JP4602006B2 - Survivin-derived HLA-A24 binding cancer antigen peptide - Google Patents

Description

The present invention relates to a peptide capable of inducing cytotoxic T cells (hereinafter referred to as CTL) targeting cancer cells.
Moreover, this invention relates to the cancer vaccine and anticancer agent containing the said peptide.
The present invention further relates to the use of the peptide for inducing CTL targeting cancer cells, the obtained CTL and an anticancer agent comprising the CTL.

Recent advances in immunology and molecular biology have had a major impact on the advancement of tumor immunity. When influenza virus infection occurs in humans, the phenomenon of establishing immunity against the infection and withdrawing from the infection can be explained by the following cellular immunity. Epithelial cells infected with influenza virus present 9-10 polypeptides from the viral genome on the major histocompatibility complex HLA molecule on the cell surface. Infected cells presenting this HLA-viral peptide complex elicit a strong allogenic response, and the infected cells are specifically recognized by CD8-positive CTLs present in peripheral blood and actively eliminated. It is understood that this mechanism of cell-mediated immunity works in the same manner against cancer cells that are generated by autologous cells becoming tumors. This was proved by isolation of the tumor antigen MAGE gene from malignant melanoma by Thierry Boon et al. In Belgium (see, for example, Non-Patent Document 1).
As a method for identifying cancer antigens recognized by T cells, a method for screening a cDNA library derived from human cancer using T cells has already been developed, and the above MAGE gene was isolated using this method. . Since then, several cancer-derived cancer antigen peptides, which are presented on class I molecules on the surface of cancer cells such as malignant melanoma and recognized by T cells, have been identified, and clinical trials using some of these have begun. And certain results have already been obtained. For example, NY-ESO-1 molecule identified from esophageal cancer as a molecule recognized by an antibody present in the serum of a cancer patient has been recognized that its synthetic peptide has an ability to induce CTL (for example, Non-Patent Documents 2 and 3).

Van der Bruggen et al., Science, 254, 1643-1647 (1991) Chen, YT. Et al., Proc. Natl. Acad. USA, 94, 1914-1918 (1997) Jager, E. et al., J. Exp. Med., 187, 265-270 (1998)

  However, few cancer antigens have been identified in epithelial cancers such as colorectal cancer, stomach cancer, breast cancer, lung cancer, and bladder cancer, which account for the majority of cancers, and immunotherapy targeting cancer antigens has not been established. . Then, this invention makes it the problem which should be solved to provide the cancer vaccine and anticancer agent which can be used for the immunotherapy of cancer.

The present inventors have focused on the fact that immunological human cancer rejection is mainly borne by CTL, in particular CD8 (+) CTL. CD8 (+) CTL recognizes and activates a complex composed of a major histocompatibility antigen complex (HLA in humans) on a cancer cell and a cancer antigen peptide presented on the HLA. The activated CTL recognizes and attacks cancer cells via the T cell antigen receptor on the cell surface. Therefore, once a cancer antigen peptide is identified, it can be used as a cancer vaccine and an anticancer agent to efficiently induce CTLs to prevent and treat cancer.
Survivin is a protein belonging to the apoptosis inhibitor (IAP) family and has a strong anti-apoptotic ability. Survivin is expressed in many cancers such as lung cancer and bladder cancer, but expression in normal tissues has been reported to be limited to fetal tissues, adult thymus, testis, etc. (Ambrosini, G. et al., Nat. Med., 3, 917-921 (1997)).
The present inventors have conducted extensive studies on cancer antigenicity, that is, CTL inducing ability of various survivin-derived peptides, and have found a specific peptide capable of inducing CTL (Japanese Patent Application Laid-Open No. 2002-284797). As a result of further studies by the present inventors, an HLA-A24 binding motif is also present in the 142 amino acid sequence encoded by the survivin gene, and a specific peptide among them can induce CTL. I found. The present invention has been made based on this finding.
That is, the present invention
(1) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and capable of inducing cytotoxic T cells targeting cancer cells by binding to HLA-A24 antigen;
(2) The 2nd position of the amino acid sequence shown in SEQ ID NO: 1 is substituted with an amino acid residue selected from tyrosine, phenylalanine, methionine and tryptophan, and / or the C-terminal amino acid of the amino acid sequence shown in SEQ ID NO: 1 Inducing a cytotoxic T cell targeting a cancer cell by binding to an HLA-A24 antigen, wherein the residue comprises an amino acid sequence substituted with an amino acid residue selected from phenylalanine, leucine, isoleucine, tryptophan and methionine Possible peptides;
(3) A cancer vaccine comprising the peptide of (1) or (2);
(4) an anticancer agent comprising the peptide of (1) or (2);
(5) Use of the peptide of (1) or (2) above for inducing cytotoxic T cells targeting cancer cells;
(6) cytotoxic T cells induced by the peptide of (1) or (2), and
(7) An anticancer agent comprising the cytotoxic T cell according to (6).

