RU2777074C2 - Antigen-binding protein recognizing peptide originating from mage-a4 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to the construction of chimeric antigen receptors (hereinafter – CAR) and production of CAR-T-cells, which can be used in CAR-infusion therapy, using an onco-specific intracellular antigen. Antigen-binding protein is proposed, which is an antibody or its antigen-binding fragment, capable of recognizing peptide GVYDGREHTV in the complex HLA-A2-MAGE-A4 and containing polypeptide of a variable domain of a heavy chain (VH) with SEQ ID NO: 36 and polypeptide of a variable domain of a light chain (VL) with SEQ ID NO: 38. CAR is also constructed, in which the specified antigen-binding protein is located in N-end position of protein and is outside the cell, and an intracellular domain is located in C-end position of protein and is inside the cell.

EFFECT: invention provides for the production of specific scFv to peptide GVYDGREHTV from MAGE-A4, recognizing the complex MAGE-A4 peptide/HLA-A2, and their use for the production of cytotoxic CAR-T-cells, which were specifically activated for cancer target cells expressing HLA-A2/MAGE-A4.

15 cl, 25 dwg, 4 tbl

Description

[Technical field]

[0001]

The present invention provides an antigen binding protein that recognizes a MAGE-A4 derived peptide and the like.

[Prior Art]

[0002]

Currently, malignant tumors cause more than 30% of deaths and are the main cause of death in developed countries. Some types of cancer can be cured with advanced medical techniques, but others are difficult to treat. The development of new cancer treatments is an urgent task. The three main therapies for cancer treatment are surgery, chemotherapy, and radiation therapy. A combination of these three therapies is used to treat cancer.

A fourth type of cancer therapy, called immunotherapy, has recently begun to develop, focusing on the non-invasiveness and high efficacy of immunotherapy. Immunotherapy is broadly divided into three types, namely vaccination, antibody-based therapy, and cell transfusion therapy. Two types of transfusion cell therapy are known. One of them is a transfusion therapy using lymphocytes transfected with the T-cell receptor gene, in which in vitro induction of the indicated T-cell receptor (TCR) gene, which specifically reacts with cancer cells, is provided in the patient's lymphocytes, and performing transfusion of said lymphocytes to the patient. Another type is T cell-specific transfusion therapy, in which a chimeric antigen receptor (CAR) containing a scFv fragment as an antigen recognition site and CD3ζ as an intracellular signaling molecule is injected into the patient's lymphocytes and infused provide the patient with the introduction of the received lymphocytes. Since a large number of cancer antigen-specific lymphocytes are obtained in a short time by gene transfer in these treatments, T cell-specific therapeutics can be used to treat various types of cancer in patients.

[0003]

TCR gene-based infusion therapy, in which T cells recognize the peptide-MHC (major histocompatibility complex) (pMHC) complex and kill cancer cells, is safe and effective and is being developed worldwide. However, there is a problem that clones of killer T cells are extremely difficult to isolate.

On the other hand, there are three advantages of CAR-based fluid therapy: (1) different patients can receive therapy because T cells transfected with a CAR containing a single-chain antibody fragment (scFv) and a T cell receptor (TCR) and signaling domain of costimulatory molecules , can independently recognize the cancer antigen-MHC complex and attack cancer cells other than the original T cells, (2) can recruit CD4 positive T cells as well as CD8 positive T cells and non-T cells, (3) higher affinity is maintained compared to TCR as the receptor acquires antibody reactivity. Indeed, anti-CD19 antibody CAR therapy has been shown to be highly clinically effective in patients suffering from leukemia and lymphoma (Non-Patent Document 1, the documents are briefly described at the end of the description). The CD19 molecule is also expressed in B cells, the disappearance of B cells can be compensated by the addition of immunoglobulin. In general, it would be ideal to use antigens that are expressed only in cancer cells. However, the problem is that no such cell surface antigens have been detected to date.

[0004]

On the other hand, cancer-specific antigens have been reported in intracellular molecules, including cancer-testicular antigen and neoantigen. However, no CAR-based fluid therapy has been described for cancer-specific intracellular antigens.

In view of these circumstances, the authors of the present invention isolated antibodies that recognize the MHC complex with intracellular peptides derived from antigenic molecules, and developed a CAR-based immunotherapy method using these antibodies. Only a few antibodies have been reported that specifically recognize the peptide/MHC complex (non-patent document 2). The production of an antibody that specifically recognizes the peptide/MHC complex and CAR-T cells that specifically kill cancer cells could provide an innovative therapeutic approach. A CAR that recognizes the peptide/MHC complex can be used to infuse CAR-T cells, and by concurrent infusion of a peptide that is specifically recognized by CAR-T cells, proliferation of CAR-T cells can be expected as a result of antigen presentation.

[SUMMARY OF THE INVENTION]

[Problems Solved by the Present Invention]

[0005]

The present invention was made in view of the problems described above, and its purpose is to provide an antigen-binding protein that specifically recognizes the complex derived from MAGE-A4 peptide/HLA-A2.

[Methods for solving these problems]

[0006]

The MAGE-A4 molecule was used as the antigen presented by the MHC. MAGE-A4 is expressed in solid tumors including malignant melanomas (Non-Patent Document 3) and is a testicular cancer antigen (cancer testis antigen, CTA) of the NY-ESO-1 type. These molecules are expressed only in the testes and placenta and are not observed in other normal tissues. After MAGA-A4 expression in cells, it is degraded in proteasomes. Degradation leads to the formation of MAGE-A4 peptides containing 10 amino acids, which bind to HLA-A*0201, beta-2 microglobulin in the endoplasmic reticulum and are presented on the cell surface as an oncospecific antigen. As the peptide presented by HLA-A*0201, p230-239 (GVYDGREHTV: SEQ ID NO:1) (Non-Patent Document 4) was selected.

Methods have been developed to obtain CAR-T cells exhibiting a specific response to target cancer cells by transfecting human lymphocytes with a CAR receptor that specifically recognizes A2-MAGE-A4 and using cell infusion therapy in humans. (1) Antibodies recognizing the MAGE-A4 derived peptide/HLA-A2 complex with high affinity were isolated, (2) the specificity of these antibodies was tested, (3) antibodies having high specificity were screened, (4) CARs were obtained using these antibodies. -T cells that were specifically activated in response to cancer target cells and confirmed the induced cytotoxicity. In a cancer therapy model using a human tumor transplanted into mice infused with CAR-T cells in vivo, specific proliferation of CAR-T cells in the mouse body and tumor infiltration were observed. Thus, the present invention has essentially been completed.

[0007]

The antigen binding protein of the present invention is characterized in that it contains the following polypeptides (A) or (B) and recognizes the HLA-A2/MAGE-A4 complex: (A) a polypeptide containing the amino acid sequence of VH (heavy chain variable region) SEQ ID NO: 36 and the amino acid sequence of VL (light chain variable region) SEQ ID NO: 38; (B) a polypeptide comprising an amino acid sequence at least 90% homologous to the VH (heavy chain variable region) sequence of SEQ ID NO: 36 and an amino acid sequence at least 90% homologous to the VL (light chain variable region) sequence of SEQ ID NO: 38. At the same time, the antigen-binding protein preferably contains the following polypeptide (C) or (D) between the VH and VL regions: (C) a polypeptide containing the amino acid sequence of the sc fragment (single-stranded fragment) of SEQ ID NO: 37; (D) a polypeptide comprising an amino acid sequence comprising at least 90% of the sequence of an sc fragment (single strand) of SEQ ID NO: 37. In addition, the antigen binding protein preferably comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 32, or a polypeptide with at least 90% homology to the sequence of SEQ ID NO: 32. The antigen binding protein is preferably a Fab, Fab', F(ab') ₂ , Fv or a single chain Fv fragment (scFv). In the present application, the antigen-binding protein comprises an antibody or an antibody derivative.

[0008]

According to the present invention, homology means the relationship between two (or three or more) amino acid sequences or nucleotide sequences, determined by comparing them. Homology means the degree of correlation between amino acid sequences or nucleotide sequences, or between a group of parts of sequences when aligned. In particular, homology is defined by identity and retention of sequence properties (substitution to maintain the physicochemical properties of a particular amino acid or amino acid sequence within an amino acid sequence). The method for determining homology is preferably a method that matches the longest alignment between sequences. To determine homology, programs available on the Internet are used, for example, BLAST (Basic Local Alignment Search Tool) <https://blast.ncbi.nlm.nih.gov/Blast.cgi>. Preferred is a polypeptide that is at least 90% (preferably at least 95%, more preferably at least 98%) homologous to the sequence of SEQ ID NO: 32,36,37,38 and recognizes the HLA-A2- MAGE-A4.

According to another embodiment of the present invention, the nucleic acid encodes an antigen-binding protein in accordance with the first embodiment of the present invention.

According to another implementation variant of the present invention, the vector contains a nucleic acid, where the specified nucleic acid includes DNA or RNA. The nucleic acid may be single-stranded or double-stranded. The vectors include a plasmid vector and a viral vector, and the like.

According to another embodiment of the present invention, the chimeric antigen receptor comprises an antigen binding protein and an extracellular domain of a signal transduction protein, wherein the signal transduction protein is preferably any of CD3-zeta chains (CD3ζ) and CD co-stimulatory molecules (GITR). Preferred is the inclusion of any of the intracellular domain of CD28 and GITR.

According to another embodiment of the present invention, the nucleic acid encodes a chimeric antigen receptor. According to another embodiment of the present invention, the vector contains a nucleic acid.

According to another embodiment of the present invention, the cell expresses a chimeric antigen receptor. According to another embodiment of the present invention, the pharmaceutical composition contains the specified cell as an active ingredient.

[Effects according to the present invention]

[0009]

The present invention provides an antigen-binding protein specifically recognizing a MAGE-A4 derived peptide/HLA-A2 complex, nucleic acids encoding said antigen-binding protein, vectors containing said nucleic acids, chimeric antigen receptors binding the antigen-binding protein , nucleic acids encoding said chimeric antigen receptor, vectors containing said nucleic acids, cells expressing said chimeric antigen receptor, and pharmaceutical compositions containing said cells. An antigen binding protein specifically recognizing the MAGE-A4 derived peptide/HLA-A2 complex can be used in cell therapy and in the field of gene therapy. This protein is extremely useful for detecting tumor cells expressing the MAGE-A4 derived peptide/HLA-A2 complex for tumor treatment, research and tumor testing. Moreover, CAR-T cells according to the present invention can be used for effective cancer therapy because their side effects are small.

[SHORT DESCRIPTION OF GRAPHICS]

[0010]

Figure 1 shows the sequence diagram of scFV MAGE#17.

Figure 2 shows a gene sequence diagram to explain how CARs are constructed.

Figure 3 ((A) and (B)) shows histograms illustrating the results of determination by ELISA of the reactivity of the selected clones against multiple antigens.

Figure 4 shows a graph of the measurement of KD values for MAGE#17. (A) - shows a graph of dynamic changes in detection sensitivity over time, (B) - shows a table with the results of calculating KD values based on data obtained from the graph, respectively.

Figure 5(A) shows a graph of the results of evaluating the magnitude of the shift obtained when the interaction of antibodies MAGE#17, #86, #209, #345 and #672 with T2 cells injected with MAGE-A4p230, CMV and DMSO, (B ) - a histogram of the results is shown.

Figure 6 shows a graph of the results of the assessment of the number of HLA using the FACS method on the surface of T2 cells, which were injected with alanine-substituted peptides.

Figure 7 shows a graph of the results of amino acid assessment associated with antibody recognition when MAGE#17 interacted with T2 cells injected with alanine-substituted peptides.

The Figure 8 shows a graph of the results of the assessment of the amount of HLA using the FACS method on the surface of T2 cells, which were injected with mimic peptides.

