions means 3-7, preferably 3-5, more preferably 3-4, even more preferably 3 amino acid residues.
• organism refers to any living entity composed of at least one cell.
• a living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal, including a human being.
• biological sample refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
• body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen.
• biological sample further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof.
• biological sample refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or polynucleotides.
• polypeptide polypeptide
• peptide protein
• protein protein
• amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• nucleic acid molecules are used interchangeably herein to refer to a polymer of nucleic acid residues and, unless otherwise specifically indicated, are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.
• chemotherapeutic agent refers to a chemical compound useful in the treatment of cancer.
• chemotherapeutic agents include, but are not limited to, the following and their pharmaceutically acceptable salts, acids and derivatives: alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, trie thy lenethiophos- phaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlor- naphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, me- chlorethamine oxide hydrochloride, melphalan, novembiehin, phenesterine, predn- imustine, tro
• paclitaxel (TAXOLO, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTEW, Rh ⁇ ne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum ; etoposide (VP- 16); ifosfamide; mitomycin C; mi- toxantrone; vincristine; vinorelbine ; navelbine ; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-I l; topoisomerase inhibitor RFS 2000; di- fluoromethylornithine (DMFO); retinoic acid; esperamicins; and capecitabine; Also included in this definition are
• the present invention relates to agents that enhance or improve the therapeutic effect of chemotherapy, more particularly antigenic peptides that target VEGFR2, induce cytotoxic T-cells against cells expressing VEGFR2, and subsequently enhance or improve the therapeutic effect of chemotherapeutic agents such as gemcitabine.
• Antigenic peptides having subsequences of VEGFR2 may be used for the method, kit, or composition of the present invention.
• Antigenic peptides suitable for use in the context of the present invention preferably have an amino acid sequence selected from those shown bellow.
• VYSSEEAEL (SEQ ID NO: 2)
• Mutated or modified peptides, peptides having amino acid sequences modified by deleting, adding and/or replacing one or more amino acid residues of a certain amino acid sequence have been known to retain the original biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. and Smith, M., Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
• the present invention contemplates variations and modifications to the above sequences.
• antigenic peptides in which one, two, or several amino acids are substituted with or added to one of the amino acid sequences mentioned above also find utility in the context of the present invention, provided the resulting modified peptides retain the requisite cytotoxic T cell inducibility.
• modified peptides, having CTL inducibility as well as an amino acid sequence as mentioned above, in which one, two, or several amino acids are substituted or added, are contemplated herein provided they do not match the amino acid sequence of another protein.
• the second amino acid from the N terminus is preferably substituted to phenylalanine, tyrosine, methionine, or tryptophan
• the C-terminal amino acid is preferably substituted to phenylalanine, leucine, isoleucine, tryptophan, or methionine; or one or two amino acids are added to the N terminus and/or C terminus.
• nonapeptides and decapeptides selected from peptides having the amino acid sequence shown below are also preferred as peptide having high CTL indu- cibility.
• AMFFWLLLV (SEQ ID NO: 7),
• VLAMFFWLL (SEQ ID NO: 13).
• peptides with cytotoxic T cell inducibility wherein one, two, or several amino acids are substituted or added to one of the amino acid sequence as mentioned above may also be used.
• the second amino acid from the N terminus is preferably substituted to leucine or methionine, or the C-terminal amino acid is preferably substituted to valine or leucine; or one or two amino acids are added to the N terminus and/or C terminus.
• modified peptide is the peptide of VIAMFFWLL (SEQ ID NO: 8), in which the second amino acid from the N terminus is substituted to leucine (VLAMFFWLL (SEQ ID NO: 13)); however, the present invention is not limited to this example.
• CTL clones obtained from stimulation with these modified peptides can recognize the original peptides, and cause damage.
• contemplated amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to several residues, as well as in- trasequence insertions of single or multiple amino acid residues.
• terminal insertions include an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
• the peptides used for the present invention may also contain modifications, such as glycosylation, side chain oxidation, or phosphorylation, so long as the modifications do not destroy the biological activity of the peptides as described herein.
• Other modifications include fusions of an enzyme or a polypeptide which increases the serum half- life of the antibody to the N-or C-terminus of the peptide. Examples of the latter include D-amino acids or other amino acid mimetics.
