KR20070019002A - Peptide vaccine for cancer therapy - Google Patents

Abstract

The present invention provides a method for treating a cancer patient, comprising administering a cancer antigen peptide to a patient in need thereof in a therapeutically effective amount, the method comprising: 1) a combination of cancer antigen peptides comprising two or more cancer antigen peptides; 2) evaluating the presence or absence of specific cytotoxic T lymphocyte progenitor cells for each peptide included in the set of cancer antigen peptides in the patient, 3) the patient having a specific cytotoxic T lymphocyte Removing the cancer antigen peptide evaluated as having no progenitor cells from the bath of the cancer antigen peptide to prepare a bath of administration cancer antigen peptide, and 4) administering the bath of administration cancer antigen peptide to the cancer patient. It provides a method comprising the.

Description

Cancer treatment peptide vaccine {PEPTIDE VACCINE FOR CANCER THERAPY}

The present invention relates to novel therapies for treating cancer.

We previously reported a group of antigenic peptides capable of inducing tumor responsive cytotoxic T lymphocytes (CTL) in HLA-A24 ⁺ or HLA-A2 ⁺ patients (1-5).

We then conducted a clinical trial of peptide vaccine administration using two different therapies (6-12). In the first treatment, lung, colon and cervical cancer patients were vaccinated with CypB, SART3 and SART2 derived peptides (6, 7, 12), respectively. Although this vaccine administration protocol was completely safe, neither the intended clinical effect nor the immune response was observed. In a second treatment called "individual" vaccine administration, it was confirmed that CTL progenitor cells were present in peripheral stromal nucleated cells (PBMC) prior to vaccination in patients with advanced epidermal cancer of HLA-A24 ⁺ or HLA-A2 ⁺ . Peptides were vaccine administered. Increases in cellular and humoral immune responses to the vaccine administered peptides were observed with high frequency in PBMCs and serum after vaccine administration, respectively (8-12). In addition, three out of five cervical cancer patients actually showed tumor regression (12), and two out of four patients with Skirus gastric cancer (GC) had invariable beds for more than two years (11).

An object of the present invention is to provide a peptide vaccine therapy which is safe and therapeutically effective for cancer patients.

[Means for solving the problem]

The present inventors have newly attempted a "predesignated vaccine" therapy in which a peptide to be administered to a patient is previously designated using a peptide (8-12) frequently selected as a vaccine candidate in the individual vaccine therapy so far. 18 cancer patients (10 cervical cancer patients and 8 gastric cancer patients participated in pre-designated vaccine therapy. Pre-designated vaccine therapy was tolerated by all patients. For most patients of pre-designated vaccine therapy, peptide specific CTL progenitor and humoral immune responses increased, but no response correlated with clinical outcome.

Three patients were pathologically intact, and prior to their vaccination, peripheral stroma nucleus cells contained peptide specific CTL progenitor cells that responded with at least two peptides in four peptides. By pre-designated vaccine therapy, in 5 of 15 patients with progressive disease, the levels of existing immunoglobulin G in response to non-vaccinated peptides were reduced and the period to worsening of the disease was very short. In the individual therapy of, such a decrease was hardly observed. From these results, pre-designated vaccine therapies can induce a primary immune response, which, if the vaccine administered contains a lack of recognition of the patient's immune system, is likely to concomitantly suppress the existing immune response. It is suggested.

One of the goals of tumor immunology is to develop safe and therapeutically effective peptide vaccine therapies for advanced cancer patients. However, despite the clinical trials of many peptide vaccine therapies over the past decade (6-12, 15-21), such treatments are not available at present. In order to achieve this goal, we conducted this study, and in the individual vaccine therapy to date, four peptides (8) were detected with high frequency of CTL progenitor cells in PBMCs prior to vaccine administration and administered to patients as vaccines (8). -12), the safety, immunological and clinical academic response of predesignated peptide vaccine administration was evaluated. As a result, cellular and humoral immune responses to vaccine administered peptides were effectively induced. Previous attempts at directed vaccine therapy are basically active studies of previous individual therapies. For pre-designated vaccine therapy, the level of peptide specific CTL progenitor cells in PBMCs prior to vaccination correlated with clinical results regarding both time to bed worsening (TTP) and mean (median) survival time (MST). It is like being. In contrast, the levels of cellular or humoral immune responses induced with peptide vaccines were strongly induced in PBMCs or serum after vaccine administration in most subjects, but they were not correlated with clinical results. From these results, the method of the present invention was completed by administering a combination of cancer antigen peptides except for those not recognized by the patient's immune system from the group of cancer antigen peptides to be administered.

This invention is based on said knowledge.

That is, the present invention provides a method for treating cancer patients, comprising administering a cancer antigen peptide to a patient in need thereof in a therapeutically effective amount,

1) providing a set of cancer antigen peptides consisting of two or more cancer antigen peptides,

2) Evaluating the presence or absence of specific cytotoxic T lymphocyte progenitor cells for each peptide included in the group of the cancer antigen peptide in the patient,

3) removing from the group of cancer antigen peptides the cancer antigen peptides assessed that the patient does not have specific cytotoxic T lymphocyte progenitor cells, and providing a group of cancer antigen peptides for administration, and

4) administering to the cancer patient a set of administration anticancer peptides,

It provides a method characterized in that.