  The peptide of the present invention can induce CTLs that damage HLA-A24-positive survivin-positive cancer cells that are thought to present the peptide of the present invention. The expression rate of HLA-A24 is high, with 20-30% being positive in Westerners and 50% or more being positive in Japanese. Therefore, the cancer antigen peptide of the present invention can be used as a cancer vaccine and anticancer agent useful for various types of survivin positive malignant tumors all over the world.

The present invention is described in detail below.
The peptide of the present invention has the following sequence:
Phe Phe Cys Phe Lys Glu Leu Glu Gly Trp (SEQ ID NO: 1)
And a peptide capable of inducing cytotoxic T cells that target cancer cells by binding to the HLA-A24 antigen.
The peptide of the present invention may be appropriately modified as long as it retains the activity of binding to the HLA-A24 antigen and inducing cytotoxic T cells targeting cancer cells. Here, “modification” of an amino acid residue means substitution, deletion, and / or addition of an amino acid residue (including addition of an amino acid to the N-terminus and C-terminus of a peptide). Substituent substitution is mentioned. In the modification related to amino acid residue substitution, the number and position of amino acid residues to be substituted are arbitrary as long as they have activity as a cancer antigen peptide, but usually the length of the peptide that binds to the HLA-A24 antigen. Is 8 to 11 amino acids, the range of 1 to several is preferred.
In the modification of the amino acid residue related to the substitution, substitution of the amino acid residue at the second position and / or C-terminal in the peptide having a binding motif structure for the HLA-A24 antigen is preferable.
A specific embodiment of the peptide according to the substitution of the present invention is an amino acid in which the amino acid residue at the second position and / or C-terminal of the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid residue A peptide consisting of a sequence, which is a peptide that binds to the HLA-A24 antigen and is recognized by CTL.
In the substitution, substitution to an amino acid residue that retains the binding motif structure of the HLA-A24 antigen is preferable. That is, as a preferred embodiment of the peptide according to the present invention, the second position of the amino acid sequence shown in SEQ ID NO: 1 is substituted with an amino acid residue selected from tyrosine, phenylalanine, methionine and tryptophan, and / or SEQ ID NO: 1 comprises an amino acid sequence in which the C-terminal amino acid residue of the amino acid sequence shown in Fig. 1 is substituted with an amino acid residue selected from phenylalanine, leucine, isoleucine, tryptophan and methionine, and is recognized by CTL by binding to HLA-A24 antigen Peptides that can be used.

Identification of the peptides of the present invention involves the following steps:
(1) providing a survivin-derived peptide having a sequence corresponding to the binding motif of HLA-A24 which is human major histocompatibility complex (MHC) class I;
(2) adding the peptide to an antigen-presenting cell expressing HLA-A24 to obtain an antigen-presenting cell presenting the peptide by HLA-A24;
(3) stimulating T cells with the antigen-presenting cells to induce CTL, and
(4) a step of measuring cancer cytotoxicity of induced CTLs;
It can carry out by the method containing.
Since the peptide of the present invention has a small number of amino acids, it can be synthesized by a general amino acid chemical synthesis method such as the Fmoc method. It can also be synthesized using a commercially available amino acid synthesizer. Since the peptide of the present invention is derived from survivin, survivin was isolated from cancer cells of cancer patients according to the method described in the literature (Suzuki, K. et al., J. Immunol., 163, 2783-2791 (1999)). The corresponding peptide can also be obtained.

