JP6699013B2 - WT1-derived peptide recognition antibody - Google Patents

Description

  The present invention provides an antibody or an antigen-binding fragment thereof that specifically recognizes a WT1 peptide/HLA-A24 complex, a nucleic acid encoding the antibody or an antigen-binding fragment thereof, a vector containing this nucleic acid, an antibody or an antigen-binding thereof. The present invention relates to a method for producing a fragment, a chimeric antigen receptor containing an antibody or an antigen-binding fragment thereof, a nucleic acid encoding the chimeric antigen receptor, a vector containing the nucleic acid, cells expressing the chimeric antigen receptor, and a composition containing the same.

  The WT1 gene is derived from a gene (Non-patent Document 1) isolated as one of the causative genes of Wilms tumor, and includes growth factor gene (PDGF α chain, CSF-1, IGF-II), growth factor receptor gene (IGF). -IR, EGFR) and transcription of other genes (RAR-α, C-myb, C-myc, bcl-2, ornithine decarboxylase, N-myc). On the other hand, the WT1 gene is known to promote transcription of RbAp46, Dax-1, and bcl-2 genes. The WT1 gene is also highly expressed in leukemia and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer. It is said that there is (Patent Document 1). It is said that these diseases are involved in tumorigenesis and play an important role in the onset and progression of solid tumors and hematopoietic tumors.

  It is known that in patients with hematopoietic tumors and the like, a humoral immune reaction against a protein derived from WT1 and a cell-mediated immune reaction by CD8-positive T cells are naturally induced. In addition, multiple cytotoxic T lymphocyte (CTL) epitope peptides recognized by WT1 to CD8-positive T cells have been identified. For example, human leukocyte antigen HLA-A0201 restricted 126-134 peptide, HLA-A2402 restricted 235-243 peptide and the like have been reported. It has been reported that CTLs that recognize these peptides damage HLA-restricted tumor cells expressing the WT1 antigen. Although the expression of the WT1 gene in normal cells excluding normal hematopoietic stem cells is extremely low, it is said that normal hematopoietic stem cells have the same level of expression as tumor cells. However, in a human in vitro colony-forming ability inhibition experiment using WT1 peptide-specific CTL and an in vivo experiment using a WT1 peptide vaccine in mice, no damage to normal hematopoietic stem cells was observed (Non-Patent Document 2). ..

  At present, many tumor antigens are found on the surface of various tumor cells, and the expression intensity in each tumor cell or normal tissue has been reported. Therapies based on cell-mediated or humoral immunity that target these tumor antigens have been studied, but there are various types of tumors and tumor antigens, and the selection and selection of appropriate tumor antigens for treatment of each tumor Trial and error continues to find therapeutic strategies based on tumor antigens.

US Patent No. 7696180

Molecular Cell Biology (Mol. Cell. Biol.), Vol. 11, p. 1707 (1991). Japanese Journal of Clinical Oncology (Jpn. J. Clin. Oncol.), Volume 40, pp. 377-387 (2010).

  An object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically recognizes the WT1 peptide/HLA-A24 complex.

  As a result of diligent efforts to solve the above problems, the present inventors have found an antibody or an antigen-binding fragment thereof that specifically recognizes the WT1 peptide/HLA-A24 complex, and completed the present invention.