• Fusion proteins are generally composed of a polypeptide or protein of interest with a polypeptide or protein of known utility. Fusion proteins can be made by techniques well known to a person skilled in the art, such as by linking a DNA encoding a peptide of the present invention with DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the protein of the present invention.
• examples of known peptides that can be used in the context of fusion proteins include, but are not limited to, FLAG (Hopp, T. P. et al., Biotechnology (1988) 6, 1204-1210), 6xHis containing six His (histidine) residues, lOxHis, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, pi 8HIV fragment, T7-tag, HSV-tag, E- tag, SV40T antigen fragment, lck tag, alpha- tubulin fragment, B -tag, Protein C fragment, and the like.
• FLAG Hopp, T. P. et al., Biotechnology (1988) 6, 1204-1210
• 6xHis containing six His (histidine) residues include, but are not limited to, 6xHis containing six His (histidine) residues, lOxHis, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment
• amino acid residue to be substituted is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution).
• amino acid side chains examples include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side- chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side- chain (H, F, Y, W).
• A, I, L, M, F, P, W, Y, V hydrophilic amino acids
• R, D, N, C, E, Q amino acids
• G, A, V, L, I, P a hydroxyl group containing side-chain
• Antigenic peptides of the present invention can be prepared using well known techniques.
• the peptides can be prepared synthetically, using either recombinant DNA technology or chemical synthesis.
• Peptides may be synthesized individually or as longer polypeptides composed of two or more peptides. These peptides are preferably isolated, i.e., substantially free of other naturally occurring host cell proteins and fragments thereof.
• the antigenic peptides of the present invention may be provided in a cocktail or may be conjugated to each other using standard techniques.
• the peptides can be expressed as a single polypeptide sequence.
• the peptides in the combination may be the same or different.
• the peptides are presented at a high density on the HLA antigens of antigen-presenting cells, which, in turn, induces CTLs that specifically react toward the complex formed between the displayed peptide and the HLA antigen.
• antigen-presenting cells having immobilized the peptides of this invention on their cell surface, obtained by removing dendritic cells from the subjects may be stimulated by the peptides of this invention. Re-administration of these cells to the respective subjects induces CTL, and, as a result, aggressiveness towards the target cells can be increased.
• the present invention provides drugs for treating and/or preventing pancreatic cancer used for in combination with a chemotherapeutic agent such as gemcitabine.
• a chemotherapeutic agent such as gemcitabine.
• the peptides used for the invention find particular utility in the treatment of pancreatic cancer.
• the antigenic peptides of the present invention can be easily performed by exposing the cells to a high concentration of the peptides, which causes these peptides to replace the peptides originally immobilized on the cells. Therefore, to be useful in the context of the present invention, the antigenic peptides must have at least a certain level of binding affinity to HLA antigens.
• the pharmaceuticals containing such peptides may be directly administered as the peptides themselves, or may be administered as pharmaceutical compositions that have been formulated by conventional formulation methods.
• the pharmaceuticals can appropriately include, in addition to the peptides, carriers, excipients, and such that are ordinarily used for pharmaceuticals, without particular limitations.
• the pharmaceuticals can be used for treatment and prevention of pancreatic cancer in combination with gemcitabine.
• compositions for treating and/or preventing pancreatic cancer including an antigenic peptide of the present invention as an active ingredient can be administered with adjuvants that effectively induce cellular immunity; can be administered with other active ingredients such as antitumor agents; and can be administered in granular forms. Suitable adjuvants are described in the literature (Clin. Microbiol. Rev., 7:277-289, 1994). Furthermore, the pharmaceuticals of this invention can be administered as liposome formulations, as granular formulations bound to beads of a few micrometers in diameter, and as formulations to which lipids are bound.
• Administration methods may be carried out, for example, orally, intradermally, or subcutaneously, or through intravenous injection, or such.
• Systemic administration or local administration to the vicinity of the target tumor or directly into the target tumor may be applicable.
• Doses of the peptides of this invention can be adjusted appropriately, depending on the disease to be treated, age and weight of the patients, administration methods, and such. Ordinarily, 0.001 mg to 1,000 mg, preferably 0.001 mg to 1,000 mg, more preferably 0.1 mg to 10 mg, of the peptides are preferably administered once in a few days to a few months.