Figure 9 shows a graph of the results of evaluating reactivity with mimic peptides when MAGE#17 interacts with T2 cells injected with said mimic peptides.

Figure 10 shows the results of tetramer staining of the expression of CAR (mRNA) introduced into peripheral blood mononuclear cells (PBMCs).

Figure 11 shows graphs of results showing that mRNA-transfected CAR-T cells specifically recognize target cells and increase IFNγ production.

Figure 12 shows the results of tetramer staining of the expression of the CAR gene introduced by a retrovirus into peripheral blood mononuclear cells (PBMCs).

Figure 13 is a graph showing that retrovirus-transfected CAR-T cells specifically recognize target cells and increase IFNγ production.

Figure 14 is a graph showing the activation of MAGE#17-CAR transfected T cells when co-cultured with T2 cells injected with the MAGE-A4 peptide.

Figure 15 is a graph showing the activation of MAGE#17-CAR transfected T cells when co-cultured with A2 positive MAGE-A4 positive cells.

Figure 16 is a graph of results showing an increase in interferon production in CAR-T cells when they are co-cultured with antigen presenting cells containing long MAGE-A4 peptides.

Figure 17 is a graph showing that T2 cells injected with the MAGE-A4 peptide have MAGE-A4 specific cytotoxic activity.

Figure 18 is a graph showing that target cells have specific cytotoxic activity against A2-positive MAGE-positive cells.

Figure 19 shows the results of CAR positivity score by tetramer staining for infused CAR-T cells.

Figure 20 (A) is a graph showing the results of the effect of CAR-T cells against an A2 positive MAGE-A4 positive tumor (NW-MEL-38), and (B) is a graph showing the results of the effect of CAR-T -cells against A2-positive MAGE-A4-negative tumor (HCT116).

Figure 21 shows the sequence diagram of the CAR zG genes.

The Figure 22 presents graphs showing the results of the effect of CAR-T cells containing various intracellular domains (intracellular domain, ICD), against A2-positive MAGE-A4-positive tumors (NW-MEL-38). (A) shows the size of the tumor, (B) shows the body weight of the animals, respectively. The arrows on the left of the graphs show the day of total body irradiation (TOT) (day 3), the arrows on the right show the day of injection of lymphocytes (day 4), respectively.

Figure 23 is an image showing tetramer staining results of CAR positivity ratio for CD8-positive T cells not injected with CAR (NGMC: control), CD8-positive T cells injected with CAR type zG (zG) , and CD8-positive T cells injected with CAR type 28z (28z).

Figure 24 is a graph showing the results of assessing the specific recognition of target cells by CAR-T cells and the increase in IFNγ production.

Figure 25 is a graph showing the results of assessing the specific recognition of target cells by CAR-T cells and the increase in IFNγ production.

[DESCRIPTION OF THE INVENTION]

[0011]

The following is a description of embodiments of the present invention with reference to figures and tables. The technical scope of the invention is not limited to these embodiments, and the present invention can be embodied in various forms without changing the gist of the present invention.

In this application, the terms "antibody" and "antigen-binding fragment" refer to an antigen-binding protein in the immune system. An antibody containing an antigen-binding site is a glycoprotein containing two heavy chains (H chains) and two light chains (L chains) linked to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). VL consists of CL domain. The VH and VL regions can be further divided into complementarity determining regions (CDRs) with hypervariable and framework regions (FRs) containing a somewhat conserved sequence. In the VH and VL regions, the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, i.e. three CDRs and four FRs are located in the direction from the amino-terminus to the carboxy-terminus, respectively. CDR3 are known to be important for antigen binding. Both VH and VL contain binding domains that interact with the antigen.

[0012]

In this application, the term "HLA" means a human leukocyte antigen (human leucocyte antigen, HLA) and refers to the molecules of the major histocompatibility complex (MHC) of a person and the complex of genes encoding cell surface molecules that are necessary for presentation antigen to cells of the immune system. HLA can be divided into class I and class II molecules. HLA class I consists of an α-chain and β2-microglobulin. HLA class I is expressed in virtually all nucleated cells and functions by antigen presentation to CD8-positive T cells. HLA class I can be divided into HLA-A, HLA-B and HLA-C.

In this application, the term "chimeric antigen receptor (CAR)" means a fusion protein (fusion protein) containing an extracellular domain that binds to an antigen, a transmembrane domain other than the extracellular domain, and at least one of the extracellular domains. The CAR may be referred to as "chimeric receptor", "T-body", or "chimeric immune receptor (CIR)". "Extracellular domain that binds to an antigen" means any oligopeptide or polypeptide that binds to an antigen, "intracellular domain" means any oligopeptide or polypeptide known to function as a signal transduction domain that activates or suppresses a biological process in cell.

As used herein, "antigen-binding protein" means the part of an antibody that binds to an antigen, or the fragment of an antibody that confers antigen specificity to the antibody.

[0013]

The following is a detailed description of the present invention.

(1) Antigen-binding fragment and nucleic acid encoding the same according to the present invention

An anti-MAGE-A4-derived peptide/HLA-A2 complex antibody (HLA-A2-MAGE-A4) is an antibody that specifically recognizes and binds to the complex of HLA-A2 and P230-239 (SEQ ID NO: 1), which is is a peptide derived from the restricted HLA-A2 antigen MAGE-A4. The antibody of the present invention is highly specific, does not bind to the complex of HLA-A2 and non-P230-239 peptide and to the complex of P230-239 and non-HLA-A2 peptide (or binds with very low activity). Thus, an antigen-binding fragment according to the present invention can specifically detect or be directed to the HLA-A2-MAGE-A4 complex.

The antigen-binding fragment of the present invention comprises (A) a polypeptide having the amino acid sequence of VH (heavy chain variable region) SEQ ID NO: 36 and the VL (light chain variable region) amino acid sequence of SEQ ID NO: 38, or (B) a polypeptide, having a VH (heavy chain variable region) amino acid sequence at least 90% homologous to SEQ ID NO: 36 and a VL (light chain variable region) amino acid sequence at least 90% homologous to SEQ ID NO: 38. The antigen-binding fragment of the present invention may comprise (C) a polypeptide having the amino acid sequence of the sc fragment (single-strand fragment) of SEQ ID NO: 37, or (D) a polypeptide having the amino acid sequence of the sc fragment (single-strand fragment) 90 homologous % or more of the sequence of SEQ ID NO: 37, between the VH and VL regions.

[0014]

An antigen-binding fragment contains one or more fragments that specifically bind to an antigen. An antibody fragment containing the antigen-binding site of an antibody specifically recognizes the antigen like a full-length antibody. Examples of antigen-binding fragments are antibodies and their fragments containing Fab, Fab', F(ab') ₂ , Fv and single chain Fv fragment (scFv). Fab is a fragment of a monovalent antibody, consisting of domains VL, VH, CL and CH1. Fab' is a fragment of a monovalent antibody, consisting of domains VL, VH, CL, CH1 and a hinge region. F(ab') ₂ is a divalent antibody fragment containing two Fab fragments linked by hinge disulfide bonds. Fv is a monovalent antibody fragment consisting of VL and VH domains. The VL and VH domains of the Fv fragment are encoded by separate genes, scFv, which is a single chain protein, can be generated using gene recombination techniques by combining two genes with a linker. scFv contains a linker (single strand) between VH and VL. sc binds the VH and VL domains and is a peptide commonly used in the art to stabilize the antigen-binding ability of an scFv antibody (see, for example, Huston et al, Methods in Enzymology, 203: 46-88 (1991), Whitlow et al, Protein Eng., 6: 989 (1993)). sc as a whole contains glycine and serine and is 15 to 18 amino acids long.

[0015]

One aspect of the present invention includes a combined antigen-binding fragment. Examples of such molecules are Fab3, diabody, triatelo, tetrabody, minibody, Bis-scFv, (scFv)2-Fc, intact IgG (Holliger et al., Nature Biotechnology, 23(9), p,1126-36 (2005)) .

One or more amino acids of the antigen-binding fragments may be modified, provided that such modification does not substantially affect the properties of said antigen-binding fragments. For example, one or more amino acids in the constant region or FR region may be replaced, deleted, added or inserted. The modification can be easily carried out by a combination of known methods, including site-directed mutagenesis (point mutation introduction and cassette mutagenesis, etc.), homologous gene recombination method, primer extension method, and PCR method.

The present invention includes a nucleic acid encoding an antigen-binding fragment. Additionally, the nucleic acid according to the present invention contains the nucleotide sequence shown in SEQ ID NO: 31 and 33-35 in the sequence listing. A nucleic acid can be codon-modified (codon optimization) to produce codons suitable for the host cell without changing the amino acid sequence encoded by said nucleic acid. The expression efficiency of a polypeptide in a host cell can be improved by codon-optimized modification.

The antigen-binding fragment according to the present invention can be obtained using known methods of genetic engineering or methods of chemical synthesis. According to the genetic engineering method, by obtaining a cloning or expression vector containing a nucleic acid according to the present invention, introducing said vector into a host cell, culturing said host cell expressing the nucleic acid, and recovering the polypeptide by purification, a polypeptide can be obtained. When multiple nucleic acids are included according to the present invention, a combination of multiple vectors each containing a nucleic acid molecule or a single vector containing multiple nucleic acids can be introduced into the host cell. If a peptide tag is attached to a polypeptide upon receipt, said polypeptide can be recovered and purified using said peptide tag.

[0016]

The vector of the present invention is operably linked to suitable control sequences for the expression of nucleic acids in a suitable host. Such control sequences include a promoter for transcription of nucleic acids, an operator sequence for controlling transcription, a sequence encoding a ribosome binding site, an enhancer, a polyadenylation sequence, and a sequence for controlling transcription and translation termination, and so on. Various known sequences (eg, a restriction enzyme cleavage site, a marker gene such as a drug resistance gene (selection gene), a signal sequence, a leader sequence, etc.) can be used in the vector. Various sequences can be selected and used appropriately according to the type of polypeptide being expressed, host cell, environmental conditions, and so on.

According to the present invention, a vector inserted or not inserted into the host genome, and an autonomously replicating episomal cytoplasmic vector, etc. can be used. An example of a vector is retroviral vector (including oncoretroviral vectors, lentiviral vectors, pseudotyped vector), adenovirus vector, adeno-associated viral (AAV) vector, simian virus vector, vaccinia vector, Sendai virus vector, virus vector Epstein-Barr (EBB), HSV-based vector, etc. As a viral vector, a virus that lacks the ability to replicate in infected cells can preferably be used. In addition, a non-viral vector can also be used by combining it with a liposome and a condensing agent such as cationic lipids. Moreover, the nucleic acid can be introduced into cells by transfection using calcium phosphate, DEAE-dectran, electroporation, particle bombardment, and the like.

[0017]

Known cells can be used as the host cell. Thus, prokaryotic cells such as E. coli cells, mammalian cells such as Chinese hamster ovary (CHO cells), human cells, and eukaryotic cells such as yeast and insect cells, etc. can be exemplified.

An antigen binding fragment of the present invention that is expressed in a host cell can be purified from media obtained from the host cell, extracts and/or lysate of the host cell. The purification process can be carried out by combining suitable known methods. For example, centrifugation, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin-sepharose chromatography, anionic or cationic resin chromatography (column with polyaspartic acid, etc.), chromatofocusing, SDS-PAGE electrophoresis, precipitation with ammonium sulfate and affinity chromatography.

[0018]

(2) Composition according to the present invention

The present invention provides a composition comprising an antibody or an antigen-binding fragment according to the present invention described in section (1), as well as a composition containing a nucleic acid encoding said antibody or antigen-binding fragment.

An antibody or antigen-binding fragment of the present invention can be used as a marker for a MAGE-A4 derived peptide/HLA-A2 complex. MAGE-A4 is known to be expressed in cancer cells and presented with HLA. T cells attack cancer cells as a result of antigen recognition. Thus, the antigen-binding fragment according to the present invention can be used as a cancer cell marker, i.e. compositions for detection or diagnosis.