That is, to outline the present invention,
[1] A heavy chain containing VH-CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8, VH-CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9, and VH-CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10 in the sequence listing. A chain, and VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 11 of the sequence listing, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 12 and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 13 An anti-WT1 peptide/HLA-A24 complex antibody comprising a light chain or an antigen-binding fragment thereof,
[2] From a heavy chain containing a heavy chain variable region (VH) consisting of the amino acid sequence shown in SEQ ID NO:6 and a light chain containing a light chain variable region (VL) consisting of the amino acid sequence shown in SEQ ID NO:7 The antibody or the antigen-binding fragment thereof according to [1],
[3] The antibody or antigen-binding fragment thereof according to [1] or [2], wherein the WT1 peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing,
[4] The antigen-binding fragment of an antibody is Fab, Fab′, F(ab′) ₂ , Fv, or single-chain Fv (scFv), according to any one of [1] to [3]. An antigen-binding fragment of
[5] An isolated nucleic acid encoding the antibody or the antigen-binding fragment thereof according to any one of [1] to [4],
[6] The nucleic acid according to [5], which comprises a base sequence encoding the amino acid sequence represented by SEQ ID NO:6 in the sequence listing and a base sequence encoding the amino acid sequence represented by SEQ ID NO:7.
[7] A vector containing the nucleic acid according to [5] or [6],
[8] A method for producing an anti-WT1 peptide/HLA-A24 complex antibody or an antigen-binding fragment thereof, comprising a step of expressing the nucleic acid according to [5] or [6] or the vector according to [7],
[9] A composition comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [4],
[10] A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [4] and an intracellular domain of a signal transduction protein,
[11] The chimeric antigen receptor according to [10], wherein the signal transduction protein is CD3ζ chain,
[12] The chimeric antigen receptor according to [11], which further comprises an intracellular domain of CD28,
[13] A nucleic acid encoding the chimeric antigen receptor according to any one of [10] to [12],
[14] A vector containing a nucleic acid encoding the chimeric antigen receptor according to [13],
[15] A cell expressing the chimeric antigen receptor according to any one of [10] to [12],
[16] A pharmaceutical composition comprising the cell according to [15] as an active ingredient.
Regarding

  According to the present invention, an antibody or an antigen-binding fragment thereof that specifically recognizes the WT1 peptide/HLA-A24 complex, a nucleic acid encoding the antibody or an antigen-binding fragment thereof, a vector containing this nucleic acid, an antibody or an antigen-binding thereof A method for producing a fragment, a chimeric antigen receptor containing an antibody or an antigen-binding fragment thereof, a nucleic acid encoding the chimeric antigen receptor, a vector containing this nucleic acid, a cell expressing the chimeric antigen receptor, and a composition containing these are provided. To be done. An antibody or an antigen-binding fragment thereof that specifically recognizes the WT1 peptide/HLA-A24 complex can be used in the fields of cell medicine and gene therapy, and a tumor cell expressing the WT1 peptide/HLA-A24 complex. It is extremely useful for the detection, treatment, research and testing therefor.

It is a figure which shows the specificity of the antibody fragment of this invention. It is a figure which shows the binding characteristic of the antibody fragment of this invention. It is a figure which shows the analysis result of the binding characteristic of the antibody fragment of this invention. FIG. 3 is a diagram showing the binding between cells displaying the WT1 peptide/HLA-A24 complex and the antibody fragment of the present invention. FIG. 3 is a diagram showing the binding between cells displaying the WT1 peptide/HLA-A24 complex and the antibody fragment of the present invention. FIG. 3 is a diagram showing the binding between cells displaying the WT1 peptide/HLA-A24 complex and the antibody fragment of the present invention. It is a figure which shows the cell surface expression of the chimeric antigen receptor of this invention. It is a figure which shows the cytokine produced by the chimeric antigen receptor of this invention. It is a figure which shows the cytotoxic activity of the cell which expresses the chimeric antigen receptor of this invention. It is a figure which shows the effect in the living body of the cell which expresses the chimeric antigen receptor of this invention.

  In the present specification, “WT1” is a protein encoded by a gene isolated as one of the genes responsible for Wilms tumor as described above. The amino acid sequence is described in NCBI RefSeq:NP_000369.3, and the base sequence is described in NCBI RefSeq:NM_000378.4. The expression of WT1 in cells can be quantified by the method described in Patent Document 1, for example.

  As used herein, "antibody" refers to an antigen binding protein of the immune system. A full-length naturally-occurring antibody having an antigen-binding region is a glycoprotein containing two heavy chains (H chains) and two light chains (L chains) interconnected 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 is comprised of three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, the complementarity determining regions (CDRs) and some conserved regions, the framework regions (FRs). In VH and VL, three CDRs and four FRs are arranged in this order from FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. It is known that CDR3 is important for binding to an antigen. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

  As used herein, “HLA (Human Leukocyte Antigen)” means human major histocompatibility complex (MHC), which is a cell surface molecule required for antigen presentation to immune cells. Refers to the complex of the encoding gene. HLA can be classified into Class I and Class II. Class I HLA is composed of α-chain and β2-microglobulin. Class I HLA is expressed by almost all nucleated cells and has been shown to function in antigen presentation to CD8+ T cells. Class I HLA can be further divided into HLA-A, HLA-B, and HLA-C.