The present invention also provides a combination of a cancer antigen peptide consisting of two or more cancer antigen peptides, and

Reagents for Evaluating the Presence of Cancer Antigen Peptide Specific Cytotoxic T Lymphocyte Progenitor Cells in Cancer Patients

It provides a kit for the treatment of cancer patients, containing.

1 is a graph showing the cellular response to vaccine administered peptides in four representative cervical cancer patients (C-1, 7, 9 and 10) and two GC patients (GC-4 and 8).

2 is a graph showing serum IgG in response to vaccine administered peptides.

FIG. 3 is a graph showing the survival time of patients under two vaccine regimens for cervical cancer and Skirus GC patients.

4 is a graph showing serum IgG responsive to non-vaccinated peptides.

Best Mode for Carrying Out the Invention

In a first aspect, the present invention provides a method for treating a cancer patient comprising administering a cancer antigen peptide to a patient in need thereof in a therapeutically effective amount,

1) providing a set of cancer antigen peptides consisting of two or more cancer antigen peptides,

2) Evaluating the presence or absence of specific cytotoxic T lymphocyte progenitor cells for each peptide included in the group of the cancer antigen peptide in the patient,

3) preparing a set of cancer antigen peptides for administration, by removing the cancer antigen peptide evaluated from that patient having no specific cytotoxic T lymphocyte progenitor cells from the set of cancer antigen peptides, and

4) administering a group of administration cancer antigen peptides to the cancer patient

It provides a method comprising a.

The term "cancer antigen peptide" used in the present specification means a tumor antigen peptide derived from a tumor antigen protein. Tumor antigen peptides are those produced by the breakdown of tumor antigen proteins, which are tumor specific proteins, by proteasomes in cells, that is, they are synthesized in cells. The tumor antigen peptide thus produced is combined with an MHC class I antigen (HLA antigen) to form a complex in the endoplasmic reticulum, and the complex migrates to the cell surface and is presented as an antigen.

Tumor antigen proteins in the present specification include human melanoma cell-derived proteins called MAGEs (Science, 254: 1643, 1991); Melanosome protein, for example, melanosite tissue specific protein gp100 (J.Exp.Med., 179: 1005, 1994), MART-1 (Proc.Natl.Acad.Sci.USA, 91: 3515, 1994 ), And tyrosinase (J. Exp. Med., 178: 489, 1993); MEGE related protein (J. Exp. Med., 179: 921, 1994); β-catenin with tumor specific amino acid mutations (J. Exp. Med., 183: 1185, 1996); And CDK4 (Science, 269: 1281, 1995); HER2-neu (J. Exp. Med., 181: 2109, 1995), p53 (variants) (Proc.

Natl. Acad. Sci. USA, 93: 14704, 1996); Tumor markers such as CEA (J. Natl. Cancer Inst., 87: 982, 1995), PSA (J. Natl. Cancer Inst., 89: 293, 1997); And viral proteins such as HPV (J. Immunol., 154: 5934, 1995) and EBV (Int. Immunol., 7: 653, 1995). A detailed description of these materials is shown in the notation of notation (see, eg, Immunol. Today, 18: 267, 1997; J. Exp. Med., 183: 725, 1996; and Curr. Opin. Immunol., 8: 628, 1996).

Typical examples of tumor antigen peptides used herein include, but are not limited to the following: Tumor antigen peptides described in WO97 / 46676, WO99 / 29715, and WO99 / 33977; Cyclophilin B-derived tumor antigen peptides (WO99 / 67288); SART-1 derived tumor antigen peptide (WO00 / 06595); SART-3 derived tumor antigen peptide (WO00 / 12701); ART-1 derived tumor antigen peptide (WO00 / 32770); SART2 derived tumor antigen peptide (J. Immunol., 164: 2565, 2000); lck derived tumor antigen peptide (Eur. J. Immunol., 31: 323, 2001); ART4 derived tumor antigen peptide (Cancer Res., 60: 3550, 2000); and ppMAPkk, Tumor antigen peptides derived from WHSC2, UBE2V, HNRPL, EIF (Cancer Res., 61: 2038, 2001).

Identification of cancer antigen peptides used in the present invention is based on whether candidate peptides comprising a portion of the tumor antigen protein can be synthesized and peptide specific cytotoxic T lymphocytes (CTLs) can be summarized and also summarized by immunoglobulin G. It can carry out by selecting them based on whether they can be recognized by (IgG). Peptide synthesis can be performed according to the method normally used in peptide chemistry. Examples of known methods include "Peptide Synthesis", Interscience, New York, 1966; "The Proteins", vol. 2, Academic Press Inc., New York, 1976; "Peptide Synthesis", Maruzen Corporation, 1975 and the like. It is described in the literature. Preferably, the peptide is synthesized based on the HLA binding motif. Selection of candidate peptides based on whether peptide specific cytotoxic T lymphocytes (CTLs) can be induced is usually performed by assays to determine whether candidate peptides presented by HLA antigens are recognized by CTL. can do. Selection of candidate peptides based on whether they can be recognized by IgG can usually be performed by ELISA.

Those skilled in the art will appreciate that the amino acid sequence includes substitution, deletion and / or addition of one or several amino acid residues relative to the amino acid sequence of the peptide of the present invention and also has properties as a cancer antigen peptide, i.e., binds to the HLA antigen. `` A derivative of a cancer antigen peptide having properties equivalent to those of the cancer antigen peptide, '' which is a mutant peptide that has the ability to be recognized by CTL and, in some cases, is recognized by a humoral immune response. It can be used. Cancer antigen peptides used herein include this derivative.