Examples of such detection methods are known detection methods such as ELISA, antibody fluorescence assay, radioimmunoassay, radioimmunoprecipitation, dot blot assay, inhibition or competition assay, sandwich assay, and latex bead agglutination assay.

Specimens for diagnosis and detection include biological samples (eg, tissue from the affected area, cells, body fluid containing blood, serum, plasma, lymph, and urine). In these samples, after preliminary purification, homogenization, centrifugation or dilution in a suitable buffer (if necessary), interaction with the antigen-binding fragment according to the present invention, an antigen-antibody complex is detected. In some cases, the antigen-binding fragment according to the present invention may be labeled, or a labeled secondary antibody may be used. The label can be an enzyme (such as horseradish peroxidase, alkaline phosphatase, etc.), radioactive isotopes (such as ³² P, ³⁵ S, ³ H, ¹²⁵ I, etc.), a fluorescent substance (such as rhodamine , fluorescamine, dansyl chloride and their derivatives, etc.) and a peptide label, etc.

[0019]

The antigen binding fragment of the present invention can be used to deliver a pharmaceutical composition to a target. An antibody or antigen-binding fragment according to the present invention, associated with a pharmaceutical agent, specifically recognizes the complex of the WT1 peptide and HLA-A24. Examples of a pharmaceutical composition for delivery to a target are cytokines (IL2, TNF, IFN-γ, etc.), their receptor proteins, cytotoxin (ricin, diphtheria toxin, gelonin, etc.), radioactive isotopes ( ⁹⁰ Y, ¹³¹ I, ²²⁵ Ac, ²¹³ Bi, ²²³ Ra and ²²⁷ Th, etc.), cells (T cells, NK cells, etc.) or low molecular weight compounds (calichimicin, doxorubicin, etc.).

The effective amount of the active ingredients of the pharmaceutical composition according to the present invention is appropriately determined depending on the purpose of treatment, the type of tumor, the medical condition, such as the area of the lesion and the size of the lesion in the patient, and the route of administration, etc. When used as a pharmaceutical composition, in addition to the active ingredient, various components may be added which may be pharmaceutically acceptable (eg, carrier, excipient, buffer, stabilizer, etc.). The pharmaceutical composition, depending on the state, can be presented in the form of a tablet, liquid substance, powder, gel, spray, microcapsule, colloidal distribution system (liposomes, microemulsions, etc.) and macroemulsions, etc. An example of the method of administration is administration as a pharmaceutical composition, intravenous, intraperitoneal, intracerebral, intrathecal, intramuscular, intraorbital, intraarterial, intralesional injection and injection through the bile duct, infusion, use of dosage forms in the form of sustained release systems, etc. The pharmaceutical composition can be administered by continuous infusion or injection.

The pharmaceutical composition of the present invention can be used to treat hematopoietic tumors such as chronic myeloid leukemia, acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML), and solid tumors such as gastric cancer, colon cancer, lung cancer, breast cancer. gland cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, mesothelioma, etc., as well as for the diagnosis and detection of tumors and solid tumors. In particular, said pharmaceutical composition is useful for treating, diagnosing and detecting MAGE-A4 positive or HLA-A2 positive hematopoietic tumor and solid tumor.

[0020]

(3) Chimeric antigen receptor of the present invention

The present invention includes a chimeric antigen receptor (CAR) containing an antigen-binding fragment from section (1) and an intracellular domain of a signaling pathway protein. CAR specifically recognizes and binds to the HLA-A2/MAGE-A4 derived peptide complex and can signal to cells expressing CAR. CAR contains an extracellular domain, a transmembrane domain, and an extracellular domain. The extracellular domain contains the antigen-binding fragment according to the present invention. When the extracellular domain specifically binds to the HLA-A2/MAGE-A4 complex, a signal is transmitted into the cell via the intracellular CAR domain and stimulates cells expressing CAR. Stimulated cells produce cytokines, etc., exhibit cytotoxicity against target cells expressing the complex, or induce cytotoxicity of other cells of the immune system.

As the extracellular domain of CAR, a domain can be used that can signal to a cell when the extracellular domain of the CAR molecule interacts (binds) with the MAGE-A4 derived peptide/HLA-A2 complex. Examples of such signaling pathway proteins are membrane proteins, signaling receptors, and cytokine receptors. Examples of an intracellular CAR domain are intracellular domains containing a primary cytoplasmic signal transduction sequence derived from CD3-zeta (CD3ζ), FCRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a and CD66d. Additionally, examples are intracellular domains containing a secondary cytoplasmic signal transduction sequence (costimulatory signal) derived from CD2, CD4, CD5, CD8α, CD8β, CD28, CD137 (also known as "4-1BB"), CD134, ICOS, GITR, and CD154. Moreover, intracellular domains containing a tertiary cytoplasmic signal transduction sequence derived from the IL-2 receptor and the IL-21 receptor are examples.

[0021]

You can use the transmembrane domain of CAR, the alpha chain or beta chain of the T-cell receptor, the transmembrane domain of the zeta chain of CD3, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (also known as "4-1BB"), ICOS, CD154 and GITR, etc. You can use an artificially constructed sequence.

The present invention includes a nucleic acid encoding a CAR. A nucleic acid encoding a CAR may be linked to another nucleic acid expressed under the control of a suitable promoter. You can use a promoter that is expressed constitutively or whose expression is induced by a drug (eg, tetracycline or doxorubicin). For example, a mammalian-derived promoter, including the phosphoglycerate kinase (PGK) promoter, the Xist promoter, the beta-actin promoter, and the RNA polymerase II promoter, a virus-derived promoter, including the early SV40 virus promoter, the cytomegalovirus promoter, the herpes simplex virus thymidine kinase promoter, can be used. , the LTR promoter of various retroviruses, etc. To achieve efficient transcription of the nucleic acid, another regulatory element may be linked to said nucleic acid that interacts with a promoter or transcription initiation site (eg, an enhancer sequence or a terminator sequence). Moreover, it is possible to insert a marker gene that can be used to confirm the expression of a nucleic acid (for example, a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein, etc.).

[0022]

A nucleic acid encoding a CAR can be introduced into cells using a vector. Moreover, a nucleic acid encoding a CAR can be used as an active ingredient in a pharmaceutical composition. A pharmaceutical composition containing a nucleic acid encoding a CAR can be prepared and used as described in section (2).

According to another aspect, the present invention includes a cell that expresses CAR. These cells can be obtained using the process of introducing CAR into the cell according to the present invention. When cells expressing a CAR bind to the HLA-A2/MAGE-A4 complex via said CAR, a signal is transmitted to the cell and the cell is activated. Cell activation can be confirmed by cytokine release, increased proliferation rate, cell surface molecular changes, etc., depending on the host cell type and intracellular domain. For example, release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells results in the destruction of tumor cells expressing the complex. Additionally, the release of cytokines and alteration of cell surface molecules stimulates other cells of the immune system, such as B cells, dendritic cells, NK cells, macrophages, etc. Thus, CAR expressing cells are useful for adoptive immunotherapy, especially directed at MAGE-A4 positive HLA-A2 positive tumors or cancers.

[0023]

The process of introducing a CAR-encoding nucleic acid into cells is carried out in vitro (ex vivo) or in vivo. As the cells into which the nucleic acid is introduced, mammalian cells such as cells of human origin and cells derived from non-human mammals including monkey, mouse, rat, pig, cow and dog, etc. can be used. Cell types that can be used are, for example, cells of body fluids such as blood (peripheral blood, cord blood, etc.), bone marrow cells, tissue or organ cells collected, isolated, purified and induced. Can be used PBMCs, immune system cells (dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes or NK cells), blood cells (neutrophils, basophils, monocytes), hematopoietic stem cells, cord blood mononuclear cells, fibroblasts, preadipocytes , hepatocytes, blood cells, dermal keratinocytes, mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, pluripotent stem cells, various cancer cell lines, or neural stem cells. According to the present invention, particularly T cells, T cell progenitors (hematopoietic stem cells, lymphoid progenitors, etc.), pluripotent stem cells or cell populations containing them are preferably used. T cells include CD8 positive T cells, CD4 positive T cells, regulatory T cells, cytotoxic T cells, tumor infiltrating lymphocytes. The cell population containing T cells and T cell precursors includes peripheral blood mononuclear cells (PBMCs). Cells useful according to the present invention may be any of cells selected from a living body, propagated by cultivation, and cell lines. When it is intended to transplant cells transfected with the nucleic acid or cells differentiated therefrom into a living body, cells derived from said living body or a living body of the same type are preferably transplanted.

[0024]

Cells expressing the CAR of the present invention may be cultured and/or stimulated with a suitable culture medium and/or stimulatory molecules prior to administration to a subject. Stimulant molecules include cytokines, appropriate proteins, and other components. Examples of cytokines are, for example, IL-2, IL-7, IL-12, IL-15 and IFN-γ, etc., preferably IL-2 is an example. The concentration of IL-2 in the medium is, for example, 0.01 - 1×10 ⁵ U/ml, preferably 1 - 1×10 ⁴ U/ml, but is not limited to specific values. In addition, examples of suitable proteins are CD3 ligand, CD28 ligand and anti-IL-4 antibody. In addition, a lymphocyte stimulant such as lectins may be added. In addition, serum or plasma can be added to the culture medium. The amount of additions to the medium is, for example, 0 to 20% by volume, but is not limited to specific values, the amount of serum or plasma can be changed according to the stage of cultivation. Serum or plasma concentrations can be reduced in steps. Serum or plasma may be intrinsic (of the same origin as the cultured cells) or non-intrinsic (of a different origin than the cultured cells). Preferably proprietary components are used for security.

Equipment that can be used for cell culture is, for example, a petri dish, a vial, a bag, a large culture bath, a bioreactor, etc., but is not limited to specific options. A CO ₂ gas permeable cell culture bag can be used as a bag. In the industrial production of cell populations in large quantities, a large culture bath can be used. In addition, cultivation can be carried out in an open system or a closed system, preferably a closed system is used for the safety of the resulting cell population.

[0025]

The present invention includes a pharmaceutical composition containing cells expressing CAR as an active ingredient. The pharmaceutical composition according to the present invention is administered parenterally. As parenteral administration, intravenous, intra-arterial, intramuscular, intraperitoneal and subcutaneous administration, etc. are included. To enhance the antitumor effect, administration to the meningioma or nearby tissue, such as subcutaneous administration, can be used. The dose to be administered is appropriately selected depending on the condition, body weight, age of the subject, and so on. Typically, cells are administered in an amount of 10 ⁷ - 10 ⁹ cells per 60 kg of body weight per administration, preferably about 5×10 ⁷ - 5×10 ⁸ cells. The pharmaceutical composition may be administered one or more times. The pharmaceutical composition may be presented in a form suitable for parenteral administration, such as injection or infusion. The pharmaceutical composition may optionally contain a pharmaceutically acceptable excipient. The pharmaceutical composition may contain saline, phosphate buffered saline (PBS), media, etc. for cell stability. In general, media are not limited to specific examples, and include media such as RPMI, AIM-V, X-Vivo 10, and so on.

[0026]

<Test method>

1. Antibody screening with a human antibody phage library

(1) Preparation of p-MHC complex-bound beads

20 μg of A2/MAGE-A4 complex as p-MHC and 200 mg of streptavidin-bound magnetic beads were mixed and reacted at 4° C. in 0.05% Tween/PBS(-). 3 μl of 2 mM biotin/PBS(-) was added and reacted for 1 hour. The solution was washed twice with 0.05% Tween/PBS(-) and suspended in 400 μl of 0.05% Tween/PBS(-). The suspension was used as antigen-containing beads.