The peptides of the present invention can be used to induce CTL targeting cancer cells. Induced CTL recognizes and attacks cancer cells. Therefore, the peptide of the present invention can be used as a cancer vaccine and an anticancer agent.
The cancer to which the cancer vaccine and anticancer agent of the present invention can be applied is a cancer composed of cancer cells presenting the peptide of the present invention by HLA-A24, for example, epithelial cancer. Examples of epithelial cancer include lung cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer, breast cancer and the like. In addition, non-epithelial cancers expressing survivin such as renal cell carcinoma, hepatocellular carcinoma, malignant lymphoma, osteosarcoma, synovial sarcoma and the like can be mentioned.
When using the peptide of this invention as a cancer vaccine and an anticancer agent, the peptide of this invention can be used by itself or with an adjuvant, and can further contain a pharmaceutically acceptable carrier suitably.
Adjuvants include adjuvants intended to enhance immune responses, such as incomplete (complete) adjuvants of floyd, aluminum adjuvants and the like.
Examples of the pharmaceutically acceptable carrier include diluents such as PBS and distilled water, and physiological saline.
The cancer vaccine and anticancer agent of the present invention can be formed into a liquid agent, oil agent, emulsion, soft capsule agent, hard capsule agent, tablet, granule, solid agent and the like by a method well known in the art.
The cancer vaccine and anticancer agent of the present invention can be administered orally, parenterally or transdermally depending on the form of use. For example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration and the like can be mentioned. The dose of the peptide of the present invention usually varies depending on the weight of the patient, the nature of the disease and the condition, but when used for an adult, it is 5 to 10 mg per day at the maximum. For example, when used by subcutaneous injection in adult cancer patients, it is 0.1 to 10 mg per week, preferably 100 to 1000 μg.

The peptides of the present invention can also be used to induce CTL targeting cancer cells. Induction can be performed according to the method described in the literature (Nabeta, Y. et al., Jpn. J. Cancer Res, 91, 616-621 (2000)).
Specifically, the following steps:
Providing a cell expressing HLA-A24;
Adding the peptide of the present invention to the cells and presenting them on HLA-A24;
Stimulating T cells with cells presenting the peptide by HLA-A24 and inducing the T cells to a cancer cell target CTL;
Can be used.
Cells expressing HLA-A24 may be collected from cancer patients, but may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.
Since the obtained CTL targets cancer cells, it can be used as an anticancer agent. In this case, similarly to the anticancer agent containing the peptide of the present invention, it may contain a pharmaceutically acceptable carrier as appropriate and can take various forms.
The anticancer agent containing the CTL of the present invention can be administered parenterally in the same manner as the cancer vaccine and anticancer agent containing the peptide of the present invention.
The dose of the CTL of the present invention usually varies depending on the weight of the patient, the nature of the disease and the condition, but when used for an adult, it is 5 × 10 ⁹ at a maximum per day. For example, when it is used by intravenous injection to an adult cancer patient, it is 1 × 10 ⁷ to 5 × 10 ⁹ , preferably 1 × 10 ⁸ to 1 × 10 ⁹ per week.
Next, the effects of the present invention will be specifically described by way of examples, but the present invention is not limited to the examples.

Example 1 Production of Survivin-Derived Cancer Antigen Peptide of the Present Invention The following amino acid sequence:
Phe Phe Cys Phe Lys Glu Leu Glu Gly Trp (SEQ ID NO: 1)
A peptide having Peptides were synthesized using a solid phase simultaneous multiplex peptide synthesizer PSSM-8 (Shimadzu) based on the 9-fluorenylmethyloxydicarbonyl (Fmoc) strategy, followed by C18 reverse phase high performance liquid chromatography (HPLC ) (Millipore). Peptide purity and identity were measured by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethyl sulfoxide at a concentration of 1.5 mg / ml and stored at −80 ° C.