The preferred length of the amino acid sequence of the cancer antigen peptide or derivative thereof is 8 to 15 amino acid residues, more preferably 8 to 11 amino acid residues.

As the cancer antigen peptide used in the present invention, any cancer antigen peptide may be used regardless of whether it is identified at this time or found in the future. Preferably, cancer antigen peptides that are predicted to be highly recognized by the patient's immune system may be selected and prepared according to the race, HLA type, sex, and type of cancer to be treated.

In the present invention, "the combination of the cancer antigen peptide which consists of two or more cancer antigen peptides", Preferably, four or more, for example, 14 cancer antigen peptides are contained.

In the method of the present invention, first, it is determined whether or not the patient has specific cytotoxic T lymphocyte progenitor cells for each cancer antigen peptide included in the group of cancer antigen peptides. Such determination may be performed by a conventionally known method, for example, according to the method described in Example 1.4 cellular immune response of the present specification.

A set of cancer antigen peptides for administration is prepared, except for the cancer antigen peptides assessed that the patient does not have specific cytotoxic T lymphocyte progenitor cells. In the method of the present invention, a set of the cancer antigen peptide for administration is administered to the patient. The group of the cancer antigen peptide for administration should just contain 1 or more cancer antigen peptide, Preferably it is good to contain 2 or more, more preferably 3-4 cancer antigen peptides. Although there is no restriction | limiting in particular in the number of cancer antigen peptides contained in the tank of administration anticancer peptide, It is preferable to set it as four in consideration of a burden on a patient.

The administration of the cancer antigen peptide for administration may be prepared as a pharmaceutical composition containing the peptide, and the composition may contain an adjuvant that is known to be used at the time of administration of the vaccine. Or you may administer the pharmaceutical composition and adjuvant separately prepared simultaneously.

The administration of the cancer antigen peptide for administration can be administered by subcutaneous, intradermal, or intravenous injection. Each peptide included in the set of administration cancer antigen peptides can be administered separately or simultaneously. Specifically, each peptide may be injected at a separate site, or all peptides may be combined at one site. The amount of tumor antigen peptide in vaccine administration can be appropriately controlled depending on, for example, the disease state, the age and the weight of a particular patient, with each peptide in the group typically being between 0.1 mg and 1 week or 2 weeks old. 10.0 mg, preferably 0.5 mg to 5.0 mg, more preferably 1.0 mg to 3.0 mg.

In a second aspect, the present invention provides a kit for carrying out the method of the present invention, specifically, a combination of cancer antigen peptides consisting of two or more cancer antigen peptides, and

Reagents for Evaluating the Presence of Cancer Antigen Peptide Specific Cytotoxic T Lymphocyte Progenitor Cells in Cancer Patients

It provides a kit for the treatment of cancer patients, containing.

The bath of the cancer antigen peptide contained in the kit is preferably freeze-dried. The group of cancer antigen peptides for evaluation and administration may be provided simultaneously, and may be divided into Article 1 for evaluation and Article 2 for administration.

The cancer antigen peptide of Article 1 used for evaluation is melt | dissolved in a suitable solvent, such as dimethyl sulfoxide, and is used, for example. After dissolving in dimethyl sulfoxide etc., when it needs to be preserved for a while until use, it is preserve | saved at low temperature, for example about 4 degreeC.

The cancer antigen peptide of Article 2 used for administration may be dissolved or suspended in a physiologically suitable solution, for example, physiological saline, prior to appropriate administration. Alternatively, sodium bicarbonate may be used as the dissolution aid. The peptide solution may also be mixed with an adjuvant, an emulsion may be prepared and administered.

Therefore, the kit of this invention may contain the solution for evaluation, administration, and the adjuvant. In addition, as the kit of the present invention, a part containing Article 1 of the cancer antigen peptide for evaluation and a reagent for evaluation, a part containing the cancer antigen peptide of Article 2 for administration, and a solution containing a dissolving solution and an adjuvant for administration It may be provided separately.

In the present invention, "a reagent for evaluating the presence or absence of cancer antigen peptide specific cytotoxic T lymphocyte progenitor cells in a cancer patient" shall contain the material required for such evaluation by any conventionally known method, For example, the cell required for evaluation, a cell culture medium, an evaluation plate, etc. shall also be included.

The present invention is further illustrated by the following examples, which are not limited in any sense. Prior to the examples, 1. patient eligibility criteria, and 2. patient characteristics are described.

1. Patient Eligibility Criteria

Ethics review boards at the facilities of Kurume University, Hokkaido University, Kouchi University, and Kyoun Hospital have approved clinical protocols for their pre-designated peptide vaccine administration. At the time of enrollment, full written consent was obtained from all patients. In the protocol, the patient needed to be HLA-A24 or -A2 positive when HLA class I expression on PBMC was determined as conventional (11, 12) by conventional serotype typing. Pathology was confirmed for all patients with gastric or cervical cancer. Eligibility criteria include age 80 or less, serum creatinine less than 1.4 mg / dl, bilirubin 1.5 mg / dl, platelet count 100,000 / μl or more, hemoglobin 8.0 g / dl or more, and total leukocyte count 3,000 / Μl or more were included. Hepatitis B surface antigen and hepatitis C RNA were negative. Patients were untreated for at least 4 weeks prior to entering the trial, and at the time of entry, the performance status of the Eastern Cooperative Oncology Group must be between 0 and 2. Patients with signs of poisoning, immunosuppression or autoimmune disease were excluded. The treatment was carried out from August 2001 to June 2003.