  As used herein, the term “chimeric antigen receptor (CAR)” means a fusion protein containing an extracellular domain that binds to an antigen, a transmembrane domain derived from a polypeptide different from the extracellular domain, and at least one intracellular domain. Say. Sometimes referred to as "chimeric receptor", "T-body", "chimeric immunoreceptor (CIR)". The "extracellular domain that binds to an antigen" refers to any oligo- or polypeptide capable of binding an antigen, and the "intracellular domain" refers to a signal that results in the activation or inhibition of a biological process within a cell. By any oligo or polypeptide known to function as a transduction domain.

  As used herein, an “antigen-binding fragment” is a partial region, portion, or protein fragment of an antibody that binds to the antigen and imparts specificity to the antigen.

Hereinafter, the present invention will be specifically described.
(1) Antibodies of the Present Invention or Antigen-Binding Fragments and Nucleic Acids Encoding These The anti-WT1 peptide/HLA-A24 complex antibodies of the present invention are HLA-A24-restricted WT1 235-243 peptides (CMTWNQMNL: SEQ ID NO: 1; hereinafter referred to as WT1 peptide), and an antibody that specifically recognizes and binds to a complex consisting of HLA-A24. Here, HLA-A24 is composed of HLA-A24 α chain (SEQ ID NO: 2) and β2-microglobulin (SEQ ID NO: 3). The antibody of the present invention recognizes these complexes more specifically than the WT1 peptide or HLA-A24 present alone. Furthermore, the antibody of the present invention does not bind to the complex of peptides other than WT1 peptide and HLA-A24 and the complex of WT1 peptide and HLA other than HLA-A24, or has low binding activity. Therefore, the antibody of the present invention can specifically detect or target the WT1 peptide/HLA-A24 complex.

  The antibody of the present invention comprises VH-CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8, VH-CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9 and VH-CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10. And a VL-CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 11, VL-CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 12, and VL-consisting of the amino acid sequence represented by SEQ ID NO: 13 in the sequence listing. It consists of a light chain containing CDR3.

  In one aspect of the present invention, the antibody of the present invention is a heavy chain containing a heavy chain variable region (VH) consisting of the amino acid sequence shown in SEQ ID NO:6 and a light chain variable consisting of the amino acid sequence shown in SEQ ID NO:7. It consists of a light chain containing the region (VL). The nucleotide sequence encoding the amino acid sequence of VH is shown in SEQ ID NO:4, and the nucleotide sequence encoding the amino acid sequence of VL is shown in SEQ ID NO:5. Further, in the amino acid sequences of the heavy chain variable region and the light chain variable region, sequences other than the complementarity determining region are replaced with other amino acid sequences, for example, amino acid sequences of antibody frameworks derived from humans or other animals. Such antibodies are also included in the present invention.

In one aspect of the invention, the invention comprises an antigen binding fragment of an anti-WT1 peptide/HLA-A24 complex antibody. Antibodies include one or more fragments that specifically bind an antigen. It has been shown that an antibody fragment containing the antigen-binding region of an antibody specifically recognizes an antigen like a full-length antibody. Examples of the antibody fragment contained in the antigen-binding fragment of the present invention include Fab, Fab′, F(ab′) ₂ , Fv, and single chain Fv (scFv). Fab is a monovalent antibody fragment composed of VL, VH, CL and CH1 domains. Fab′ is a monovalent antibody fragment composed of VL, VH, CL, CH1 domain and hinge region. F(ab′) ₂ is a divalent antibody fragment containing two Fab fragments linked by a hinge disulfide bond. Fv is a monovalent antibody fragment composed of single-armed VL and VH of the antibody. The two domains of the Fv fragment, VL and VH, are encoded by separate genes, and these domains can be linked by a linker by a gene recombination technique to produce a single molecule of protein scFv. The scFv additionally contains a linker between VH and VL. The linker is a peptide commonly used in the art for artificially linking VH and VL to stabilize the antigen-binding ability of scFv antibody (Reference: For example, GS linker is Huston et al., Methods). in Enzymology, 203:46-88 (1991), EK linker is Whitlow, et al., Protein Eng., 6:989 (1993)). The linker generally comprises glycine and serine and is 15-18 amino acids in length.