• 0.5 mg to 2.0 mg of the peptides may be administered once in a few days to a few months, more preferably in a week (7 days), in combination with gemcitabine.
• One skilled in the art can appropriately select suitable doses.
• intracellular vesicles which present complexes formed between the peptides of this invention and HLA antigens on their surface may be used for the purpose of the present invention. These intracellular vesicles are called exosomes.
• Exosomes can be prepared, for example, according to the methods specifically described in Published Japanese Translation of International Publication Nos. Hei 11-510507 and 2000-512161. Exosomes can preferably be prepared using antigen-presenting cells obtained from subjects who are to be the target of therapy or prophylaxis.
• the exosomes of this invention can be inoculated as cancer vaccines, as for the peptides of this invention.
• HLA- A24 or HLA-A02 particularly HLA-A2402 or HLA-0201, is often appropriate.
• isolated cytotoxic T cells that are induced by the peptides may also be used for the purpose of the present invention.
• the cytotoxic T cells which have been induced by stimulation with antigen-presenting cells that present the peptides of this invention, are preferably derived from subjects to be the target of therapy and/or prophylaxis.
• the cytotoxic T cells can be administered alone or, for the purpose of antitumor effect, in combination with other drugs, including the peptides, exosomes and so on of this invention.
• the obtained cytotoxic T cells act specifically against target cells presenting the peptides of this invention, or preferably, against target cells presenting the same peptides used for induction.
• the target cells may be cells that endogenously express KDR, or cells forced to express KDR.
• cells that present the peptides of this invention on their cell surface due to stimulation by these peptides can also be targeted.
• antigen-presenting cells that present complexes formed between HLA antigens and the peptides may also be used for the purpose of the present invention.
• the antigen-presenting cells that are obtained by contact with the peptides, or with nucleotides encoding the peptides, are preferably derived from subjects to be targeted for therapy and/or prophylaxis.
• the antigen-presenting cells can be administered as vaccines alone, or in combination with other drugs such as the peptides of this invention, exosomes, and cytotoxic T cells.
• the peptides are preferably administered in combination with gemcitabine.
• Gemcitabine is a common name given to the compound 2'-deoxy-2',2'-difluorocytidine monohydrochloride (b-isomer).
• a pharmaceutical composition composed of the hydrochloride salt of gemcitabine (gemcitabine HCl) is commonly commercially available as Gemzar (trade name).
• gemzar trade name
• at least one of gemcitabine, a pharmaceutically acceptable salt of gemcitabine, or a prodrug thereof may be administered in combination with the aforementioned peptides. Accordingly, unless otherwise stated, references herein to gemcitabine include its salt or prodrug.
• Gemcitabine is a chemotherapeutic agent that is already in clinical use as a therapeutic agent for several cancers including pancreatic cancer.
• the standard therapeutic protocol for administering gemcitabine to an adult for the treatment of pancreatic cancer involves the administration of 1000 mg/m 2 of gemcitabine per week for up to seven weeks.
• Gemcitabine is typically administered by intravenous infusion.
• pancreatic cancer therapy generally, a schedule of three weeks of administration followed by one week of no treatment is set as one cycle, and the treatment is continued and repeated as needed. During this period, the dose of gemcitabine can be adjusted using hematologic toxicity or such as an indicator.
• the peptides are administered in accordance with such an administration schedule for gemcitabine.
• the antigenic peptides of the present invention can be administered at any stage during the gemcitabine administration period. Alternatively, so long as the CTLs that are induced by the antigenic peptides of the present invention maintain their activity in vivo, such peptides can be administered prior to gemcitabine administration. Generally, it is reasonable to administer them with the same schedule as for gemcitabine administration, thereby keeping the time commitment of the patient to a minimum.
• VEGFR2-derived peptides having amino acid sequences such as RFVPDGNRI can improve the therapeutic effects of gemcitabine.
• amino acid sequences of exemplary VEGFR2-derived peptides that can be used in the context of the present invention are described again below.
• modified or mutated versions of these amino acid sequences can also be used in the present invention, so long as they retain the desired CTL-inducing ability.