2. Patient Characteristics

Eighteen patients (10 patients with recurrent cervical cancer and eight patients with advanced gastric cancer (GC)) were enrolled in this study. The detailed characteristics of these patients are shown in Table 1 below. The average age of the patients was 50 years and ranged from 27 to 74 years. 10 and 8 patients were HLA-A24 and -A2 positive, respectively. The sites of recurrence of cervical cancer were vaginal tip (n = 4), lymph nodes (n = 4), lungs (n = 1), and vulvar epithelium (n = 1). The histology of cervical cancer was squamous cell carcinoma (n = 8) or adenocarcinoma (n = 2). Treatments to date have included surgical resection (n = 7), chemotherapy (n = 6), and radiation therapy (n = 10). Five, two and one GC patients were Stage IV, IIIB and IIIA, respectively, based on the Japanese Gastric Cancer Classification 13th Edition. The histology of GC was all adenocarcinoma, six (GC-3 to -8) were Scyrus (obesity), and the remaining two were Bskirus. All GC patients are undergoing surgical resection of the primary lesion, six of whom previously failed chemotherapy.

^a C (uterine cervix cancer) / CG (stomach cancer)

^b Performance status by EOCG score

^c Japanese Gastric Cancer Classification 13th Edition and UISS and FIGO Classification of Cervical Cancer (1997)

^d SSC squamous cell carcinoma, adeno adenocarcinoma

^f RH Extensive Uterine Whole, TH Uterile, DG Distal Gastrectomy, TG Potential, BSO Bilateral Fallopian Ovarian Extraction

^g DDP Cisplatin: CPT11 hydrochloric acid irinotecan BOMP hydrochloric acid preomycin + sulfate vincristine + mytomycin C + cisplatin: CBDCA carboplatin: TS1 1M tegapur-0.4M5-chloro 2.4 dihydroxypyridine-1M Ota Start Potassium, 5'DFUR Doxyfluidine: 5FU Fluorouracil: LV leucoborin calcium: MTX methotrexite

Example  One

Predesignated Vaccine Administration

1.1 Peptide

The peptide used in this invention was prepared by Multiple peptide Systems (San Diego, CA) on condition of a suitable manufacturing standard (GMP). The arrangement of peptides is as follows: Peptides used in HLA-A24 ⁺ patients were: SART3 ₁₀₉ (VYDYNCHVDL;

SEQ ID NO: 1), SART3 ₃₁₅ (AYIDFEMKI; SEQ ID NO: 2), lck ₂₀₈ (HYTNASDGL; SEQ ID NO: 3), lck ₄₈₈ (DYLRSVLEDF; SEQ ID NO: 4), and SART2 ₉₃ (DYSARWNEI; SEQ ID NO: 5). HLA-A2 ⁺ patients were used with CypB ₁₇₂ (VLEGMEVV; SEQ ID NO: 6), lck ₂₄₆ (KLVERLGAA;

SEQ ID NO: 7), ppMAPkkk ₄₃₂ (DLLSHAFFA; SEQ ID NO: 8), and UBE2V ₄₃ (RLQEWCSVI; SEQ ID NO: 9). All of these peptides have shown the ability to induce HLA-A24- or HLA-A2 binding and tumor specific CTL activity in cancer patients' PBMCs (2-12).

They are frequently administered to cancer patients in individual vaccine therapies up to now confirming cellular responses to peptides prior to vaccine administration (8-12). Peptides for HLA-A2 ⁺ patients were all selected based on binding motifs for HLA-A * 0201 molecules, but these peptides were used in HLA-A * 0201 patients as well as other HLA-A2 subtypes, eg For example, there is also immunogenicity in patients with HLA-A * 0206 or HLA-A * 0207 (2, 11, 12).

1.2 Peptide Vaccine Administration

Each peptide was dissolved in a small amount of dimethylsulfoxide and diluted to 4 mg / ml in physiological saline. Montanide ISA-51, an incomplete Freund's adjuvant, was manufactured by Seppeic, Inc. (Franklin Lakes, NJ). 1 ml (2 mg / peptide / injection) of each of the four different peptide emulsions obtained was injected subcutaneously into each site of the lateral abdominal wall for patients with gastric cancer (GC) using free syringes as shown (11, 12), Cervical cancer patients were injected subcutaneously into each site of the lateral thigh. The interval between vaccine administrations was one week, in total six injections.

1.3 Hazards

All 18 patients were evaluated for adverse events. Tolerance of vaccine administration was generally good. Fever of grades I and II with mild cold symptoms was observed in 4 patients and 1 patient, respectively; Their fever is transient and was natural in nature. Local redness and swelling of grades I and II at the injection site were observed in 12 and 4 patients, respectively. Nausea, vomiting, abdominal pain, and diarrhea were observed in two patients each. This peptide vaccine administration was not accompanied by moderate (grade III or IV) toxicity. Grade I, bleeding from bladder infiltrates and bleeding from the vaginal hem was observed in one patient each, while hematocrit of grade IV due to tumor progression was observed in one GC patient (GC-7). Judgment by symptom, physical diagnosis or clinical examination revealed no clinical or academic evidence indicating an autoimmune response.