(2) Human antibody library

VH and VL were amplified by PCR from tonsil tissue, cord blood, peripheral blood, and bone marrow from dozens of healthy subjects. The PCR products were inserted by recombination into the pUC119 phagemid vector to create scFv with a repertoire of 3.4×10 ¹² . scFv was presented on cp3 of the M13 phage. The product was used as a human antibody library.

[0027]

(3) Screening with human antibody library, antibody isolation, obtaining cp3-containing culture supernatant

A reaction mixture was prepared containing a solution containing 3.4×10 ¹² cfu of human antibody libraries, 100 μg of streptavidin (Pierce), 30 μg of A2-CMV tetramer, 30 μg of A2-Foxp69 tetramer, 30 μg of A2-IDOp41 tetramer, 30 μg of tetramer A2-IDOp195, 20 µg Gamma guard reagent, 100 µl 2% BSA solution, 65 µl 10% Triton X100 solution, 1000 µl 1% Triton X100/PBS solution, and mixed and swirled for 1 hour at room temperature and 100 µl added solution of magnetic beads associated with tetramer A2/MAGE-A4, and mixed and twisted for 1 hour. The magnetic beads were then captured using a magnetic trap (Toyobo) with five washings with 1% Triton X100/PBS. Magnetic beads were added to cultured XL1-blue cells, infected for 1 hour at 37°C, centrifuged and suspended in 600 μl of 2×YTAG medium (200 μg/ml ampiillin, 1% glucose/2×YT). The suspension was placed on a YTAG agar plate (200 μg/ml ampicillin, 2% glucose/nutrient agar (Nissui)), and cultured for 15 hours at 37°C. Colonies were collected with 30 ml of 2×YTAG medium, 200 µl of the collected solution was added to 20 ml of 2× YTAG medium and 30 µl of VCSM helper phage was added. The mixture was incubated for 30 minutes at 37°C for infection and incubated for 90 minutes at 37°C. 80 ml of 2x YTAG medium, 60 μl of kanamycin at a concentration of 50 μg/ml and 50 μl of 1M IPTG were added to the cultured mixture and incubated for 20 hours at 28°C. After centrifugation of the incubated mixture, the supernatant was mixed with 25 ml of PEG solution (20% PEG#600, 2.5M NaCl). The precipitate was collected by centrifugation, suspended in 1 ml of PBS and sterilized with a filter. The operation was repeated twice. Reconstituted E. coli were placed on YTAG agar medium. Then incubated at 30°C, the colonies were collected and cultured in LB medium at 30°C overnight. DNA was extracted with a Miniprep DNA kit (QIAGEN) using a portion of the LB culture medium and sequenced. 50 μl culture was mixed with 1.5 ml 2×YTAI (2×YT medium containing 0.5 mm IPTG) and cultured at 30°C overnight. The supernatant was separated by centrifugation. The supernatant was used as the cp3-containing supernatant. Conducted ELISA by using cp3-containing supernatant.

[0028]

2. Enzyme immunoassay (ELISA)

(1) MHC-peptide complex (monomer)

Complex resulting from the binding of HLA-A*0201 to MAGE-A4p230, MAGE-A4 P286, MAGE-A3 P195, CMV, MelanA, GP3, HTLV-1, NY-ESO-1 p157, NY-ESO-1 p159, Foxp3 p69, Foxp3 p127, Foxp3 p390, IDO p41, IDO p41, IDO p195 and IDO p199 were used as the MHC-peptide complex.

(2) Immobilization of p-MHC

500 ng of neutravidin (PIERCE Co.) in 50 μl of PBS were suspended in a Maxisorp loose plate (Nunc) and shaken at 4° C. overnight. After removal of the solution, 200 μl of a 2% BSA/PBS solution was added, the solution was left to block overnight. After removal of the solution, 300 ng of HLA-A2/MAGE-A4 monomer in 50 μl of PBS was added and shaken at 4° C. overnight. The solution was washed with PBS.

Immobilization of HLA-A2/CMV as a negative control was performed in the same manner.

(3) Measurement of ELISA reaction

100 μl of the supernatant of the cp3-containing culture was poured into the well of the plate with the immobilized antigen, after shaking for 1 hour at room temperature, the well of the plate was washed with PBS, 100 μl of rabbit antibodies against cp3, diluted 2000 times with 0.05% Tween 20 solution, were poured into the well. /PBS, and the tablet was shaken for 1 hour at room temperature. Then the well was washed with PBS, 100 μl of horseradish peroxidase (HRP) labeled antibodies against rabbit IgG (MBL Co., Ltd.) at 4000-fold dilution in 0.05% Tween 20/PBS was poured into the well, and the plate was shaken in for 1 hour at room temperature. The well was then washed with PBS, OPD (WAKO Co., Ltd.) was suspended in a solution containing 0.01% H ₂ O ₂ , 0.1 M Na ₂ PO ₄ , 0.1 M citric acid (pH5.1), poured out and subjected to reaction, after the appearance of color was confirmed, 2N H ₂ SO ₄ was added to stop the reaction. Absorbance was measured at 490 nm using a SpectraMaxM2 reader (Molecular Devices).

[0029]

3. Antibody purification

(1) Transfer (transfection) into a vector for purification

The antibody gene was transferred to an expression vector containing a His/FLAG tag to obtain said gene in the form of scFv-FLAG/His. The FLAG tag was used for detection using the Fluorescence-Activated Cell Sorting (FACS) method, the His tag was used for column purification, respectively.

(2) Transfection

After the cloned DNA of the isolated antibody was treated with SalI (Takara Bio Inc.), the DNA fragment was subjected to interaction with the vector using T4 DNA ligase. Competent DH5α cells (TOYOBO Co. Ltd.) were transfected with DNA and plated on an LBGA plate. After incubation at 30° C. overnight, the colonies were cultured in 2x YTAG medium and part of the culture solution was cultured in 2x YTAI medium, the supernatant was obtained by centrifugation.

(3) Confirmation of scFv-FLAG/His expression

After 100 μl of the culture supernatant was poured into the well of the antigen plate, the plate was shaken for 1 hour at room temperature and the well was washed with PBS. To confirm the expression of His-tags, horseradish peroxidase (HRP)-labeled anti-His antibodies (MBL Co., Ltd.) were poured in a 4000-fold dilution in 0.05% Tween 20/PBS solution and shaken for 1 hour at room temperature. temperature. To confirm the expression of the FLAG tag, a reaction was performed in turn with anti-FLAG(DDDK) antibodies and HRP-labeled antibodies.

After washing with PBS of the antigen-bound plate, OPD (WAKO Co., Ltd.) suspended in a solution containing 0.01% H ₂ O ₂ , 0.1M Na ₂ PO ₄ , 0.1M citric acid (pH 5 ,1) and subjected to interaction. After color appearance was confirmed, 2N H ₂ SO ₄ was added to stop the reaction. Absorbance was measured at 490 nm using a SpectraMaxM2 reader (Molecular Devices).

[0030]

(4) Coarse purification of the antibody

2 ml of the bacterial solution after pre-culture was placed in 100 ml of 2×YTAI and cultured at 30° C. overnight. The solution was centrifuged (8000 rpm, 10 min, 4°C), the supernatant was collected, 100 ml of saturated sulfuric acid was added to this supernatant and stirred. The solution was centrifuged (8000 rpm, 20 min, 4°C) and suspended in 10.5 ml of PBS solution. After centrifugation of the PBS solution (12,000 rpm, 1 h, 4°C), 1 ml of nickel-sepharose medium (Ni Sepharose excel, GE Healthcare) equilibrated with PBS was added to the supernatant and incubated at 4°C for nights. The column was loaded with sample, washed with 0.5 M NaCl-PBS containing 0.5 M NaCl-PBS and 20 mM imidazole, and eluted with 0.5 M NaCl-PBS containing 250 mM imidazole. The eluent was dialyzed against PBS, after dialysis it was concentrated using an Amicon Ultra 10K concentrator (Millipore). A portion of the sample was subjected to SDS-PAGE to confirm band presence by Coomassie Brilliant Blue (CBB) staining and to determine the protein concentration of the crudely purified antibody.

(5) His tag protein purification using the AKTA Prime plus system

The antibody after coarse purification was further purified using an AKTA Prime plus chromatography system (GE Healthcare) using a HisTRAP column. The procedure was carried out according to the purification protocol using the attached His-tag. The recovered peak fractions were detected by SDS-PAGE electrophoresis.

[0031]

4. Measurement of the dissociation constant (KD values)

The KD values of the purified antibodies were measured using the BiacoreX100 system (GE Healthcare). Ligands were immobilized using a Biotion capture kit (GE Healthcare).

(1) Ligand immobilization

HLA-A2/MAGE-A4p230 was immobilized on the CAP sensor chip. The concentration was adjusted to 200 nM with HBS buffer and the ligand was immobilized as a suitable PE (resonance unit).

(2) Obtaining the analyzed solution (analyte solution)

The concentration of purified antibody was adjusted to 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM with HBS EP + buffer.

(3) Measurement of KD values

The binding reaction was measured for 180 seconds as the analyzed solution passed through the flow cell of the sensor chip. After the measurement, the analyzed solution was replaced with HBS buffer and the dissociation reaction was measured for 300 seconds. The measurement was carried out using the single-cycle method of the program for a continuous flow of the analyzed solution from low concentration to high concentration.

After completion of the reactions with all concentrations, the binding constant was calculated using a point curve fitting program.

[0032]

5. Analysis by flow cytometry (FACS)

(1) Hardware

The FACS Calibur/FACS Cant/FACS CantII (BD) system was used for flow cytometry measurement.

(2) Fluorescently labeled antibody

Fluorescently labeled antibodies were used as described in Table 1.

[0033]

[Table 1]

(3) PV tetramer

PE-tetramer means streptavidin-phycoerythrin (PE) to which four biotinylated monomers are attached. The PE tetramer was used to detect antigen-specific T cells. The tetramers used in the application were the A2/MAGE-A4p230 tetramer and the A2/NY-ESO-1 p157 tetramer.

[0034]

6. HLA stability assay and scFv binding assay

(1) T2 cells (hybrid human lymphoblastic cell line HLA-A*02 ⁺ BT)

T2 cells are a type of cell line that lacks Transporter Associated with Antigen Processing (TAP) and lacks intracellular protein presentation with HLA.

(2) Introduction of the peptide into T2 cells

The peptide was added to T2 cells to achieve a final concentration of 10 μM in RPMI1640 medium for human cells, left for 15 minutes at room temperature, RPMI1640 for human cells containing 20% EBS was added to achieve a final concentration of EBS of 10%. Cells were incubated in CO ₂ -incubator for 45 min at 37°C. After two washes, the cells were used for analysis.

(3) HLA stability analysis

Anti-HLA-A2 antibodies coupled to Alexa Fluor 488 were reacted for 1 hour with T2 cells injected with the peptide. The amount of HLA stabilized on the surface of T cells was determined by detection using a fluorescence activated cell sorting (FACS) method.

[0035]

(4) scFv binding assay

T2 cells injected with the peptide were reacted with the obtained antibodies for 1 hour, with anti-FLAG antibody for 1 hour, with FITC-conjugated anti-mouse IgG antibody for 1 hour, and detected by FACS.

(5) Peptides with one amino acid substitution and mimic peptides

As shown in Table 2, peptides with one amino acid substitution were synthesized, where one of the 10 amino acids MAGE-A4p230-239 was replaced by alanine (A), methionine (M), phenylalanine (F), and tryptophan (W), and the concentration value was evaluated half maximal inhibitory concentration (IC50) for each resulting peptide. The concentration of each peptide was adjusted to a final concentration of 10 mM in DMSO.

The IC50 for MHC is a theoretical value calculated using the Immune Epitope Database (IEDB, https://tools.immuneepitope.org/processing/). The lower the value, the stronger the relationship between HLA-A*0201 and the peptide.

Table 2 shows the amino acid sequence of peptides with one amino acid substitution and IC50 values.