In one aspect of the invention, the invention comprises a combination of antigen binding fragments. Such molecules are exemplified by Fab ₃ , Diabody, Triabody, Tetrabody, Minibody, Bis-scFv, (scFv) ₂ -Fc, intact-IgG, Holliger et al., Nature Biotechnology 9, Vol. 23, 23. ． 1126-36 (2005).

  The antibody or antigen-binding fragment of the present invention may be modified by one, several or 1-9 amino acids, as long as it does not substantially affect its properties. For example, amino acids can be substituted, deleted, added or inserted in the constant region or FR region. Such modifications include, for example, site-specific mutagenesis (point mutation and cassette mutagenesis), gene homologous recombination method, primer extension method, PCR method, and other modifications of nucleic acids well known to those skilled in the art. A combination of methods can be easily implemented.

  The present invention further includes a nucleic acid encoding the above-described antibody or antigen-binding fragment of the present invention. As one aspect of the present invention, the nucleic acid of the present invention comprises the base sequence shown in SEQ ID NO:4 and the base sequence shown in SEQ ID NO:5 in the sequence listing. The nucleic acid of the present invention can be modified to use a codon suitable for a host cell (optimal codon) without changing the encoded amino acid sequence. By thus changing the optimal codon, the expression efficiency of the polypeptide in the host cell is improved.

  The antibody or antigen-binding fragment of the present invention can be produced by a method known to those skilled in the art, for example, various means such as genetic engineering techniques or chemical synthesis. As a genetic engineering technique, for example, a replicable cloning vector or expression vector containing the nucleic acid of the present invention is prepared, and this vector is introduced into a host cell by an appropriate method known to those skilled in the art, and the host cell is cultured. Then, the nucleic acid can be expressed, and the resulting polypeptide can be recovered and purified. When the nucleic acid of the present invention comprises a plurality of nucleic acid molecules, a combination (set) of a plurality of vectors containing each nucleic acid molecule is introduced into a host cell, or one vector containing a plurality of nucleic acids is introduced into a host cell. You may. In addition, when producing a polypeptide, a peptide tag can be linked to the desired polypeptide and purified using the peptide tag.

  The vector usable in the present invention is operably linked to appropriate control sequences so that the nucleic acid of the present invention is expressed in a suitable host. The control sequence includes a promoter for transcription of the nucleic acid of the present invention, an arbitrary operator sequence for controlling transcription, a sequence encoding a ribosome binding site, an enhancer, a polyadenylation sequence, and termination of transcription or translation. The control sequence is included. Further, various sequences known to those skilled in the art, for example, restriction enzyme cleavage site, marker gene (selection gene) such as drug resistance gene, signal sequence, leader sequence and the like may be used in the vector, if necessary. These various sequences or sites can be appropriately selected and used by those skilled in the art depending on the type of polypeptide to be expressed, the host cell used, the culture medium, and other conditions.

  As the vector usable in the present invention, any of a vector that is integrated into the host genome, a vector that is not integrated, and an episomal vector that exists in the cytoplasm and autonomously replicates can be used. For example, retrovirus vector (including oncoretrovirus vector, lentivirus vector, pseudotype vector), adenovirus vector, adeno-associated virus (AAV) vector, simian virus vector, vaccinia virus vector or Sendai virus vector, Epstein-Barr vector. Viral vectors such as virus (EBV) vector and HSV vector can be used. As the above-mentioned viral vector, one lacking the replication ability so that the virus cannot self-replicate in infected cells is preferable. In addition, a non-viral vector can also be used in combination with a liposome and a condensing agent such as a cationic lipid. Furthermore, the nucleic acid of the present invention can be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation, particle bombardment.

  As the host cell into which the nucleic acid of the present invention is introduced, any cell known to those skilled in the art can be used. For example, typical host cells include prokaryotic cells such as E. coli, and Examples thereof include mammalian cells such as Chinese hamster ovary cells (CHO cells) and human-derived cells, and eukaryotic cells such as yeast and insect cells.