1.4 Cellular Immune Response

To measure cellular response, as in the notation (11, 12), PBMCs were separated by Ficoll-Conray specific gravity centrifugation and then used for CTL progenitor cell assays. Evaluation of the cellular immune response to the dosing peptide is shown in Table 2. To assess the cellular immune response to the vaccine-administered peptide (12), PBMCs (1 × 10 ⁵ cells / well) before and after the vaccine administration (sixth time), as indicated, were replaced with interleukin (IL) -2 Incubated in four different wells of each peptide 10 μM and 96 well microculture plates (Nunc, Roskilde, Denmark) in 200 μl of 100 units / ml) containing culture medium. On day 14 of culture, peptide stimulation PBMCs (8-12 × 10 ⁵ / well) were separately collected from each well and divided into quarters. As shown (10-12), two of them are C1R-A2402 cells (Oiso M, Eura M, Katsura F, Takiguchi M, Sobao Y, Masuyama K, Nakashima M, Itoh, respectively, for HLA-A24 ⁺ PBMC). K, Ishikawa TA newly identified MAGE-3-derived epitope recognized by HLA-A24-restricted cytotoxic T lymphocytes.Int. J. Cancer 81: 387-394, 1999), and T2 cells (HLA for HLA-A2 ⁺ PBMC). Two cases were tested for interferon (IFN) -γ production ability against the -A * 0201 expressing cell line, ATCC: CRL-1992, and the other two were negative control peptides (HIV peptide, RYLRDQQLL and HLA for HLA-A24). For -A2, SLYNTVATL) was tested. Background IFN- [gamma] production (<50 pg / ml) in response to HIV peptide was subtracted. After incubation for 18 hours, the supernatant was recovered to measure IFN-γ by enzyme immunoassay (ELISA) (detection limit: 10 pg / ml). For statistical analysis, two-sided Student's t-test was used. Representative four cervical cancer patients (C-1, 7, 9, and 10) and two GC patients (GC-4 and 8) are shown in FIG. 1. The detailed results are shown in Table 2. Criteria for assessing cellular response are also described in the legends of Table 2.

^a CTL progenitor cell induction assay was performed in four rows and the background response to the HIV peptide was subtracted from the value.

The results were evaluated by the following criteria: Ar (p value ≤ 0.1 and IFN-γ production ≥500 pg / ml), A (p value ≤ 0.05 and IFN-γ production> 50),

B ((p values <0.05 and 25 <IFN-γ production <50),

C (0.5 <p << 0.1 and IFN-γ production> 50) and

D (0.05 <p-value <0.1 and 25 <IFN- [gamma] generation <50). The classification is represented by alphabetic characters, with each character representing the result of each well.

For example, AAB means that three wells were determined to be A, A, and B, and one well was negative.

^{b A} net OD value (subtracting the OD value responding to the HIV peptide in serum dilution 1: 100 from the OD value responding to the corresponding peptide)> 0.02 was judged to be positive for peptide-reactive IgG.

^c Number of vaccine doses when DTH for the peptide is first detected.

^d SD (hospital constant), PD (hospital deterioration).

^e TTP (time to exacerbation),

^f OS (overall survival), + (patient survival, September 15, 2003)

^g NT (not tested).

In order to avoid the effect of in vitro manipulation on peptide specific CTL progenitor cell assays, PBMCs before and after the vaccine administration (the sixth time) were thawed simultaneously.

PBMCs prior to vaccine administration from two patients (GC-1 and GC-3) cannot be used for analysis because of limited cell numbers, and from three patients (C-3, GC-5 and GC-7). PBMC was not available because the disease rapidly worsened and died after vaccination. Peptide specific CTL progenitor cell assays were performed in four cases and subtracted the amount of IFN-γ in response to HIV peptide. The results were evaluated from the point of two parameters, P value and IFN-γ production as described in the legend of the table. Overall, peptide specific CTL progenitor cells were induced in 9 of 13 patients (69%). For example, in HLA-A24 ⁺ GC patients (GC-4), PBMC did not respond to any of the four vaccine dose peptides prior to vaccine administration, but each well of PBMC after vaccine administration had a SART3 ₁₀₉ peptide, SART3 ₃₁₅ Respond to peptide or lck ₄₈₈ peptide. In HLA-A2 ⁺ cervical cancer patients (C-9), one well of PBMC responded to UBE2V ₄₃ peptide, two wells responded to lck ₂₄₆ peptide, and two wells responded to ppMAPkkk ₄₃₂ peptide before vaccine administration. In response, 4, 4, and 3 wells of PBMCs after vaccine administration responded to UBE2V ₄₃ peptide, lck ₂₄₆ peptide and ppMAPkkk ₄₃₂ peptide, respectively. These results mean that all directed vaccine therapies have the ability to effectively induce peptide specific CTLs.