[0036]

[Table 2]

Table 3 shows amino acid sequences and IC50 values for mimetic peptides that were selected by BLAST search from MAGE family peptides and amino acids recognizing the MAGE#17 antibody.

[0037]

[Table 3]

[0038]

7. Creation of the CAR construct and vector

(1) MAGE#17-28z CAR

Figure 1 shows the sequence of the CAR construct. The construct contains the leader sequence, VH, VL, CL, CD28TM, CD28ICD (CD28 intracellular domain) and CD3-zeta (CD3ζ) chains from the 5' end. VH and VL are the MAGE#17 scFv fragment in the indicated chains.

(2) CAR design

Figure 2 schematically shows the steps involved in constructing a CAR. For clarification, each step was as follows.

First, a PCR reaction was performed using scFv-cp3 as a template, and a leader sequence was added with an EcoRI recognition site at the 5' end of VH-VL and CL with an AscI recognition site at the 3' end, respectively. Then, the fragment of the PCR product treated with EcoRI and AscI and the fragment of the CAR 28z vector treated with EcoRI and AscI were subjected to interaction with ligase to obtain CAR 28z.

[0039]

8. Obtaining human lymphocytes

Human lymphocytes were obtained from the blood of a healthy donor using Ficoll-PaqueTM PLUS solution (17-1440-03, GE Healthcare). These lymphocytes were obtained by separation of peripheral blood mononuclear cells (PBMC). Collection of samples containing human peripheral blood used in the study and analysis were performed in accordance with the Declaration of Helsinki and in accordance with the protocol approved by the Ethics Committee of the Faculty of Medical Research at Mie University with the written consent of the subjects.

Collected samples were stored in a freezer or liquid nitrogen vessel in encrypted form to prevent the identification of subjects and as a measure to protect against data theft. The personal information of the subjects was anonymized, with particular attention paid to the privacy of personal data and the prevention of data leakage of gene analysis results.

[0040]

9. Introduction of mRNA into cells by electroporation

(1) Obtaining mRNA

The CAR DNA was cut into single stranded molecules to obtain a linear DNA template. DNA was purified using the Wizard SV Gel DNA Purification System and the PCR Clean-UP DNA Purification System (Promega). mRNA was obtained from purified DNA using the mMESSAGEmMACHINE T7 Ultra kit (Life Technologies).

(2) Verification with the Bioanalyzer system

The addition of polyA to mRNA was confirmed using a Bioanalyzer system (Agilent Technologies).

(3) Obtaining a culture of PBMCs in a plate

Anti-CD3 antibodies (OKT-3, eBioscience) were diluted with ACD-A anticoagulant solution (TERUMO) to reach a final concentration of 5 µg/ml, RetroNectin reagent (Takara Bio) was diluted with ACD-A solution to reach a final concentration of 25 µg/ml, 400 μl of both solutions were poured into each well of a 12-well plate (Nunc) and incubated for 5-10 hours at 37°C in a CO ₂ incubator.

[0041]

(4) Obtaining CM (culture medium)

A PBMC culture was prepared containing 50 ml of GT-T503 lymphocyte medium, 50 μl of human IL-2 (600 IU), 400 μl of Albuminar solution (25% human serum albumin, PSA) and 300 μl of donor plasma lymphocytes.

(5) Isolation and cultivation of PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of a healthy person using Ficoll solution and suspended in CM at a concentration of 2×10 ⁵ cells/ml, 2.5 ml of the suspension was poured into a plate and cultured for 4 days at 37°C in CO ₂ - incubator.

(6) Electroporation

The resulting mRNA was introduced into PBMCs by electroporation 10 days after cell isolation. mRNA-introduced PBMCs were used for analysis within 24 hours.

[0042]

10. Transfer of genes into cells using a retrovirus (experiment on infection)

(1) Production of retrovirus with Plat-A packaging cells

The CAR gene was introduced into plat-A packaging cells using the FuGENE reagent (Promega). After incubation for two days, the supernatant was recovered and used as a virus solution.

(2) Isolation and cultivation of PBMCs

Isolation and cultivation of PBMCs was carried out in accordance with the above method in section 9, (3)-(5).

(3) Obtaining a tablet for retroviral infection

The RetroNectin reagent was diluted with ACD-A solution at a final concentration of 20 µg/mL. 500 μl of the solution was poured into each well of a 24-well plate and stored at 4° C. overnight. The plate was washed twice with ACD-A before loading the virus solution.

[0043]

(4) Obtaining a tablet containing a retrovirus for infection, infection of cells

The virus solution prepared above (10.(1)) was poured into a 1 ml volume plate for retroviral infection, and the plate was centrifuged at 2000×g for 2 hours at 32°C to coat the plate with virus. The plate was washed twice with 1.5% Alubuminar/PBS.

(5) Infection with retrovirus PBMC, obtaining CAR-T cells

Cultured PBMCs (10.(2), above) were regenerated. After dilution with the culture medium to a concentration of 4×10 ⁵ , 950 μl of the suspension for retrovirus infection was seeded into the plate, then the plate was centrifuged at 1000×g for 10 minutes at 32°C and incubated at 37°C in a CO ₂ incubator.

The resulting CAR-T cells were used for analysis 12-14 days after PBMC isolation.

(6) Obtaining control cells

Some of the cultured cells (10.(2), above) were used as control cells (without gene transfer procedures).

[0044]

11. Characteristics of tumor cell lines used in in vitro experiments

The characteristics of the cell lines used in the in vitro experiments are shown in Table 4.

[Table 4]

[0045]

12. IF-γ release assay

The concentration of IF-γ in culture was determined by ELISA using the eBioscience kit.

10× immobilization buffer was diluted 10 times with purified water to obtain 1× immobilization buffer. 48 μl of primary antibodies were added to 12 ml of immobilization buffer. Buffer was added to a 96 well flat bottom plate at a volume of 100 μl per well. After maintaining the tablet at a temperature of 4°C during the night, it was washed five times with a 0.05% solution of FSB-T. The 5× analytical diluent solution was diluted 5 times with purified water to obtain a 1× analytical diluent solution. Analytical diluents were added at a concentration of 200 μl per well. After 1 hour of incubation of the tablet at room temperature to block the tablet was washed 5 times with a 0.05% solution of FSB-T. IFN-γ was prepared at a maximum concentration of 1000 pg/ml and a 7-step two-fold dilution was performed to prepare standards. Samples and standards were added to the plate, allowed to react for 2 hours at room temperature and the plate was washed 5 times with 0.05% PBS-T solution. 48 μl of secondary antibodies were added to 12 ml of analytical diluents, the resulting diluents were added in a volume of 100 μl per well. After incubation of the tablet for 1 hour at room temperature was carried out five times washing with 0.05% solution of FSB-T. To 12 ml of analytical diluents, 48 μl of horseradish peroxidase (HRP) conjugated streptavidin was added, the resulting solution was added in a volume of 100 μl per well. The tablet was left to react in the dark for 30 minutes at room temperature and washed seven times with 0.05% PBS-T. A solution of tetramethylbenzidine (tetramethylbenzidine, TMB) substrate was added in a volume of 100 μl, allowed to react in the dark for 15 minutes at room temperature, and 50 μl of 0.18 M H ₂ SO ₄ was added to stop the reaction. The plate was immediately analyzed at a wavelength of 450 nm using a plate reader (Model 680, Bio-Rad).

[0046]

13. Intracellular Cytokine Staining (ICS)

Target cells were adjusted to a concentration of 1×10 ⁵ cells/ml, effector cells were adjusted to a concentration of 1×10 ⁵ cells/ml. 0.5 µl of a solution of human CD107a-targeted antigen presenting cells (APCs) was added per sample, target cells and effector cells were added in a 1:1 ratio to a 96-well plate (U-shaped bottom), co-cultured for 1 hours at 37°C. Then 0.7 μl of Golgistop reagent (protein transport inhibitor) was added to each well and incubated for 4 hours at 37°C. Cells from each well were transferred to a 96-well V-bottom plate, centrifuged at 1200 rpm for 5 minutes at 4°C, and then washed twice with 0.5% bovine serum albumin (BSA) in PBS . 0.5 µl of a solution of human CD4-targeted APC-cy7 and 0.5 µl of fluorescein isothyocyanate (FITC) conjugated anti-human CD8 antibodies were added to each well and placed on ice for 20 minutes in the dark and the plate was washed twice with 0. 5% BSA/PBS solution. 100 µl of cell fixative solution (Cytofix/Cytoperm: BD) was added, placed on ice for 20 minutes in the dark, 100 µl of Perm/Wash permeabilization/wash buffer (BD) was added and centrifuged. The plate was then washed twice with Perm/Wash permeabilizing/wash buffer. 0.5 µl of IFN-γ V450 and 0.5 µl of TNF-α PE-Cy7 were added to each well. The plate was placed on ice in the dark for 30 minutes, washed twice with 0.5% BSA/PBS solution. Thereafter, the plate was analyzed using a FACSCanto II (BD) flow cytometer and data analysis was performed using the FACS Diva (BD) software.

[0047]

14. Long Peptide APC Capture, Cross Presentation

(1) MAGE-A4 long peptide

The amino acid sequence of the MAGE-A4 long peptide was NYKRCFPVIF GKASEGVYDG REHTVYGEPR KAETSYVKVL EHVVRVNARV RI (SEQ ID NO: 28), the molecular weight of the peptide was 6006.811. The long peptide was generated by joining the MAGE-A4 A24 (143-151) epitope (NYKRCFPVI: SEQ ID NO: 29), the MAGE-A4 A2 (230-239) epitope (GVYDGREHTV: SEQ ID NO: 1) and the helper epitope (AETSYVKVLE HVVRVNARVR I: SEQ ID NO: 30).

(2) CHP-long peptides

CHP-long peptide refers to an entity in which a MAGE-A4 long peptide is packaged in a nanogel based on a cholesteryl group-bearing pullulan ( CHP) as a drug delivery system. It is expected that the use of this object can cause the induction of cellular immunity (non-patent document 5).

(3) Selection of CD3-negative granules

1×10 ⁷ human lymphocytes were suspended in 80 μl of a 2% EBS/PBS solution. 20 µl CD3-bound microbeads (Miltenyi Biotec) were added to the suspension and mixed thoroughly. After incubation for 15 minutes at 4° C. with light shielding, the suspension was washed with 10 volumes of 2% EBS/PBS. The cells were suspended in 3 ml of a 2% EBS/PBS solution for use as a cell suspension. The cell suspension was applied to a MACS separator equipped with a MACS column. The fractions that passed through the column were CD3-negative fractions used as APC.

[0048]

15. Cytotoxicity assay

(1) Cr release analysis

After the target cells were collected by centrifugation, they were suspended in 100% FBS at a final concentration of 1×10 ⁶ cells/50 μl. 50µCi (3.7×10 ⁶ Bq) ⁵¹ Cr was added to the cells and incubated for 1 hour at 37°C in a CO ₂ incubator. The concentration of effector cells was adjusted with RPMI1640 medium containing 10% EBS according to the E/T ratio and the cells were plated in a 96-well U-bottomed plate. After cultivation, the cells were washed three times with RPMI1640 medium containing 10% FBS. Cells were suspended in RPMI1640 medium containing 10% EBS at a final concentration of 1×10 ⁵ cells/ml, seeded at a concentration of 1×10 ⁴ cells/100 μl/well in a 96-well plate with a U-shaped bottom and cultured for 8 hours in a CO2 incubator at 37°C. After culturing, the plate was centrifuged at 1200 rpm for 5 minutes at 4°C, 30 μl of the supernatant was transferred to a reading plate and air dried overnight. The amount of ⁵¹ Cr was determined using a MicroBeta2 scintillation counter (Perkin Elmer). The calculation of cytotoxic activity was determined in accordance with the following equation. The maximum release value was the value measured when adding 100 μl of 1% NP-40.