• VYSSEEAEL SEQ ID NO: 2
• GYRIYDVVL SEQ ID NO: 3
• SYMISYAGM SEQ ID NO: 4
• RFVPDGNRI SEQ ID NO: 1
• KWEFPRDRL SEQ ID NO: 5
• DFLTLEHLI SEQ ID NO: 6
• AMFFWLLLV SEQ ID NO: 7
• VIAMFFWLL SEQ ID NO: 8
• AVIAMFFWL SEQ ID NO: 9
• KLIEIGVQT SEQ ID NO: 10
• YMISYAGMV SEQ ID NO: 11
• IQSDVWSFGV SEQ ID NO: 12
• VLAMFFWLL SEQ ID NO: 13
• the present invention provides agents that enhance the pancreatic cancer therapeutic effect of gemcitabine, such agents including the above-described VEGFR2-derived peptides as an active ingredient.
• the present invention provides uses of the VEGFR2-derived peptides in the production of pharmaceutical compositions that enhance the therapeutic effects of gemcitabine on pancreatic cancer.
• the present invention provides combined uses (combination) of the VEGFR2-derived peptides during pancreatic cancer therapy using gemcitabine.
• the VEGFR2-derived peptides can be used in combination with gemcitabine for pancreatic cancer therapy. More specifically, the present invention provides a kit for treating pancreatic cancer which is composed of a pharmaceutical composition that contains as active ingredients each of a pharmaceutically acceptable carrier, the aforementioned VEGFR2-derived peptides, and gemcitabine. Furthermore, the present invention provides anticancer agents for treating pancreatic cancer, which include a combination of the aforementioned VEGFR2-derived peptides and gemcitabine.
• the present invention provides a kit for treating pancreatic cancer which includes the aforementioned VEGFR2-derived peptides and an instruction sheet stating that the therapeutic effect of gemcitabine is enhanced when the peptides are administered in combination with gemcitabine to pancreatic cancer patients.
• the present invention also provides a use of a combination of the VEGFR2-derived peptides and one or more chemotherapeutic agents selected from the group consisting of gemcitabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof, in manufacturing a pharmaceutical composition for treating a cancer including pancreatic cancer.
• the present invention provides a use of the VEGFR2-derived peptides in manufacturing a pharmaceutical composition for treating a cancer including pancreatic cancer, wherein the pharmaceutical composition is used in combination with one or more chemo- therapeutic agents selected from the group consisting of gemcitabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof.
• the present invention further provides a use of the VEGFR2-derived peptides in manufacturing a pharmaceutical composition for enhancing a therapeutic effect of one or more chemotherapeutic agents selected from the group consisting of gemcitabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof, for treating a cancer including pancreatic cancer.
• the present invention further provides a method or process for manufacturing a pharmaceutical composition for enhancing a therapeutic effect of one or more chemotherapeutic agents selected from the group consisting of gemcitabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof, for treating a cancer including pancreatic cancer, wherein the method or process comprises the step of formulating a pharmaceutically or physiologically acceptable carrier with the VEGFR2-derived peptides as active ingredients.
• the present invention further provides a method or process for manufacturing a pharmaceutical composition for enhancing a therapeutic effect of one or more chemotherapeutic agents selected from the group consisting of gemcitabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof, for treating a cancer including pancreatic cancer, wherein the method or process comprises the step of admixing the VEGFR2-derived peptides with a pharmaceutically or physiologically acceptable carrier.
• the present invention also provides a method or process for manufacturing a kit for treating a cancer including pancreatic cancer, wherein the method or process comprises the step of combining or packaging a pharmaceutical composition comprising the VEGFR2-derived peptides and a pharmaceutically or physiologically acceptable carrier, together with one or more chemotherapeutic agents selected from the group consisting of gemcitabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof.
• the present invention also provides a use of a combination of the VEGFR2-derived peptides and one or more chemotherapeutic agents selected from the group consisting of gemcitabine, a pharmaceutically acceptable salt thereof, and a prodrug thereof, in manufacturing a kit for treating a cancer including pancreatic cancer.
• a subject in which a cancer is to be treated, may be HLA-A24-positive or HLA-A02-positive.
• the cancer to be treated includes pancreatic cancer.
• VEGFR2/KDR Vascular Endothelial Growth Factor Receptor 2
• IFA Incomplete Freund's Adjuvant
• the present clinical trial is intended to verify the safety of the novel vaccine chemotherapy aimed at inhibiting new tumor blood vessels and yielding anti-tumor effects through chemotherapy by performing a dose escalation of the administered epitope peptides in a cohort of three patients.