  The antibody or antigen-binding fragment of the present invention expressed in a host cell can be purified from the culture medium of the host cell, an extract and/or a lysate of the host cell. The purification method can be carried out by appropriately combining arbitrary methods known to those skilled in the art. For example, centrifugation, hydroxyapatite chromatography, gel electrophoresis, dialysis, fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, anion or cation. It is preferably purified by resin chromatography (polyaspartic acid column etc.), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and affinity chromatography. Affinity chromatography is one of the preferred purification techniques with high efficiency, which utilizes the affinity of a polypeptide with a peptide tag.

(2) Composition of the present invention The present invention provides a composition comprising the antibody or antigen-binding fragment of the present invention described in (1) above, and a composition comprising a nucleic acid encoding the antibody or antigen-binding fragment of the present invention. I will provide a.

  The antibody or antigen-binding fragment of the present invention can be used as a probe for the complex of WT1 peptide and HLA-A24. In particular, it has been clarified that WT1 is presented to HLA on the surface of cancer cells, and T cells attack cancer cells based on their antigen recognition. Therefore, the antibody or antigen-binding fragment of the present invention of the present invention can be used as a probe for tumor (cancer) cells, that is, a composition for detection or a composition for diagnosis.

  Examples of the detection method include conventional common detection methods such as enzyme-linked immunosorbent assay (ELISA), fluorescent antibody assay, radioimmunoassay, radioimmunoprecipitation method, dot blot assay, inhibition or competition assay, sandwich assay, latex beads. Examples include, but are not limited to, agglutination assays.

  The sample to be diagnosed or detected is a biological sample, for example, tissue or cells at a disease site, body fluid such as blood, serum, plasma, lymph, urine, and the like. An antibody of the present invention is brought into contact with these samples, or samples that have been subjected to preliminary purification, homogenization, centrifugation, or dilution as necessary, in an appropriate buffer to form a complex of the antigen and the antibody as described above. It is detected by the detection method of. In this case, the antibody or antigen-binding fragment of the present invention may be labeled, or a labeled secondary antibody may be used. Labels include enzymes (eg horseradish peroxidase, alkaline phosphatase etc.), radioisotopes (eg 32P, 35S, 3H, 125I etc.), fluorescent substances (eg rhodamine, fluoresamine, dansyl chloride, their derivatives etc.), tag peptides And so on.

The antibody or antigen-binding fragment of the present invention can be used to deliver a drug to a target. The antibody or antigen-binding fragment of the present invention linked to a drug specifically recognizes the complex of WT1 peptide and HLA-A24, and delivers the drug to the target. Examples of drugs that can be linked include proteins such as cytokines (IL2, TNF, IFN-γ) and their receptors, cytotoxins (ricin, diphtheria, gelonin, etc.), radionuclides ( 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 (calicheamicin, doxorubicin, etc.).

  The effective amount of the active ingredient of the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art depending on, for example, the therapeutic purpose, the type of tumor, the disease state in the administration subject such as site and size, various conditions of the patient, and the administration route. it can. A typical single dose or daily dose will depend on the above conditions, first in vitro, and then, if possible, for example using assays for tumor cell survival or growth known in the art. In addition, the appropriate dose range can be determined in a suitable animal model that allows the dose range for human patients to be extrapolated.

  When the composition of the present invention is used as a pharmaceutical composition, it is well known to those skilled in the art in addition to the active ingredient, depending on various conditions such as the type of active ingredient, drug form, administration method/purpose, and pathological condition of the administration subject. Various pharmaceutically acceptable components (eg, carrier, excipient, buffer, stabilizer, etc.) can be appropriately added. The pharmaceutical composition of the present invention is a tablet, liquid, powder, gel, spray, or microcapsule, colloidal distribution system (liposome, microemulsion, etc.), macroemulsion, etc., depending on the above various conditions. It can take various drug forms.

  Examples of the administration method include intravenous, intraperitoneal, intracerebral, intraspinal, intramuscular, intraocular, intraarterial, in particular bile duct, or intralesional injection or injection, and sustained release system preparation. Be done. The pharmaceutical composition of the present invention can be administered continuously by infusion or by bolus injection.