Cytotoxic activity (%) = 100 × (measured value (cpm) - spontaneous release value (cpm)) / (maximum release value (cpm) - spontaneous release value (cpm))

[0049]

16. Experiment in an in vivo cancer treatment model using NOG mice

(1) Used mice

NOG mice (NOD/Shi-scid, IL-2RγKO Jic) were obtained from CLEA Japan, Inc. The experiment used female mice aged 7-8 weeks. The T-cell infusion therapy in laboratory animals and the gene immunotherapy study were approved by the Committee for Recombinant DNA Experiments at Mie University, the Ethics Committee of the Faculty of Medical Research at Mie University, and the Committee for Animal Research. The mice were bred at the Center for Life Sciences for Research and Support of the Animal Research Department of Mie University.

(2) Human tumor implantation and total body irradiation (TOT)

As a pre-treatment, NOG mice received total body irradiation (TOT, 2.5 Gy) the day before cell infusion. The recipient's lymphocyte count was reduced by pre-treatment to facilitate engraftment of the donor's transferred lymphocytes. The concentration of CAR-T cells and human lymphocytes for infusion was 1×10 ⁷ cells/animal. Cell transfusion was performed through the tail vein of NOG mice. Body weight was measured every 2-3 days.

Tumor transplantation into NOG mice was performed 7 days prior to CAR-T cell infusion. NW-MEL-38 cells (A2-positive MAGE-A4-positive) were injected into mice in the right flank at a concentration of 2.5×10 ⁶ cells/mouse, HCT116 cells (A2-positive MAGE-A4-negative) were injected subcutaneously into mice in the left side at a concentration of 2.5×10 ⁶ cells/mouse. Tumor diameter was measured every 2-3 days.

[0050]

(3) Obtaining cells for infusion

CAR-T cells for infusion were prepared according to the method in section “10. Transfer of genes into cells using a retrovirus.

(4) Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Mouse Peripheral Blood by Orbital Blood Extraction

Peripheral blood was collected from the eye socket of a mouse, and peripheral blood mononuclear cells (PBMCs) were separated using the Ficoll reagent. Cells were stained with A2/MAGE-A4 tetramer and fluorescently labeled anti-human CD4CD8 antibodies and cell infusion confirmed by FACS.

(5) Confirmation of IOL (tumor infiltrating lymphocytes)

The mouse tumor was crushed, stained with A2-MAGE-A4 tetramer and fluorescently labeled anti-human CD4CD8 antibodies, and cell transfusion confirmed by FACS.

(6) Immunostaining

Tumors extracted from mice were frozen in liquid nitrogen, and frozen tissue sections were prepared on a cryostat. These sections were stained with DAPI, FITC-conjugated antibodies against human CD4 and FITC-conjugated antibodies against human CD8 and observed using a fluorescent microscope.

[0051]

17. In vitro experiments on CD8 positive T cells injected with zG type CAR and CD8 positive T cells injected with 4-1BBz type CAR

The effects of CAR-T cells associated with differences in the ICD region (intracellular domain) were confirmed. In particular, GITR type (zG) and CD137 type (4-1BBz) type CARs were obtained from CD28 (28z) type CARs, and their effects were confirmed. As shown in Figure 21, glucocorticoid-induced tumor necrosis factor receptor (GITR) was used in place of CD28 as the ICD in zG type CARs. Similarly, CD137 (4-1BBz) was used instead of CD28.

CD8-positive T cells that did not transfect with CAR (NGMC) were used as controls.

[0052]

<Results>

1. Isolation and evaluation of an antibody recognizing HLA-A2/MAGE-A4

MAGE-A4 (HLA-A2-MAGE-A4) is known to be highly expressed in melanomas. Thus, a plurality of antibodies recognizing MAGE-A4 have been isolated by screening with human antibody libraries. Phage display was used to isolate antibodies. According to the method, scFv was presented as part of the coat protein of the cp3 phage. Clones that bind to the antigen with high affinity can be selected using ELISA. After collecting approximately 1500 clones, ELISA was performed to detect clones in the form of scFv-CP3. Clones were selected responding to the HLA-A2/MAGE-A4 complex, which was used as a positive target, and not responding to the A2/CMV complex, which was used as a negative target. To assess the binding specificity of selected antibodies, ELISA was performed using complexes of several peptides with MHC (p-MHC). Each p-MHC monomer containing HLA-A2/MAGE-A4p230, MAGE-A4 p286, MAGE-A3 p195, CMV, MelanA, GP3, HTLV-1 p11, NY-ESO-1 p157, NY-ESO-1 p159, Foxp3 p69, Foxp3 p127, Foxp3 p390, IDO p41, IDO p41, IDO p195 and IDO p199 were immobilized and ELISA was performed using selected antibodies. Some ELISA results are shown in Figure 3.

[0053]

Fifteen clones were generated that showed a specific reaction with HLA-A2/MAGE-A4 by comparing the sequences of clones that showed strong binding to HLA-A2/MAGE-A4. Of these, 5 clones for which strong binding was observed, namely MAGE#17, #86, #209, #345 and #672, were selected as candidate clones and subjected to analytical methods.

The KD value for the MAGE#17 antibody was then measured using a BiacoreX100 instrument. KD values were determined using Biacore by measuring dynamic changes in detection sensitivity (resonance, reflecting the change in mass on the matrix) over time. The dissociation rate constant (Kd) and the binding rate constant (Ka) were determined using a trend curve, the binding constant was determined from the ratio of the two constants. As measured, the binding constant of MAGE#17 was 22 nM (Figure 4).

[0054]

2. Study of the specificity of recognition of the A2/MAGE-A4 complex and mimic antigen by the obtained antibodies

T2 cells lack an antigen processing-associated transporter (TAP) and do not present intracellular protein to HLA. Since HLA is stabilized on T2 cells, and its expression level has been confirmed by fluorescent anti-HLA antibodies, when the peptide is introduced into T2 cells, the degree of binding of this peptide to HLA can be determined.

Selected antibodies, i.e. MAGE#17, #86, #209, #345, and #672 antibodies were reacted with T2 cells injected with MAGE-A4p230, CMV, and DMSO, and the shift was determined by fluorescence-activated cell sorting (FACS). Only in T2 cells that were injected with MAGE-A4p230, for selected MAGE antibody clones #17, #86, #209, #345 and #672, a change in the magnitude of the shift was observed (Figure 5).

Thereafter, to search for amino acids associated with the recognition of the antibody, peptides were obtained with the replacement of one amino acid by another amino acid (mainly alanine) in MAGE-A4p230 (GVYDGREHTV: SEQ ID NO: 1), the binding strength between HLA-A*0201 and the peptide was evaluated using the database of immune epitopes (immune epitope database, IEDB) and determined the IC50 for MHC (table 2). The IC50 for MHC is a theoretical value calculated using IEDB, the lower the value, the stronger the association between HLA-A*0201 and the peptide. Peptides with amino acid substitution for alanine were introduced into a T2 cell, the amount of HLA on the surface of said T2 cell was determined using the FACS method (Figure 6). The stronger the peptide bound to HLA, the more stable the peptide-HLA structure was, since the amount of shift in the FACS method increased. The value of the average fluorescence intensity (MFI) was an indicator of the binding strength of the peptide and HLA. With the exception of some peptides, the SIF value and the IC50 value for MHC were consistent with each other.

[0055]

The alanine amino acid peptide was then introduced into a T2 cell and interacted with MAGE#17 and the amino acids involved in antibody recognition were evaluated (Figure 7). Since the SIF value decreased with the substitution of 1A, 2A, 3A, 4A, 5A, 6A and 8A, seven amino acids were determined to be involved in recognition. The amino acid sequence of GVYDGRxHxx was deduced by BLAST search and a mimic peptide was selected (Table 3). After the introduction of the stimulating peptide into T2 cells, the amount of HLA on the surface of T2 cells was determined using the FACS method. Similar to the results shown in Figure 6, the SIF value and the IC50 value for the MHC matched each other (Figure 8).

The mimic peptide was then introduced into T2 cells, interacted with MAGE#17, and it was confirmed that the T2 cell did not recognize said mimic peptide (Figure 9). Similar analyzes were performed in practice using other clones #86, #209, #345, and #672, the results showed that clone #17 contained the most amino acids involved in antigen recognition. For this reason, the MAGE#17 antibody was selected as the final candidate.

[0056]

The nucleotide sequence (SEQ ID NO: 31) and amino acid sequence (SEQ ID NO: 32) of MAGE#17 were shown.

Нуклеотидная последовательность MAG#17 (SEQ ID NO: 31): CAGGTCCAGC TGGTACAGTC TGGGGCTGAG GTGAAGAAGC CTGGGTCCTC GGTGAAGGTC TCCTGCAAGG CTTCTGGAGG CACCTTCAGC AGCTATGCTA TCAGCTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TCTTTGGTAC AGCAAACTAC GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGCGGACA AATCCACGAG CACAGCCTAC ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTACTGTGC GAGATCCCCC CGGCGGGCAT ATCATGATGC TTTTGATATC TGGGGCCAAG GGACAATGGT CACCGTCTCT TCAGGTGGAG GCGGTTCAGG CGGAGGTGGC AGCGGCGGTG GCGGGAGTTC CTATGAGCTG ACTCAGCCAC CCTCGATGTC AGTGGCCCCA GGAAAGACGG CCAGCATTAC CTGTGGCGGA GACCATATTG GAAGTAAAAG TGTTCACTGG TACCAGCAGA AGCCAGGCCA GGCCCCTGTA CTGGTCGTCT ATGATGATAG CGACCGGCCC TCAGGGATCC CTGAGCGATT CTCTGGCTCC AACTCTGGGA ACACAGCCAC TCTGACCATC AGCGGGACCC AGGCTATGGA TGAGGCTGAC TATTACTGTC TGGCGTGGGA CAGCAGCACT GCGATCTTCG GCGGAGGGAC CAAGCTGACC GTCCTC.

[0057]

Аминокислотная последовательность MAGE#17 (SEQ ID NO: 32): QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGG IIPIFGTANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARSP RRAYHDAFDI WGQGTMVTVS SGGGGSGGGG SGGGGSSYEL TQPPSMSVAP GKTASITCGG DHIGSKSVHW YQQKPGQAPV LVVYDDSDRP SGIPERFSGS NSGNTATLTI SGTQAMDEAD YYCLAWDSST AIFGGGTKLT VL.

The above sequences show that the nucleotide sequence contains a VH region (SEQ ID NO: 33), an sc region (SEQ ID NO: 34) and a VL region (SEQ ID NO: 35), and the amino acid sequence contains a VH region ( SEQ ID NO: 36), sc region (SEQ ID NO: 37), and VL region (SEQ ID NO: 38) as shown below.

Нуклеотидная последовательность VH-участка (SEQ ID NO 33): CAGGTCCAGC TGGTACAGTC TGGGGCTGAG GTGAAGAAGC CTGGGTCCTC GGTGAAGGTC TCCTGCAAGG CTTCTGGAGG CACCTTCAGC AGCTATGCTA TCAGCTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TCTTTGGTAC AGCAAACTAC GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGCGGACA AATCCACGAG CACAGCCTAC ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTACTGTGC GAGATCCCCC CGGCGGGCAT ATCATGATGC TTTTGATATC TGGGGCCAAG GGACAATGGT CACCGTCTCT TCA.

[0058]

The nucleotide sequence of the sc region (SEQ ID NO 34): GGTGGAGGCG GTTCAGGCGG AGGTGGCAGC GGCGGTGGCG GGAGTTCCTA T.