• the secondary purpose is to evaluate the response rate, survival time, and immune responses.
• Surgical resection is the required therapy for curing pancreatic cancer; however, early detection is difficult and at the time of diagnosis, approximately 60% of the patients are in a situation where resection is not possible [Matsuno S et al. Int J Clin Oncol. 5:153-157, 2000., Pantalone D et al. 18:41-46, 2001.].
• gemcitabine is used as the standard therapy against unresectable pancreatic cancer; however, although the median survival period and the one-year survival rate have improved compared to groups administered with 5-FU alone, they are 5.7 months and 18% respectively and are by no means satisfactory. Furthermore, the response rate is 5.4% to 14.3% which is not high [Burris HA et al.
• tumor antigens recognized and attacked by cytotoxic T cells were found, and thereafter, tumor- specific antigens were identified one after another and clinical trials for cancer vaccine therapy using epitope peptides, which is a specific immunotherapy targeting these antigens, are in progress.
• CTLs cytotoxic T cells
• new problems have also been revealed. Even if potent CTLs can be induced, decreased or lost expression of MHC molecules in tumor cells, lack of target molecules in the tumor cells, and such may cause the loss of the CTL antitumor effects.
• the tumor antigen peptides that are identified to date are present in certain types of tumors, but they do not encompass all tumors.
• the target cells of CTLs in vaccine therapy were set to tumor neovascular endothelial cells instead of the tumor cells themselves, and vaccine therapy targeting new tumor blood vessel-derived molecules was formulated.
• As the target molecule attention was focused on Vascular Endothelial Growth Factor Receptor 2 (VEGFR2/KDR) which is hardly expressed in normal endothelial cells, but is highly expressed in tumor neovascular endothelial cells, and is a vascular endothelial cell growth factor receptor indispensable for the growth of these cells.
• VEGFR2/KDR Vascular Endothelial Growth Factor Receptor 2
• VEGFR2 is known to be expressed in the tumor tissues of many solid tumors such as breast cancer, colon cancer, kidney cancer, malignant melanoma, and lung cancer [Folkman J. Nature Biotechnol. 15, 510, 1997., Folkman J. EXS 79, 1-8, 1997.]. VEGFR2 expression has also been revealed to be strongly related to cancer cell proliferation [Kranz A, et al. Int J Cancer 84: 293-298, 1999., Nakopoulou L, et al. Hum Pathol 33:863-870, 2002., Reden L, et al. Breast Cancer Res. and Treat. 82:147-154, 2003. ].
• VEGFR2 may become a target for immunotherapy
• results of basic research on vaccination with VEGFR2 protein and DNA showed that antitumor effects were recognized through suppression of new tumor blood vessels regardless of the type of tumor; thus, it was confirmed that VEGFR2- specific cytotoxic T cells are responsible for this antitumor effect.
• the above showed that VEGFR2 may become a target for tumor immunotherapy [Yiwen, Li. et al. J. Exp. Med. 195, 1575-1584, 2002. Niethammer, A.G. et al. Nature Med. 8, 1369-1375, 2002.].
• CTL precursor cells were found to exist in cancer patients as well [Wada S, Cancer Res. 65, 4939-4946, 2005.]. According to the above, by administering the present peptides and by inducing VEGFR2- specific CTLs in patients, tumor neovascularization can be inhibited, and potent antitumor effects may be obtained.
• a new vaccine chemotherapy was devised, in which a peptide vaccine expected to show antitumor effects through inhibition of tumor neovascularization is used in combination with gemcitabine which is currently standard chemotherapy against pancreatic cancer.
• RFVPDGNRI SEQ ID NO: 1
• VYSSEEAEL SEQ ID NO: 2
• GYRIYDVVL SEQ ID NO: 3
• SYMISYAGM SEQ ID NO: 4
• KWEFPRDRL SEQ ID NO: 5
• DFLTLEHLI SEQ ID NO: 6
• AMFFWLLLV SEQ ID NO: 7
• VIAMFFWLL SEQ ID NO: 8
• AVIAMFFWL SEQ ID NO: 9
• KLIEIGVQT SEQ ID NO: 10
• YMISYAGMV SEQ ID NO: 11
• IQSD VWSFGV SEQ ID NO: 12
• VLAMFFWLL SEQ ID NO: 13
• VEGFR2 is an important molecule involved in the growth of tumor neovascular endothelial cells, and this peptide can be used to induce specific CTLs in vitro [Wada S, Cancer Res. 65, 4939-4946, 2005., WO2004/024766 ].