  The composition of the present invention is, for example, a hematopoietic tumor such as chronic myelogenous leukemia, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, It is used for the treatment, diagnosis and detection of solid cancers such as skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer and mesothelioma. In particular, it is useful for the treatment, diagnosis, and detection of WT1-positive and HLA-A24-positive tumors and cancers.

(3) Chimeric antigen receptor of the present invention The present invention includes the antibody or antigen-binding fragment of the present invention described in (1) above and a chimeric antigen receptor (CAR) containing the intracellular domain of a signal transduction protein. The CAR of the present invention can specifically bind to the WT1 peptide/HLA-A24 complex and transmit a signal to cells expressing CAR. The CAR of the present invention comprises an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain includes the antibody or antigen-binding fragment of the present invention. When the extracellular domain and the complex specifically bind to each other, a signal is transduced into the cell via the intracellular domain of CAR and stimulates cells expressing CAR. The stimulated cells can produce cytokines and the like, exert cytotoxicity to target cells expressing the complex, or induce cytotoxicity of other immune cells.

  As a CAR intracellular domain, a signal that is a molecule capable of transmitting a signal intracellularly when an extracellular domain existing in the same molecule binds (interacts) with the WT1 peptide/HLA-A24 complex The intracellular domain of the transfer protein can be used. Examples of the signal transduction protein that can be used in the CAR of the present invention include proteins selected from membrane proteins, signal receptors and cytokine receptors. The intracellular domain of CAR is exemplified by an intracellular domain containing primary cytoplasmic signaling sequences derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Further, for example, an intracellular domain containing a secondary cytoplasmic signal (costimulatory signal) transduction sequence derived from CD2, CD4, CD5, CD8α, CD8β, CD28, CD137, CD134, ICOS, GITR and CD154 is exemplified. Furthermore, the intracellular domain containing a tertiary cytoplasmic signaling sequence derived from the IL-2 receptor and the IL-21 receptor is exemplified.

  The transmembrane domain of CAR is, for example, α, β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, of the T cell receptor. The transmembrane domains of CD134, CD137, ICOS, CD154, GITR can be used. Also, an artificially designed array may be used.

  The invention includes nucleic acids encoding the CARs of the invention. A nucleic acid encoding a CAR can be linked to another nucleic acid so that it is expressed under the control of a suitable promoter. As the promoter, any promoter that constitutively promotes expression, one that is induced by a drug (for example, tetracycline or doxorubicin), and the like can be used. For example, phosphoglycerate kinase (PGK) promoter, Xist promoter, β-actin promoter, mammalian-derived promoter such as RNA polymerase II promoter, SV40 early promoter, cytomegalovirus promoter, herpes simplex virus thymidine kinase promoter, various retrovirus Viral promoters such as the LTR promoter can be used. Also, in order to achieve efficient transcription of the nucleic acid, a nucleic acid containing a promoter or another regulatory element cooperating with the transcription initiation site, for example, an enhancer sequence or a terminator sequence may be linked. In addition, a gene that can be a marker for confirming the expression of nucleic acid (for example, a drug resistance gene, a gene encoding a reporter enzyme, a gene encoding a fluorescent protein, etc.) may be incorporated.

  The nucleic acid encoding the CAR of the present invention can be introduced into cells by the vector usable in the present invention described in (1) above. Further, the nucleic acid encoding CAR can be used as an active ingredient of a pharmaceutical composition. A pharmaceutical composition containing a nucleic acid encoding CAR can be produced and used as described in (2) above.

  In another aspect of the invention, the invention includes cells that express the CAR of the invention. The cell can be prepared by the step of introducing a nucleic acid encoding the CAR of the present invention into the cell. A cell expressing CAR is activated by transmitting a signal into the cell by binding to the WT1 peptide/HLA-A24 complex via CAR. Cell activation varies depending on the type of host cell or intracellular domain, but can be confirmed using, for example, cytokine release, improvement in cell growth rate, changes in cell surface molecules, and the like as indicators. For example, release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells results in destruction of tumor cells expressing the complex. In addition, it stimulates other immune cells such as B cells, dendritic cells, NK cells, macrophages, etc. due to cytokine release and changes in cell surface molecules. Therefore, the CAR-expressing cells of the present invention are useful in adoptive immunotherapy, particularly in adoptive immunotherapy against WT1-positive, HLA-A24-positive tumors or cancers.