1.5 Humoral Immune Response

We have recently reported that the induction of IgG in response to a dosing peptide correlates frequently with the clinical response and outcome of patients of several cancer types (8, 10, 11). Therefore, we dynamically examined the level of peptide specific IgG. Briefly, serum samples before and after administration of 100 μl / well of vaccine (6 times) were diluted with 0.05% Tween 20-Block Ace and added to peptide (20 μg / well) solidification plates. After incubation at 37 ° C. for 2 hours, the plates were washed and further incubated with 1: 1000 diluted rabbit anti human IgG antibody (γ chain specific, DAKO, Glostrup, Denmark) for 2 hours. Plates were washed and 100 μl of 1: 100 diluted goat anti rabbit Ig antibody binding Western mustard peroxidase-dextran polymer (En Vision, DAKO) was added to each well and the plate was incubated for 40 minutes. After washing, 100 μl / well of tetramethyl-benzidine substrate solution (KPL, Guildford, UK) was added, and 1 M phosphoric acid was added to stop the reaction. The absorbance (OD) of each sample is expressed in OD units / ml. To determine the cutoff, the serum of the patient was simultaneously measured for responsiveness to both of the corresponding peptides and HIV peptides used as negative control peptides. It was determined that the peptide-reactive IgG was positive when the OD value (subtracted the OD value in response to HIV peptide at serum dilution 1: 100 from the OD value in response to corresponding peptide) was> 0.02.

As shown in Table 2, IgG responsive to peptide was detected before vaccine administration, and peptide specific IgG increased after peptide vaccine administration (C-4, GC-1, 4, and 6). In addition, IgG responsive to the administration peptide was induced in serum after vaccine administration from 6 of the 15 patients tested (C-1, 4, 6, 7, 8, and 10). Overall, induction or increase in vaccine-induced peptide specific IgG was observed in serum after vaccine administration of 9 (60%) of the 15 patients tested. The results of 9 patients whose serum has significant levels of IgG responding to at least one peptide are shown in FIG. 2. In HLA-A2 ⁺ GC patients (GC-6), IgG specific for UBE2V ₄₃ and lck ₂₄₆ peptides can be detected at significant levels (serum dilution 1: 100 at <0.02) in serum prior to vaccination (The IgG specific for the other two peptides could not be detected), and the peptide vaccine administration increased the level of the peptide specific IgG. The peptide specificity of each of these IgGs is shown to be different (6-12).

1.6 Delayed Hypersensitivity (DTH) Reaction

A skin test was conducted by intradermal injection of 50 μg of each peptide using a tuberculin syringe equipped with a 26 gauge needle. Physiological saline was used as a negative control. Immediate response and delayed-type hypersensitivity (DTH) response were measured at the time points 20 minutes and 24 hours after injection, respectively. If at least 10 mm of cure or 20 mm erythema appeared 24 hours after injection, the DTH skin test was determined positive.

Delayed-type hypersensitivity (DTH) response to the peptide was not observed in any of the patients tested prior to vaccine administration. By the sixth vaccine administration, peptide specific DTH responses were induced in four cervical cancer patients (C-1, 2, 6 and 7); These details are shown in Table 2. For example, in patient C-1, DTH responses to lck ₂₀₈ and lck ₄₈₈ peptides were observed after 3 and 4 vaccine administrations, respectively.

1.7 Evaluation of Clinical Responses

Before and after administration of each of the third to sixth vaccines, all known disease sites were evaluated by CT scan or X-ray examination. Patients were classified according to the Response Assessment Criteria for Solid Tumors (revised edition of the WHO Standard, published in June 1999 as a WHO Handbook to Report the Results of Cancer Treatment).

The clinical response is summarized in Table 2. Noticeable tumor regression (> 50% reduction) was not observed in any patients. Four patients (C-3, GC-3, -5 and -7) rapidly worsened and died of cancer before the sixth dose of vaccine. At the time of the sixth vaccine administration, out of 14 patients eligible for evaluation, eight patients were diagnosed as pathological invariant (SD), and the remaining six patients were diagnosed as bed worsening (PD). All of these 14 patients decided to go into individual vaccine regimens based on the selection of CTL progenitor cells for peptides with PBMCs after vaccination (the sixth time). At the time of the twelfth vaccine administration, out of eight patients eligible for evaluation, three patients (C-4, -9 and GC-8) were diagnosed with SD and the remaining five patients were diagnosed with PD. In one (C-9) patient, tumor regression by size reduction of 16% of the aortic lymph nodes was observed at the time of the tenth vaccine administration. The mean (median) observation period, mean duration to worsening (mean (median) TTP) and mean (median) survival (MST) of 18 patients were 11, 1.5 (47 days), and 5 months (156 days, respectively). )

Interpretation of three SD patients will assist in the evaluation of prognostic factors. In patient C-4, peptide specific IgG was induced after peptide vaccine administration, but no peptide specific CTL and DTH were observed. For patients C-9 and CG-8, peptide specific CTLs were induced but no peptide specific IgG and DTH were observed. Common to these three SD patients compared to the other 15 patients was that peptide specific CTL progenitor cells for two or more peptides were detected in PBMC prior to vaccine administration. That is, CTL progenitor cells for three of the four vaccine dose peptides can be detected in PBMCs prior to vaccine administration of patient C-9, and four vaccines in PBMCs prior to vaccine administration of patients C-4 and GC-8. CTL progenitor cells for two of the administered peptides could be detected. The results are summarized in Table 3.

1.8 Shortening of Patient Survival in Predesignated Vaccine Therapy

In this study, we have newly attempted a "predesignated vaccine" therapy in which a patient is vaccinated with a peptide frequently selected as a vaccine candidate in individual therapy. However, the clinical response lags behind individual therapies. We therefore determined the overall survival rate (n = 16) of cervical cancer and GC (hard) patients under pre-designated vaccine therapy, to date under individual vaccine therapy (n = 9, 5 cervical cancer patients, and hard GC patients). 4)) (FIG. 3). As a result, the overall survival rate of the patients in this therapy was significantly shorter (p = 0.0033 by the Log-rank test) than the individual therapies so far.