• Specific CTLs can be induced in vitro also from cancer patient peripheral blood mononuclear cells using VEGFR2-derived HLA- A24 or HLA- A02 restricted epitope peptides [Wada S, Cancer Res. 65, 4939-4946, 2005.].
• Gemcitabine is known to enhance immunocompetence such as CTL inducibility [Correale P, et al. J Immunol. 175, 820-828, 2005., Dauer M, et al. J Im- munother.28, 332-342, 2005.], and effects from combined use with vaccine formulations can be expected.
• Subject patients were selected according to the following selection criteria and exclusion criteria.
• pancreatic cancer for which radical resection was judged to be impossible due to: distant metastasis such as liver metastasis, peritoneal metastasis, and bone metastasis from various diagnoses such as image diagnoses by CT or ultrasound examination; distant lymph node metastasis as defined by the Japan Pancreas Society- edited Classification of Pancreatic Carcinoma, 5th Ed; or infiltration into the great vessels (abdominal aorta, proper hepatic artery, left and right hepatic arteries, superior mesenteric artery, and superior mesenteric veins that cannot be reconstructed); or recurrent pancreatic cancer.
• distant metastasis such as liver metastasis, peritoneal metastasis, and bone metastasis from various diagnoses such as image diagnoses by CT or ultrasound examination
• distant lymph node metastasis as defined by the Japan Pancreas Society- edited Classification of Pancreatic Carcinoma, 5th Ed
• infiltration into the great vessels
• the expected life prognosis must be three months or more at the start of the gem- citabine and vaccine therapy.
• the patient should have recovered from the effects of the operation. Alternatively, four weeks or more must have elapsed since the previous therapy.
• bone marrow function white blood cell count of 2000/mm 3 or more and 15000/mm 3 or less, and platelet count of 7.5/mm 3 or more
• hepatic function GPT of 150 IU/L or less, GPT of 150 IU/L or less, and T-bil 3.0 g/dL or less
• renal function Cr of 3.0 or less
• Treatment Plan 9. Patients determined by a physician or a principle physician to be inappropriate.
• Subject patients were those carrying suitable HLA and having primary pancreatic cancer for which radical resection was judged to be impossible or having recurrent pancreatic cancer. Cases where pancreatic cancer was most highly suspected by image diagnosis were also included in the subject patients. [0065] Method for examining HLA Expression:
• the dosage and administration of gemcitabine (1,000 mg/m 2 , three weeks of administration and one week of no administration) were fixed, and vaccine administration was dose escalated in terms of peptide dose to 0.5 mg, 1 mg, and 2 mg.
• 0.5 mg of peptide is administered to three patients. If not a single individual shows undeniably correlated hematologic toxicity of Grade 4 (NCI-CTC version 3.0) or higher (excluding nausea/vomiting) or hematologic toxicity of Grade 3 (NCI-CTC version 3.0) or higher, the peptide at the next dose (1 mg) is administered to the three patients. If side effects are expressed in two or more individuals, this clinical trial is discontinued.
• cGMP grade peptides (Neo-MPS, San Diego) were a gift from Human Genome Center, Institute of Medical Science, University of Tokyo.
• GMP grade-compliant incomplete Freund's adjuvant (MONTANIDE*ISA51VG) was purchased from SEPPIC Co., France. Storage of the peptides and preparation of the peptide vaccines were carried out at the Pharmaceutical Department of Wakayama Medical University Hospital.
• 10 mg of the peptide is subcutaneously administered as a preliminary test administration before the first vaccine administration to a site other than the site of the actual administration, and monitoring is carried out for 30 minutes. If a Grade 3 or higher local reaction or systemic adverse event could not be observed, the actual administration was carried out. With regard to the "preliminary test administration", local adverse event and systemic adverse event were not observed in any of the cases.