Example 2 HLA-A24 binding property of survivin-derived cancer antigen peptide of the present invention T2-A24 cells are cell lines in which HLA-A2402 gene is introduced into human lymphoblastoid cell T2 cells and expressed. A low level of HLA-A24 molecule is expressed on the cell surface of this cell, and it can be detected using an HLA-A24-specific monoclonal antibody and a flow cytometer. The expression level is quantified as mean fluorescence intensity (MFI). When a synthetic peptide is added to the cells in vitro, when the added peptide binds to the HLA-A24 molecule, the cell surface HLA-A24 expression level increases in correlation with the binding affinity. Using this experimental system, the HLA-A24 binding affinity of the survivin-derived cancer antigen peptide (survivin C peptide) of the present invention produced in Example 1 was analyzed.
Three types of peptides already proven to bind to T2-A24 cells with survivin C peptide or HLA-A24 (cytomegalovirus CMV-pp65-derived peptide, AIDS virus HIV-env-derived peptide, survivin 2B peptide), HLA Each peptide of a peptide not binding to -A24 (VSV virus-derived VSV8 peptide) was added to a final concentration of 100 μg / ml. Immediately thereafter, the cell surface HLA-A24 expression level was stained with a monoclonal anti-HLA-A24 antibody and a FITC-labeled anti-mouse IgG antibody, and analyzed with a flow cytometer (FACScan, Becton Dickinson). The expression level is quantified by the MFI value,
(MFI shift) = (MFI value after addition of peptide) − (MFI value before addition of peptide)
As a result, the degree of expression change was quantified.
As shown in FIG. 1, the survivin C peptide of the present invention has a binding affinity almost comparable to that of the HIV-env peptide.

Example 3 In Vitro Induction of CTL Using Survivin-derived Cancer Antigen Peptide of the Present Invention Peripheral blood mononuclear cells (PBMC) were collected by centrifugation in human peripheral blood and Ficoll-Conlay density gradient, Separated into non-adherent cells. Adherent cells were incubated with 100 ng / ml GM-CSF (Novartis Pharma ceuticals) and 10 IU / ml IL-4 (Ono Pharmaceutical) in AIM-V (Gibco Co.). This cell was used as an antigen presenting cell (APC). Non-adherent cells were incubated with 30-100 IU / ml recombinant IL-2 (Ajinomoto) in AIM-V. On the 5th to 6th days, the peptide of the present invention (final concentration 30 μg / ml) was added to APC, and after irradiation, this APC and CD8 positive cells separated from autologous non-adherent cells were treated with IL-2 ( Takeda Pharmaceutical Co., Ltd.) was co-cultured in an AIM-V culture medium supplemented with a final concentration of 50 to 100 IU / ml. After 14 days, the peptide of the present invention (final concentration 30 μg / ml) was added to autologous PHA blasts (PHA-stimulated T cells) stimulated with T cell mitogen PHA, and the irradiated cells were used as APC. CD8 positive cells were stimulated every day. At each stimulation, fresh medium containing 50-100 IU / ml IL-2 was added to the culture. CTL on day 28 (after 4 stimulations) was used for the following activity tests.

Example 4 Cytotoxicity of CTL Induced Using Peptide of the Present Invention (1) Test Method CTL cytotoxicity was evaluated by ⁵¹ Cr cytotoxicity test. CTL target cells were labeled with 100 μCi sodium chromate ( ⁵¹ Cr) for 2 hours at 37 ° C., washed and resuspended. The labeled target cells on which the test peptide was presented were obtained by labeling the target cells (5 × 10 ⁵ cells / ml) with ⁵¹ Cr and incubating with 30 μg / ml test peptide for 2 hours at room temperature. Effector cells (CTL) were placed in each well of a V-shaped microtiter plate Costar3894 (Corning Incorporated), and the labeled target cells were added thereto at a concentration of 5 × 10 ³ cells / well to a volume of 0.2 ml. After 6 hours of incubation, 0.1 ml of the supernatant was collected and ⁵¹ Cr release was measured by an automated gamma counter (LKB Wallac). The measurement was performed in triplicate and the standard deviation was calculated. The percentage of specific cytotoxicity is expressed as the percentage of specific ⁵¹ Cr release as follows:
[(Experimental value) − (Spontaneous release value) / (Maximum release value) − (Spontaneous release value)] × 100
It was determined by calculating using Spontaneous release values were obtained from release values when target cells were incubated alone in the absence of effector cells. The maximum release value was obtained by the maximum release amount when incubated with the surfactant 10% Nonidet-P40 (Nacalai Chemical Co.).