1.9 Concomitant Inhibition of Existing Immune Responses by Predesignated Vaccine Administration

As a mechanism for shortening the survival time of patients in pre-designated vaccine therapies, in pre-designated vaccine therapies, existing immune responses are concomitantly suppressed by induction of a primary immune response to the administration peptide where no CTL progenitor cells were present. There is a possibility. To investigate this possibility, we compared the level of IgG responsive to vaccine administered peptides to the level of IgG responsive to non-vaccinated peptides. In HLA-A24 ⁺ patients (n = 8), in six different HLA-A24 binding CTL epitope peptides (CypB ₈₄ , ART1 ₁₇₀ , ART4 ₁₃ , ART4 ₇₅ , MRP3 ₅₀₃ , and MRP3 ₁₂₉₃ ), HLA-A2 ⁺ patients Vaccine from 15 patients for (n = 7) IgG levels specific for five different HLA-A2 binding CTL epitope peptides (WHSC ₁₀₃ , WHSC ₁₄₁ , HNRPL ₁₄₀ , HNRPL ₅₀₁ , and EIF ₅₁ ) Serum was tested before and after administration (time 6). None of the peptides were administered as vaccines in all designated vaccine therapies, all of which have been administered in individual therapies so far, and elsewhere, peptide specificity of serum IgG has been reported (8-14). The results are shown in FIG. As shown in the left column of FIG. 4, in the sera of 5 of 15 patients (C-5, 7, 8, GC-2 and 6), the level of peptide specific IgG decreased after administration of the predesignated vaccine. . It should be noted that the TTP of these five patients was very short (39, 41, 47, 53 and 65 days) and the average (median) TTP was 43 days. Serum from individual vaccine regimens was also prepared to investigate responsiveness to non-vaccinated peptides, and the results of six patients whose sera showed an increase or decrease in response to specific peptides after vaccine administration were shown in the right side of FIG. 4. Shown in the column. Detailed results for individual vaccine administration are described elsewhere (11, 12). In the individual therapy, except for the MRP3-1293 peptide of patient HS-001, peptide specific IgG was increasing after vaccine administration.

1.10 Consideration

Regarding the patient characteristics, no distinct difference is seen between the two treatments except for the number of cervical cancer patients (for 10 in all designated vaccine therapies and 5 in individual therapies so far). An increase in cellular response to the vaccine administered peptide is observed in PBMC after administration of 11 (85) of 13 patients in this therapy and in 26 peptides (50) out of 52 peptides, while to date In individual therapy, 11 of 18 patients (61) and 9 of 48 peptides (18.8) were observed (11, 12). Humoral responses to peptides were observed in sera after vaccine administration in 9 of 60 patients (60) and in 17 peptides (28.3) of 60 peptides tested in pre-designated vaccine therapy, while In therapy, 13 of 15 patients (87) and 20 of 56 peptides (35.7) were observed. These results suggest that induction of a cellular response to the administered peptide is superior to that of the individual designated therapies, whereas in the case of a humoral response, the individual therapies seem to be superior. However, noticeable differences in clinical response were observed between these treatments. That is, the mean (median) TTP (n = 16, 1.5 months) (uterine cervical cancer patient (n = 10, 2 months) and Scyrus GC (n = 6, 1 month)) of patients under this therapy are It was significantly shorter than that of patients receiving individual therapy (n = 9, 15 months) (uterine cervical cancer patients (n = 5, 15 months) and Skirus GC (n = 4, 7 months)) (11, 12). The overall survival of these patients under pre-specified vaccine therapy was significantly shorter than that under individual therapy to date (FIG. 3).

The immunological mechanisms involved in the shortening of survival time associated with predesignated vaccine therapy are not clear at this time. However, for one thing, there is a possibility that the induction of a strong primary immune response to the administration peptide inhibits the existing specific immune response to tumor cells. To investigate this possibility, we examined the level of serum IgG in response to CTL epitope peptides. These levels were frequently expressed in both cancer patients and normal donors before the vaccine administration, as reported (8-12, 14, 15). As a result, it was clear that the peptide-specific IgG level in serum before vaccine administration from 5 out of 15 patients was lower than that in serum after vaccine administration and was related to the shortening of TTP of these patients. . As for the peptide, it was found that IgG in response to five of the eleven kinds of peptides decreased. In contrast, a decrease in the humoral response to these non-vaccinated peptides was hardly seen after the vaccine administration of patients under individual therapy.

[literature]

The following documents form part of the present specification by reference.

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3.Yang D, Nakao M, Shichijo S, et al .: Identification of a gene coding for a protein possessing shared tumor epitopes capable of inducing HLA-A24-restructed Cytotoxic T lymphocytes in cancer patients. Cancer Res 59: 4056-4063, 1999.

Yamada A, Kawano K, Koga M, Takamori S, Nakagawa M, Itoh K: Gene and peptide analyses of newly defined lung cancer rejection antigens recognized by HLA-A2402-restricted tumor-specific cytotoxic T lymphocytes. Cancer Res 63: 2829-2835, 2003.