• peripheral blood mononuclear cells PBMC
• the CTL reaction resulting from the administered peptides was measured using IFN- gamma ELISPOT assay (ELISPOT Human IFN-gamma set, BD). More specifically, Stimulator was prepared by pulsing VEGFR2-169 (SEQ ID NO: 1) and HIV- A24 peptides into A24-LCL (HLA- A*2402-positive), and HIV peptide was used as the negative control. The assay was performed three wells at a time for each of R/S ratio and Stimulator and the average value was calculated in terms of the number of spots per single well. The number of spots was read on an ELISPOT reader (IMMUNO SPOT, Cellular Technology Ltd.).
• the value obtained by subtracting the number of spots from HIV pulsing from the number of spots from VEGFR2-169 pulsing was taken to be the specific IFN-gamma production spots with respect to VEGFR2-169 (Specific Spots). If an increase in the specific IFN-gamma production spots was observed after one course and after two courses, it was considered that there has been an immune response due to vaccine administration.
• the CD8-positive fraction was further developed using CD27(BD) and CD45RA(SD) to obtain the effector fraction (CD27-negative / CD45RA-positive), the effector memory fraction (CD27 -negative / CD45RA-negative), the memory fraction (CD27 -positive / CD45RA-negative), and the naive fraction (CD27 -positive / CD45RA-positive).
• the functional lymphocyte fractions were determined by perforin staining (Cytofix/Cytoperm kit, BD).
• CD25high and Foxp3-positive cells among CD4-positive T cells were specified as regulatory T cells and their numerical change before and after vaccine administration was measured by flow cytometry (FACS Calibur, BD) after four color staining. More specifically, as in Fig. 3, lymphocyte fraction of PBMC was gated, and after development using CD4 and CD25, the proportion of CD25high and Foxp3-positive cells (Human Regulatory Staining kit, eBioscience) among CD4-positive T cells was calculated.
• Case 1 is a sixty-eight-year-old female who was previously treated with chemotherapy using TS-I, became unresponsive, and registered thereafter in the instant trial. Two courses were carried out but systemic adverse events were absent, and local adverse events at the site of vaccination were also absent (Table 1, see below). Image assessment was PD. Tumor markers also increased. According to immunologic monitoring, specific CTL reaction against the administered peptide was not observed (Table 2, see below). Before vaccination, it was within the normal range of regulatory T cells (normal average is 3.9 +/- 1.2%), but an increase beyond the normal range was observed after one course and also after two courses (Table 6, see below). The patient died due to aggravation of the original disease 3.3 months after the initial vaccination.
• Case 2 is a sixty-six-year-old male who received two vaccinations but was transferred to another hospital at his own request, and subsequently dropped out of the trial (Table 1, see below). Systemic adverse events were absent, and local adverse events at the site of vaccination were also absent.
• Case 3 is a sixty-four- year-old male.
• Grade 3 neutropenia and hepatic dysfunction were observed as systemic adverse events (Table 1, see below).
• Withdrawal of gemcitabine for one week allowed recovery from both conditions, and administration was then continued.
• Grade 2 induration and redness at the site of inoculation were observed and lymphadenopathy (inguinal swelling) near the inoculation site was observed as local adverse events.
• Image showing strong accumulation was observed at the swollen site using PET (Fig. 4), and histopathologically strong inflammation was observed as a result of biopsy, suggesting at an immune response in response to peptide inoculation.
• Image assessment was SD and the tumor marker (CA19-9) decreased (Fig. 5).
• Case 4 is a sixty-one-year-old male. Two courses were carried out. Systemic adverse events were absent, and as local adverse events, Grade 2 induration and redness were observed at the site of inoculation (Table 1, see below). Image assessment was Objective Response. More specifically, the primary focus in the pancreatic tail region was found to be SD after one course, and after two courses it was clearly reduced, and that effect was maintained for nearly two months (Fig. 7). Liver metastatic focus in the hepatic portal region became completely inapparent after two courses (Fig. 8). Liver metastatic focus near the gallbladder became inapparent after one course, and almost disappeared after two courses (Fig. 9).
• tumor markers CA19-9 and CEA
• Fig. 10 tumor markers
• Fig. 11 specific CTL reaction against the administered peptides was observed (Fig. 11).
• Regulatory T cells were within the normal range before the start of vaccination, after one course, and after two courses (Table 6, see below). 6.3 Months since the first vaccination the subject was alive.