  The step of introducing the nucleic acid encoding CAR into a cell is carried out ex vivo or in vivo. As the cells into which the nucleic acid is introduced, cells derived from mammals such as humans or cells derived from non-human mammals such as monkeys, mice, rats, pigs, cows and dogs can be used. As the cell type, for example, cells collected, isolated, purified, or derived from blood (peripheral blood, umbilical cord blood, etc.), body fluid such as bone marrow, tissue or organ can be used. PBMC, immune cell [dendritic cell, B cell, hematopoietic stem cell, macrophage, monocyte or NK cell, hematopoietic cell (neutrophil, basophil, monocyte)], hematopoietic stem cell, cord blood mononuclear cell, fiber Blast cells, preadipocytes, hepatocytes, blood cells, skin keratinocytes, mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, pluripotent stem cells, various cancer cell lines or neural stem cells can be used. In the present invention, it is particularly preferable to use T cells, T cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.), pluripotent stem cells, or a cell population containing these. T cells include CD8-positive T cells, or CD4-positive T cells, regulatory T cells, cytotoxic T cells, and tumor infiltrating lymphocytes. The cell population containing T cells and T cell progenitor cells includes PBMCs. The cells used in the present invention may be either cells collected from a living body, cells that have been expanded and cultured, or cells that have been established as cell lines. When it is desired to transplant a cell into which a nucleic acid has been introduced or a cell differentiated from the cell into a living body, it is preferable to introduce the nucleic acid into the living body itself or a cell collected from a living body of the same species.

Cells expressing the CAR of the invention may be cultured and/or stimulated with an appropriate medium and/or stimulatory molecule prior to administration to a subject. Stimulatory molecules include cytokines, appropriate proteins, and other components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, IFN-γ, etc., and a medium containing IL-2 is preferably used. The concentration of IL-2 in the medium is not particularly limited, but is, for example, preferably 0.01 to 1×10 ⁵ U/mL, more preferably 1 to 1×10 ⁴ U/mL. Examples of suitable proteins include CD3 ligand, CD28 ligand and anti-IL-4 antibody. In addition to these, a lymphocyte stimulating factor such as lectin can be added. Furthermore, serum or plasma may be added to the medium. The amount of these added to the medium is not particularly limited, but is more than 0 to 20% by volume, and the amount of serum or plasma to be used can be changed depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced before use. The origin of serum or plasma may be either autologous (meaning that it has the same origin as the cells to be cultured) or non-self (meaning that it has the same origin as the cells to be cultured), but is preferably Is self-derived from the viewpoint of safety.

The cell culture equipment used for culturing cells is not particularly limited, and for example, a petri dish, a flask, a bag, a large culture tank, a bioreactor or the like can be used. A CO ₂ gas permeable bag for cell culture can be used as the bag. Further, in the case of industrially producing a large amount of cell population, a large culture tank can be used. The culture can be performed in either an open system or a closed system, but it is preferable to perform the culture in a closed system from the viewpoint of the safety of the obtained cell population.