5.Harashima N, Tanaka K, Sasatomi T, et al .: Recognition of the Lck tyrosine kinase as a tumor antigen by T cells of metastatic cancer patients. Eur J Immunolo 31: 323-332, 2001.

6.Gohara R, Imai N, Rikimaru T, et al .: Phase 1 clinical study of cyclophilin B peptide vaccine for lung cancer patients. J Immunother 25: 439-444, 2002.

7.Miyagi Y, Imai N, Sasatomi T, et al .: Induction of cellular immune responses to tumor cells and peptides in colorectal cancer patients by vaccination with SART3 peptides. Clin Cancer Res 7: 3950-3962, 2001.

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Tanaka S, Harada M, Mine T, et al .: Peptide vaccination for patients with melanoma and other types of cancer based on pre-existing peptide-specific cytotoxic T lymphocyte precursors in the periphery. J Immunother 26: 357-366, 2003.

10.Noguchi M, Kobayashi K, Suetsugu N, et al .: Induction of cellular and humoral immune responses to tumor cells and peptides in HLA-A24 positive hormone-refractory prostate cancer patients by peptide vaccination. Prostate 57: 80-92, 2003.

11. Sato Y, Shomura H, Maeda Y, et al .: Immunological evaluation of peptide vaccination for patients with gastric cancer based on pre-existing cellular response to peptide. Cancer Sci 94: 802-808, 2003.

12. Tsuda N, Mochizuki K, Harada M, et al .: Vaccination with pre-designated or evidence-based peptides for patients with recurrent gynecologic cancers. J Immunother (in press), 2003.

13. Ohkouchi S, Yamada A, Imai N, et al .: Non-mutated tumor-rejection antigen peptides elicit type-I allergy in the majority of healthy individuals. Tissue Antigens 59: 259-272, 2002.

14.Kawamoto N, Yamada A, Ohkouchi S, et al .: IgG reactive to CTL-directed epitopes of self-antigens is enter lacking or unbalanced in atopic dermatitis patients. Tissue Antigens 61: 352-361, 2003.

15. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al .: Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nature Med 4: 321-327, 1998.

Rosenberg, SA ,: A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity 10: 281-287, 1999.

17. van Driel WJ, Ressing ME, Kenter GG, et al .: Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: clinical evaluation of a phase I-II trial. Eur J Cancer 35: 946-952, 1999.

18. Nestle FO, Alijagic S, Gilliet M, et al .: Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4: 328-332, 1998.

19.Gjertsen MK, Buanes T, Rosseland AR, et al .: Intradermal ras peptide vaccination with granulocyte-macrophage colony-stimulating factor as adjuvant: Clinical and immunological responses in patients with pancreatic adenocarcinoma. Int J Cancer 92: 441-450, 2001.

20. Lau R, Wang F, Jeffery G., et al .: Phase I trial of intravenous peptide-pulsed dendritic cells in patients with metastatic melanoma. J Immunother 24: 66-78, 2001.

21. Jager E, Gnjatic S, Nagata Y, et al .: Induction of primary NY-ESO-1 immunity: CD8 + T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1 + cancers. Proc Natl Acad Sci USA 97: 12198-12203, 2000.

<110> Kurume University <120> Peptide vaccines for treatment of cancers <130> 665260 <150> JP 2004-182811 <151> 2004-06-21 <160> 9 <170> Patent In Ver. 2.1 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> SART3 109 <400> 1 Val Tyr Asp Tyr Asn Cys His Val Asp Leu   1 5 10 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> SART3 315 <400> 2 Ala Tyr Ile Asp Phe Glu Met Lys Ile   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> lck 208 <400> 3 His Tyr Thr Asn Ala Ser Asp Gly Leu   1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> lck 488 <400> 4 Asp Tyr Leu Arg Ser Val Leu Glu Asp Phe   1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> SART2 93 <400> 5 Asp Tyr Ser Ala Arg Trp Asn Glu Ile   1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CypB 172 <400> 6 Val Leu Glu Gly Met Glu Val Val   1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> lck 246 <400> 7 Lys Leu Val Glu Arg Leu Gly Ala Ala   1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ppMAPkkk 432 <400> 8 Asp Leu Leu Ser His Ala Phe Phe Ala   1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> UBE2V 43 <400> 9 Arg Leu Gln Glu Trp Cys Ser Val Ile   1 5  

Claims (4)

A method for treating a cancer patient comprising administering a cancer antigen peptide to a patient in need thereof in a therapeutically effective amount, 1) providing a set of cancer antigen peptides consisting of two or more cancer antigen peptides, 2) Evaluating the presence or absence of specific cytotoxic T lymphocyte progenitor cells for each peptide included in the group of the cancer antigen peptide in the patient, 3) preparing a set of cancer antigen peptides for administration by removing the cancer antigen peptides assessed that the patient did not have specific cytotoxic T lymphocyte progenitor cells from the set of cancer antigen peptides, and 4) administering a group of administration cancer antigen peptides to the cancer patient Method comprising a. The method of claim 1, A set of cancer antigen peptides comprises four or more cancer antigen peptides. The method of claim 1, The crude of the antigenic peptide for administration contains 3 or 4 types of cancer antigen peptides. A combination of cancer antigen peptides consisting of two or more cancer antigen peptides, and Reagents for Evaluating the Presence of Cancer Antigen Peptide Specific Cytotoxic T Lymphocyte Progenitor Cells in Cancer Patients Kit for the treatment of cancer patients containing.

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