• Case 5 is a sixty-five-year-old male. Two courses were carried out. Systemic adverse events and local adverse events were absent (Table 1, see below). Image assessment was PD. Tumor markers also increased. According to immunologic monitoring, specific CTL reaction against the administered peptides was observed (Table 2, see below), and in CD8-positive T cell fraction analysis, an increase was observed in the naive T cell fraction and effector T cell fraction (Table 4, see below). Regulatory T cells were within the normal range before the start of vaccination, after one course, and after two courses (Table 6, see below). Thereafter, the patient died due to aggravation of the original disease 4.5 months after the initial vaccination.
• Case 6 is a fifty-seven-year-old female. Two courses were carried out. Grade 3 neutropenia was observed as the systemic adverse event (Table 1, see below). Withdrawal of GEM for one week allowed recovery, and administration was then continued. Grade 2 induration and redness at the site of inoculation were observed and lymphadenopathy (inguinal swelling) near the inoculation site were observed as local adverse events. Image assessment was Objective Response. More specifically, the primary focus in the pancreatic head region was reduced after two courses, and this effect was maintained for nearly 2.5 months (Fig. 12). PET Scans obtained before vaccination and after completion of two courses were compared. Accumulation to the tumor clearly decreased after two courses (Fig. 13).
• Case 7 is a sixty -nine-year-old male. Two courses were carried out. Grade 3 neutropenia was observed as the systemic adverse event (Table 1, see below). Withdrawal of GEM for one week allowed recovery, and administration was then continued. Grade 2 induration and redness at the site of inoculation were observed and lymphadenopathy (inguinal swelling) near the inoculation site was observed as local adverse events. Image assessment was SD (Fig. 15). More specifically, the primary focus in the pancreatic head region did not show any change in size after two courses, and this effect was maintained for nearly two months (Fig. 15). Furthermore, the tumor marker (tumor markers other than CA 125 were normal from the time of registration) decreased after one course, and the decreasing effects continued even two months later (Fig. 16).
• Case 8 is a fifty -eight- year-old male.
• One course was completed, and while the second course was being carried out, gastrointestinal bleeding occurred from the enlarged tumor.
• the cause was judged to be enlargement of the tumor, and the trial was discontinued (Table 1, see below).
• Hepatic dysfunction was observed as the systemic adverse event. Local adverse events were absent.
• Image assessment as of the completion of one course was PD.
• the tumor markers also increased.
• Case 9 (Level III / 2 me) Case 9 is a seventy -three-year-old male. After one administration, the study was deferred due to gastrointestinal constriction caused by enlargement of the tumor (Table 1, see below).
• Case 9 is a sixty -two-year-old male.
• Systemic adverse events were absent, and as local adverse events, induration and redness of Grade 2 or less were observed (Table 1, see below).
• Image assessment was SD (Fig. 18).
• the tumor marker tumor markers other than CA 125 were normal from the time of registration) decreased, and the decrease in the tumor marker continued for approximately one month (Fig. 19).
• specific CTL reaction against the administered peptides was observed (Table 2, see below), and in CD8-positive T cell fraction analysis, an increase was observed in the Naive T cell fraction and a decrease was observed in the Effector T cell fraction (Table 5, see below).
• Regulatory T cells were at a high value of 6.4 before the start of vaccination, but decreased to a normal range at 2.1 after one course (Table 6, see below).
• pancreatic cancer is intractable and is a tumor with the worst prognosis.
• the only pharmaceutical therapy against pancreatic cancer is gemcitabine, but clinically, it is still unsatisfactory.
• VEGFR2 which is highly expressed in tumor neovascular endothelial cells and identified epitope peptides that can be used as vaccines [Wada S, Cancer Res. 65, 4939-4946, 2005., WO 2004/024766 ].
• the therapeutic effect of chemotherapeutic agents against pancreatic cancer can be significantly improved or enhanced when combined with an appropriate antigenic peptide, for example, the KDR peptide identified as a cancer vaccine in WO 2004/024766, the contents of which are incorporated by reference herein in their entirety.
• an appropriate antigenic peptide for example, the KDR peptide identified as a cancer vaccine in WO 2004/024766, the contents of which are incorporated by reference herein in their entirety.
• the present invention provides an improved method for treating pancreatic cancer in a subject in need thereof.

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