(2) Peptide-specific cytotoxicity of CTL Using the peptide of the present invention, CTL was induced using the peripheral blood of one HLA-A24-positive breast cancer patient A in the same manner as in Example 3.
As T2-A24 cells in which HLA-A2402 gene was introduced into a T2 cell line (obtained from ATCC) that does not have HLA-A2402 gene and HLA-A24 molecule was expressed on the cell surface as CTL target cells, -C1R-A31 cells in which HLA-A3101 gene was introduced into C1R cell lines (obtained from ATCC) that do not have A2402 gene, and K562 cells that did not express HLA at all were used. According to the test method of (1), a test peptide was added to each target cell to present the peptide on the target cell, and this was used for the test. In addition to the survivin C peptide of the present invention, the synovial sarcoma antigen SYT-SSX peptide presented by HLA-A24 was used as the test peptide.
The results of the CTL ⁵¹ Cr cytotoxicity test are shown in FIG. The vertical axis represents the percentage of target cell thawing, and the horizontal axis represents the E / T ratio (effector cell (CTL) / target cell) in each target cell shown in the figure. CTL induced by the peptide of the present invention (survivin C) injures only the target cell (A24 + SUVwild) added with survivin C peptide to T2-A24 cells expressing HLA-A24, and does not add any peptide Cells (T2A24, C1RA31), cells added with SYT-SSX peptide (T2A24 + SYT), C1R-A31 cells not expressing HLA-A24 and cells added with survivin C peptide (A31 + SUVwild), K562 not expressing HLA The cells were hardly damaged.
These results indicate that the CTL induced by the peptide of the present invention specifically recognizes cells having HLA-A24 positive and survivin C peptide on the cell surface and exerts high cytotoxic activity.

(3) Cancer Cytotoxicity of CTL Induced by Peptide of the Present Invention Cytotoxicity of CTL induced by the peptide of the present invention from peripheral blood of breast cancer patient A in (2) targeting various human cancer cells Sex was tested. The four cancer cell lines used as target cells are all cancer cell lines expressing survivin protein, but HMC2 cells and HMC1 cells are cancer cell lines expressing HLA-A24 on the cell surface, MCF7 cells and K562 cells are HLA-A24 negative cancer cell lines. The results of the ⁵¹ Cr cytotoxicity test are shown in FIG. The test was performed at three E / T ratios (10, 3 and 1) per target cell. CTLs induced with the peptides of the present invention injured only HLA-A24 positive cancer cell lines.
Next, using the peripheral blood of HLA-A24-positive oral cancer patient B different from (2) above, CTL was induced using the peptide of the present invention in the same manner as in Example 3. This CTL was tested for cytotoxicity by targeting HLA-A24 positive cancer cells and HLA-A24 negative cancer cells. All three types of cancer cell lines used as target cells are cancer cell lines expressing survivin protein. OSC20 cells are oral squamous cell carcinoma cell lines that do not have the HLA-A24 gene, and OSC20A24 cells are cell lines that are expressed by introducing the HLA-A2402 gene into OSC20 cells. The results of the ⁵¹ Cr cytotoxicity test are shown in FIG. The test was performed under the conditions of three E / T ratios (30, 10, and 3) per one type of target cell. CTLs induced with the peptides of the present invention injured only HLA-A24 positive cancer cell lines.
From these experiments, the CTL induced by the peptide of the present invention specifically shows not only cells to which survivin C peptide is added from the outside of the cells, but also HLA-A24 positive and cancer cells expressing survivin in the cells. It has been shown to recognize and exert high cytotoxicity.

It is a graph showing the result of having tested the binding property of survivin C peptide of this invention, and HLA-A24 molecule. It is a graph which shows the result of the target cytotoxicity test about the cytotoxic T cell (CTL) induced | guided | derived using the survivin C peptide of this invention. Figures A and B were tested using CTL derived from the same cancer patient. It is a graph which shows the result of the target cytotoxicity test about the cytotoxic T cell (CTL) induced | guided | derived from the cancer patient peripheral blood different from FIG. 2 using the survivin C peptide of this invention.

Claims (6)

  A peptide comprising the amino acid sequence shown in SEQ ID NO: 1 and capable of inducing cytotoxic T cells targeting cancer cells by binding to HLA-A24 antigen. A cancer vaccine comprising the peptide according to claim 1 . An anticancer agent comprising the peptide according to claim 1 . Use of the peptide according to claim 1 for in vitro induction of cytotoxic T cells targeting cancer cells. A cytotoxic T cell induced by the peptide of claim 1 . An anticancer agent comprising the cytotoxic T cell according to claim 5 .

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