Нуклеотидная последовательность VL-участка (SEQ ID NO 35): GAGCTGACTC AGCCACCCTC GATGTCAGTG GCCCCAGGAA AGACGGCCAG CATTACCTGT GGCGGAGACC ATATTGGAAG TAAAAGTGTT CACTGGTACC AGCAGAAGCC AGGCCAGGCC CCTGTACTGG TCGTCTATGA TGATAGCGAC CGGCCCTCAG GGATCCCTGA GCGATTCTCT GGCTCCAACT CTGGGAACAC AGCCACTCTG ACCATCAGCG GGACCCAGGC TATGGATGAG GCTGACTATT ACTGTCTGGC GTGGGACAGC AGCACTGCGA TCTTCGGCGG AGGGACCAAG CTGACCGTCC TC.

VH region amino acid sequence (SEQ ID NO 36): QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGG IIPIFGTANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARSP RRAYHDAFDI WGQGTMVTVS S.

The amino acid sequence of the sc region (SEQ ID NO: 37): GGGGSGGGGS GGGGSSY.

VL region amino acid sequence (SEQ ID NO: 38): ELTQPPSMSV APGKTASITC GGDHIGSKSV HWYQQKPGQA PVLVVYDDSD RPSGIPERFS GSNSGNTATL TISGTQAMDE ADYYCLAWDS STAIFGGGTK LTVL.

[0059]

3. MAGE#17-CAR specifically recognizes target cancer cells and increases IFN-γ production

Since the specificity of the MAGE#17 antibody clone was confirmed, MAGE#17 was transferred to the CAR vector. Then, after obtaining the mRNA, its inclusion was confirmed using the Bioanalyzer system, the CAR gene was introduced into human peripheral blood lymphocytes (PBMC) using the electroporation method. Normal expression of the introduced CAR was confirmed by tetramer staining. The resulting CAR positivity ratio for CAR T cells was 98.8% (CD8) and 98.5% (CD4) (Figure 10). CAR-T cells and peptide-introduced T2 cells were co-cultured for 24 hours and the level of IFN-γ in the culture supernatant was measured. A specific increase in IFN-γ production was observed in T2 cells injected with MAGE-A4. A similar analysis was performed using control cells (cells not subject to gene transfer). In addition, tumor-specific IFN-γ production was observed in target cells, A2-positive MAGE-A4-positive NW-MEL-38 and LB-23 cell lines, compared to LC-1/sq and MEL72 cell lines, not being targets (Figure 11).

[0060]

Because CAR expression was transient as measured by mRNA, gene transfection was performed with a retrovirus to permanently express CAR. After a retroviral vector was constructed to introduce CAR, a retrovirus was generated using plat-A packaging cells, CAR-T cells were generated by infecting peripheral blood mononuclear cells (PBMCs) with the retrovirus. Gene transfer was carried out in accordance with the method from the above section “10. Transfer of genes into cells using a retrovirus. CAR expression was confirmed by tetramer staining. The resulting CAR positivity ratio for CAR T cells was 59.3% (CD8) and 49.5% (CD4) (Figure 12).

CAR-T cells subjected to retroviral gene transfer and T2 cells into which the peptide was introduced were co-cultured for 24 hours, the level of IFN-γ in the culture supenant was measured. A specific increase in IFN-γ production was observed in T2 cells injected with MAGE-A4. A similar analysis was performed using control cells (cells not subjected to gene transfer). In addition, tumor-specific IFN-γ production was observed in target cells, A2-positive MAGE-A4-positive NW-MEL-38 and LB-23 cell lines, compared with non-MAGE-A4 LC-1/sq and MEL72 cells. targets (Figure 13). CAR expression was confirmed by introducing mRNA and introducing the CAR gene with retroviral vectors, the cells specifically recognized the tumor and produced IFN-γ.

[0061]

4. CAR-T cells injected with the MAGE#17-CAR gene specifically recognize target cancer cells and exhibit cytotoxic activity

It was expected that CAR-T cells would recognize A2/MAGE-A4 expressed as an antigen on cancer cells, be activated, and exhibit cytotoxic activity. IFN-γ, TNF-α, and CD107a are known to be indicators of T-cell activation and are upregulated as T-cells are activated. Thus, intracellular cytokine staining (IVS) was performed on T2 cells injected with the peptide and MAGE#17-CAR-T cells co-cultured with target cells.

As a result, the percentage of CAR-T cells stained with IFN-γ, TNF-α, and CD107a increased when co-cultured with T2 cells injected with MAGE-A4p230 (Figure 14). Moreover, the percentage of CAR-T cells stained with IFN-γ, TNF-α, and CD107a increased when they were co-cultured with target cells, A2-positive MAGE-A4-positive NW-MEL-38 and LB-23 cells. (Figure 15).

[0062]

5. IFN-γ production in CAR-T cells was increased as a result of antigen presentation by antigen presenting cells (APCs) that captured the MAGE-A4 long peptide antigen.

Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of a healthy person, negative selection was carried out using magnetic beads associated with CD3, CD3-negative fractions were used as antigen-presenting cells (APCs). A2-APK was used as APC with MAGE-A4p230 presentation and A24-APK was used as negative control APC. MAGE-A4 long peptide and MAGE-A4 long peptide CHP were added to achieve a final concentration of 10 μM, cells were cultured overnight and co-cultured with CAR-T cells for 24 hours. Gene transfer to CAR-T cells was carried out in accordance with the method from the above section “10. Transfer of genes into cells using a retrovirus. As a result, the concentration of IFN-γ in the culture supernatant increased when co-cultivated with A2-APK, which introduced the long peptide MAGE-A4 (Figure 16).

MAGE-A4 long peptides were inserted into APCs, MAGE-A4p230 was presented with class I MHC molecules, and IFN-γ production by CAR-T cells that recognized the MAGE-A4p230/MHC class I complex was specifically increased. On the other hand, specific production of IFN-γ was not observed in the case of MAGE-A4 CHP-long peptides packaged in CHP-nanogel used as a drug delivery system.

[0063]

6. CAR-T cells, into which the MAGE#17-CAR gene was transferred, specifically lysed cancer target cells

The presence of specific cytotoxic activity containing MAGE#17-CAR cells was confirmed by analysis of the release of ⁵¹ Cr. When the ratio of CAR-T cells and cancer target cells was an E/T ratio (effector/target ratio), Cr-capturing cancer cells and CAR-T cells were co-cultured for 8 hours at an E/T ratio of 10:1 , 3:1, 1:1, the level of cytotoxicity (% lysis) was determined based on the radioactivity of ⁵¹ Cr in the supernatant. Gene transfer in CAR-T cells was carried out in accordance with the method from the above section “10. Transfer of genes into cells using a retrovirus.

As a result, MAGE-A4 specific cytotoxicity was observed in T2 cells injected with the MAGE-A4p230 peptide (Figure 17). In addition, specific cytotoxicity against target cells was confirmed on NW-MEL-38 cells, which were A2-positive MAGE-A4-positive cells (Figure 18).

[0064]

7. Mouse model of cancer therapy

(1) Model using NW-MEL-38 and HCT116 cells

Because the NOG mouse lacks a receptor containing a common γ chain, these mice do not have NK cells. Thus, engraftment of human cells and human tissue is significantly better in NOG mice compared to NOD-SCID mice, human tumors and human tissues can easily engraft in NOG mice. In addition, since human T cell differentiation occurs after transplantation of human hematopoietic stem cells, the mouse model of NOG was expected to be used as a model of the human immune system. NOG mice have features including absence of T and B cells, absence of natural killer (NK) cells, decreased dendritic cell function, decreased macrophage function, loss of complement activity, absence of aging-associated leakage of B cells and T- cells (permeability).

NOG mice were injected in the right flank with NW-MEL-38 cells (A2-positive MAGE-A4-positive) at a concentration of 2.5×10 ⁶ cells/mouse and subcutaneously injected in the left flank with HCT116 cells (A2-positive MAGE-A4-negative ) at a concentration of 2.5×10 ⁶ cells/mouse. As a pretreatment, NOG mice received total body irradiation (TOT, 2.5 Gy) 6 days after tumor transplantation. Because TOT causes a decrease in lymphocytes in the recipient, transplanted donor lymphocytes should readily engraft. On day 7, human T cells and lymphocytes containing MAGE#17-CAR were infused via the tail vein at a concentration of 1×10 ⁷ cells/animal. Gene transfer to CAR-T cells was carried out in accordance with the method from the above section “10. Transfer of genes into cells using a retrovirus. The CAR positivity ratio was 36.3% (CD8) and 38.5% (CD4) (Figure 19).

Animal experiments were performed, including a group of animals that were infused with CAR-T cells (n=2), a group of animals that were infused with human lymphocytes (no CAR transfection, n=2), and a group of animals that were transfused with PBS ( n=2), tumor size and body weight were measured every 2-3 days. Measurements were taken up to day 42 after tumor implantation.

As a result, in the group of animals that were infused with CAR-T cells, mouse #1 and mouse #2 were observed to suppress the growth of NW-MEL-38 cells (A2-positive MAGE-A4-positive). On the other hand, growth suppression of HCT116 cells (A2-positive MAGE-A4-negative) was not observed. Mouse #1 died 34 days after tumor transplantation. The cause of death was not a tumor, but probably an infection (Figure 20).

[0065]

(2) Experiments to study the effects of differences in intracellular domains (ICD)

Then, the effects of CAR-T cells associated with differences in the ICD region were confirmed. Figure 21 shows the sequence image of the CAR zG genes. In CAR zG ICD GITR was used instead of ICD CD28. GITR is a glucocorticoid-inducible tumor necrosis factor receptor. The use of CAR with ICR is known to increase the level of cytokine expression compared to CAR (WO2013051718). In CAR zG, the CD28 intracellular domain of the CAR 28z receptor (Figure 2) was changed to ICD GITR, and the order of the CD3ζ and ICD domains of the CAR 28z receptor was reordered. The sequences of the other parts used were identical to those of CAR 28z.

NW-MEL-38 cells (A2-positive MAGE-A4-positive) at a concentration of 2.5×10 ⁶ cells/mouse were injected subcutaneously into NOG mice. Three days after tumor implantation, total body irradiation (TOT, 2.0 Gy) was performed as a preliminary treatment. On day 4, cells or PBS and human lymphocytes were infused via the tail vein at a concentration of 4×10 ⁶ cells/mouse in each group A - C (n=4), as shown below. Gene transfer to CAR-T cells was carried out in accordance with the method from the above section “10. Transfer of genes into cells using a retrovirus. PBS was infused in group A, MAGE#17-CAR-T cells in group B (28z) and CAR-T cells (zG) in group C.

Tumor diameter and body weight were measured every 2-3 days for groups A - C. Measurements were made up to 24 days after tumor implantation.

As a result, growth inhibition of NW-MEL-38 cells (A2-positive MAGE-A4-positive) was observed upon infusion of CAR-T cells containing 28z ICT or zG ICT (Figure 22). According to an embodiment of the present invention, it was found that changing the intracellular domain produced the same effect.

[0066]

8. Experiments to evaluate the effects of CD8-positive T cells injected with CAR type zG and CD8-positive T cells injected with CAR type 4-1BBz.

(1) CAR type zG and CAR type 28z were introduced using a constructed retroviral vector. After producing a retrovirus using plat-A packaging cells, CAR-T cells were obtained by infecting peripheral blood mononuclear cells (PBMCs) with said retrovirus. Gene transfer was carried out in accordance with the method from the above section “10. Transfer of genes into cells using a retrovirus. CAR expression was confirmed by tetramer staining. As a result, the CAR positivity ratio for zG type CAR T cells was 99.1% (CD8) and for 28z type CAR T cells 99.8% (CD8) (Figure 23).

(2) Retrovirally gene-transferred CAR-T cells (4×10 ⁴ cells) and T2 cells injected with the peptide, NW-MEL-38, SK-MEL-37 and HCT116 cells, respectively (2×10 ⁵ cells ) were co-cultured for 18 hours, the level of INF-γ in the culture supernatant was measured. As shown in Figure 24, IFN-γ production was specifically upregulated in CAR-T cells transfected with CAR type zG and CAR type 28z, T2 cells injected with MAGE-A4, and NW-MEL-38 and SK- MEL-37.

Similar analyzes were performed using control cells (cells without gene transfer). IFN-γ production was not observed in A2 positive MAGE-A4 containing HCT116 cells used as target cells. IFN-γ production was not observed in cells containing only effector cells (only effectors) and cells containing only target cells (only target).

Thus, CD8-positive T cells injected with zG-type CARs induced INF-γ production in vitro. The data obtained can successfully explain the results obtained in vivo.

(3) Then, analyzes similar to those in (1) and (2) were performed using CD8-positive T cells injected with CAR type 4-1BBz to characterize the cells. As shown in Figure 25, in CAR-T cells transfected with CAR type 4-1BBz, an increase in IFN-γ production was observed, specific for T2 cells injected with MAGE-A4.

T2 cells injected with the control peptide did not show IFN-γ production. In cells containing only effector cells (only effectors) and cells containing only target cells (only target), no IFN-γ production was observed.

Thus, even when the ICD was changed to a 4-1BBz type domain, the same effect was observed. The in vitro data can easily explain the results obtained in vivo.

Thus, even when the ICD was changed, the same effect was observed as for the zG type domain.

[0067]

Thus, in accordance with these embodiments of the present invention, an antigen-binding protein has been provided that specifically recognizes a complex derived from MAGE-A4 peptide/HLA-A2, nucleic acids encoding said antigen-binding protein, a vector containing said nucleic acids, a chimeric an antigen receptor containing said antigen-binding protein, nucleic acids encoding said chimeric antigen receptor, a vector containing said nucleic acids, a cell expressing said chimeric antigen receptor, and a pharmaceutical composition containing said cells.

An antigen binding protein specifically recognizing the MAGE-A4 derived peptide/HLA-A2 complex can be used in cell therapy and in the field of gene therapy. The present invention is particularly useful for identifying tumor cells expressing the MAGE-A4 derived peptide/HLA-A2 complex, treating and conducting research and experiments on tumors. The CAR-T cells of the present invention can provide a highly effective cancer therapy because they cause very few side effects.

[prior art]

[Non-Patent Documents]

[0068]

Non-Patent Document 1: Brentjens RJ, et al: Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-leukemias. Blood 2011,118:4817-4828.

Non-Patent Document 2: Dao T, Yan S, Veomett N, Pankov D, Zhou L, Korontsvit T, et al: Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med 2013; 5:176ra133.

Non-Patent Document 3: Van Der Bruggen P, Zhang Y, Chaux P, Stroobant V, Panichelli C, Schultz ES, Chapiro J, Van Den Eynde BJ, Brasseur F, Boon T. Tumor-specific shared antigenic peptides recognized by human T cells. Immunological Reviews 2002. Vol. 188:51-64.

Non-Patent Document 4: Duffour MT, Chaux P, Lurquin C, Cornelis G, Boon T, van der Bruggen P. A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T Lymphocytes. Eur. J. Immunol 1999. 29: 3329-3337.

Non-Patent Document 5: Muraoka D, Harada N, Hayashi T, Tahara Y, Momose F, Sawada S, Mukai SA, Akiyoshi K, Shiku H. Nanogel-based immunologically stealth vaccine targets macrophages in the medulla of lymph node and induces potent antitumor immunity . ACS Nano.Sep 23.2014;8(9):9209-18.

Non-Patent Document 6: Hillig RC, Coulie PG, Stroobant V, Saenger W, Ziegler A, Huelsmeyer M. High-resolution Structure of HLA-A*0201 in Complex with a Tumour-specific Antigenic Peptide Encoded by the MAGE-A4 Gene. J. Mol. biol. 2001,310,1167-1176.

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SEQUENCE LIST

<110> Mie University

<120> ANTIGEN-BINDING PROTEIN RECOGNIZING ORIGIN

MAGE-A4 PEPTIDE

<130>JP2018001MIU

<150>JP2017-111157

<151> 2017-06-05

<160> 38

<170> PatentIn version 3.5

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<212> Protein

<213> Homo sapiens

<400> 25

His Val Tyr Asn Gly Arg Thr His Gln Trp

1 5 10

<210> 26

<211> 10

<212> Protein

<213> Homo sapiens

<400> 26

Arg Val Tyr Asp Gly Arg Glu Ile Leu Thr

1 5 10

<210> 27

<211> 10

<212> Protein

<213> Homo sapiens

<400> 27

Gly Val Tyr Ser Gly Arg Ala His Pro Lys

1 5 10

<210> 28

<211> 52

<212> Protein

<213> Artificial sequence

<220>

<223> MAGE-A4 long peptide

<400> 28

Asn Tyr Lys Arg Cys Phe Pro Val Ile Phe Gly Lys Ala Ser Glu Gly

1 5 10 15

Val Tyr Asp Gly Arg Glu His Thr Val Tyr Gly Glu Pro Arg Lys Ala

20 25 30

Glu Thr Ser Tyr Val Lys Val Leu Glu His Val Val Arg Val Asn Ala

35 40 45

Arg Val Arg Ile

fifty

<210> 29

<211> 9

<212> Protein

<213> Homo sapiens

<400> 29

Asn Tyr Lys Arg Cys Phe Pro Val Ile

fifteen

<210> 30

<211> 21

<212> Protein

<213> Homo sapiens

<400> 30

Ala Glu Thr Ser Tyr Val Lys Val Leu Glu His Val Val Arg Val Asn

1 5 10 15

Ala Arg Val Arg Ile

twenty

<210> 31

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> MAGE#17

<400> 31

caggtccagc tggtacagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180

240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagatccccc 300

cggcgggcat atcatgatgc ttttgatatc tggggccaag ggacaatggt caccgtctct 360

tcaggtggag gcggttcagg cggaggtggc agcggcggtg gcgggagttc ctatgagctg 420

actcagccac cctcgatgtc agtggcccca ggaaagacgg ccagcattac ctgtggcgga 480

gaccatattg gaagtaaaag tgttcactgg taccagcaga agccaggcca ggcccctgta 540

ctggtcgtct atgatgatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc 600

aactctggga acacagccac tctgaccatc agcgggaccc aggctatgga tgaggctgac 660

tattactgtc tggcgtggga cagcagcact gcgatcttcg gcggagggac caagctgacc 720

gtcctc 726

<210> 32

<211> 242

<212> Protein

<213> Artificial sequence

<220>

<223> MAGE#17

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Arg Arg Ala Tyr His Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gly Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro

130 135 140

Ser Met Ser Val Ala Pro Gly Lys Thr Ala Ser Ile Thr Cys Gly Gly

145 150 155 160

Asp His Ile Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Val Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly

180 185 190

Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

195 200 205

Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Leu

210 215 220

Ala Trp Asp Ser Ser Thr Ala Ile Phe Gly Gly Gly Thr Lys Leu Thr

225 230 235 240

Val Leu

<210> 33

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> VH section MAGE#17

<400> 33

caggtccagc tggtacagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180

240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagatccccc 300

cggcgggcat atcatgatgc ttttgatatc tggggccaag ggacaatggt caccgtctct 360

tca 363

<210> 34

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> sc region MAGE#17

<400> 34

ggtggaggcg gttcaggcgg aggtggcagc ggcggtggcg ggagttccta t 51

<210> 35

<211> 312

<212> DNA

<213> Artificial sequence

<220>

<223> VL section MAGE#17

<400> 35

gagctgactc agccaccctc gatgtcagtg gccccaggaa agacggccag cattacctgt 60

ggcggagacc atattggaag taaaagtgtt cactggtacc agcagaagcc aggccaggcc 120

cctgtactgg tcgtctatga tgatagcgac cggccctcag ggatccctga gcgattctct 180

ggctccaact ctgggaacac agccactctg accatcagcg ggacccaggc tatggatgag 240

gctgactatt actgtctggc gtgggacagc agcactgcga tcttcggcgg agggaccaag 300

ctgaccgtcc tc 312

<210> 36

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> VH section MAGE#17

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Arg Arg Ala Tyr His Asp Ala Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 37

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> sc region MAGE#17

<400> 37

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

1 5 10 15

Tyr

<210> 38

<211> 104

<212> Protein

<213> Artificial sequence

<220>

<223> VL section MAGE#17

<400> 38

Glu Leu Thr Gln Pro Ser Met Ser Val Ala Pro Gly Lys Thr Ala

1 5 10 15

Ser Ile Thr Cys Gly Gly Asp His Ile Gly Ser Lys Ser Val His Trp

20 25 30

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp

35 40 45

Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser

50 55 60

Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu

65 70 75 80

Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Ser Ser Thr Ala Ile Phe Gly

85 90 95

Gly Gly Thr Lys Leu Thr Val Leu

100

<---

Claims (19)

1. An antigen-binding protein capable of recognizing the GVYDGREHTV peptide in the HLA-A2-MAGE-A4 complex, said antigen-binding protein being an antibody or antigen-binding fragment thereof, which contains: (A) a heavy chain variable region (VH) polypeptide comprising the amino acid sequence of SEQ ID NO: 36; and (B) a light chain variable region (VL) polypeptide containing the amino acid sequence of SEQ ID NO: 38. 2. An antigen-binding protein according to claim 1, wherein said antigen-binding protein is Fab, Fab', F(ab') ₂ or Fv. 3. An antigen binding protein capable of recognizing the GVYDGREHTV peptide in the HLA-A2-MAGE-A4 complex, said antigen binding protein being a single chain Fv fragment (scFv) containing A) a heavy chain variable region (VH) polypeptide comprising the amino acid sequence of SEQ ID NO: 36; and B) a light chain variable region (VL) polypeptide comprising the amino acid sequence of SEQ ID NO: 38. 4. Antigen-binding protein according to claim 3, containing a polypeptide containing the amino acid sequence of the sc-fragment (single-stranded fragment) SEQ ID NO: 37 between VH and VL. 5. Antigen-binding protein according to any one of paragraphs. 3, 4 containing the polypeptide of SEQ ID NO: 32. 6. Nucleic acid encoding an antigen-binding protein according to any one of paragraphs. 3-5. 7. A vector for ensuring the expression of an antigen-binding protein according to any one of paragraphs. 3-5 containing the nucleic acid according to claim 6. 8. Chimeric antigen receptor capable of recognizing the GVYDGREHTV peptide in the HLA-A2-MAGE-A4 complex, said receptor comprising an antigen-binding protein according to any one of paragraphs. 3-5 and an intracellular domain of a signal transduction protein, wherein said antigen-binding protein is located at the N-terminal position of the protein and is located outside the cell, and said intracellular domain is located at the C-terminal position of the protein and is located inside the cell. 9. A chimeric antigen receptor according to claim 8, wherein said signal transduction protein is any of CD3-zeta (CD3ζ) and CD co-stimulatory molecules (GITR). 10. The chimeric antigen receptor of claim 9, further comprising any of the intracellular domain of CD28 and GITR. 11. Nucleic acid encoding a chimeric antigen receptor according to any one of paragraphs. 8-10. 12. A vector for ensuring the expression of a chimeric antigen receptor according to any one of paragraphs. 8-10 containing the nucleic acid according to claim 11. 13. A cell expressing a chimeric antigen receptor according to any one of paragraphs. 8–10, while said cell contains the nucleic acid according to claim 11. 14. A pharmaceutical composition containing an effective amount of a cell according to claim 13 as an active ingredient to provide cytotoxic activity directed towards a cell expressing the GVYDGREHTV peptide in the HLA-A2-MAGE-A4 complex. 15. The pharmaceutical composition according to claim 14, characterized in that said cell expressing the GVYDGREHTV peptide in the HLA-A2-MAGE-A4 complex is a cancer cell and said pharmaceutical composition is for the treatment of cancer.

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