The present invention includes a pharmaceutical composition containing CAR-expressing cells as an active ingredient. The pharmaceutical composition of the present invention is parenterally administered to a subject before use. Parenteral administration methods include methods such as intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. It may also be administered to meningioma or nearby tissue, eg subcutaneously, to enhance its antitumor effect. The dose is appropriately selected depending on the condition of the subject, body weight, age, etc., but usually, as a cell number, about 10 ⁷ to 10 ⁹ cells per subject per 60 kg body weight, preferably, About 5×10 ^{7 to} 5×10 ⁸ cells are administered. The pharmaceutical composition of the present invention can be administered once or multiple times. The pharmaceutical composition of the present invention can be in a known form suitable for parenteral administration, for example, injection or infusion. The pharmaceutical composition of the present invention may optionally contain a pharmacologically acceptable excipient. The pharmaceutical composition of the present invention may also contain physiological saline, phosphate buffered saline (PBS), medium and the like in order to stably maintain cells. The medium is not particularly limited, but mediums such as RPMI, AIM-V and X-VIVO10 are generally used.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples.
Example 1 Screening of scFv specific to WT1 peptide/HLA-A24 complex (1) Preparation of WT1 peptide/HLA-A24 complex A WT1 peptide having the amino acid sequence shown in SEQ ID NO: 1 was synthesized (Hokkaido Biosystems). In addition, the extracellular fragment of HLA-A24 heavy chain having a biotinylated site at the C-terminus (SEQ ID NO: 2) and β2-microglobulin (β2M) having the amino acid sequence shown in SEQ ID NO: 3 were expressed in E. coli and purified. 10 mg WT1 peptide, 13.2 mg β2M, 18.6 mg HLA-A24 heavy chain extracellular fragment in 200 mL solution (100 mM Tris-HCl (pH 8), 400 mM L-Arginine-HCl, 2 mM Na2EDTA, 5 mM red. Glutathione, 0.5M oxid.glutathione, 0.1 mM PMSF, 0.2 mg pepstatin, 0.2 mg leupeptin), and then dialyzed with distilled water, Labscale TFF system (Millipore) and Amicon Ultra 10k (Millipore). The product was concentrated. The concentrate was subjected to fractionation with AKTA (manufactured by GE) to obtain the WT1 peptide/HLA-A24 complex. Similar to WT1 peptide, hTERT (human telomerase reverse transcriptase), SAGE (meat seed antigen), Her2 (human epidermal growth factor receptor type 2), CMV (cytomegalovirus), EBNA (Epstein-Barr virus nuclear antigen) )-Derived HLA-A24-restricted peptide and a complex of HLA-A24 were prepared. Then, the WT1 peptide/HLA-A24 complex was biotinylated with biotin ligase (manufactured by AVIDITY). The biotinylated product was purified again with AKTA and designated as A24-WT1.

(2) Screening for scFv 20 μg of A24-WT1 prepared in Example 1-(1) was mixed with 200 mg of streptavidin-conjugated magnetic beads, and the mixture was reacted overnight in a 0.05% Tween/PBS solution at 4° C. After that, 3 μL of 2 mM biotin/PBS was added thereto and reacted for 1 hour. This was washed twice with a 0.05% Tween/PBS solution, and then suspended in 400 μL of a 0.05% Tween/PBS solution, which was used as an antigen bead solution.
As a reaction solution, a phage library solution of an antibody derived from human B lymphocytes equivalent to 1×10 ¹³ cfu, 100 μg of streptavidin (manufactured by PIERCE), 50 μg of A24-CMV prepared in Example 1-(1), 50 μg A24-Her2, 1% Triton X-100/PBS was prepared, rotatically mixed at room temperature for 1 hour, then 100 μL of the antigen bead solution was mixed, and tumbled for 1 hour.

  Then, the magnetic beads trapped by a magnet trapper (manufactured by Toyobo Co., Ltd.) were washed 5 times with 1% Triton X-100/PBS and then once with PBS. The magnetic beads recovered after washing were added to the cultured Escherichia coli DH12S, and the cells were infected at 37°C for 1 hour. It was cultured overnight at 30° C. in 500 mL of 2×YT medium (2×YTAG) containing 200 μg/mL ampicillin and 1% glucose.

  5 mL of 2×YT medium (2×YTA) containing 200 mM ampicillin and 50 μL of helper phage M13KO7 were added to 1 mL of the culture solution and incubated at 37° C. for 1 hour, and then 2×YT containing 50 μM kanamycin and 200 μM ampicillin (2×). (YTAK) (500 mL) was added, and the mixture was cultured at 30° C. overnight. The supernatant obtained by centrifugation was mixed with 100 mL of 20% PEG#600, 2.5 M NaCl solution, and the mixture was centrifuged to collect the precipitate. This was suspended in 10 mL PBS and then filter sterilized. The procedure from the incubation with the antigen bead solution to the filter sterilization was repeated 4 times, and after the 4th time, the recovered Escherichia coli was treated with LBGA plate (200 μg/mL ampicillin, 0.1% glucose in an ordinary agar medium (Nissui Co., Ltd.). Made)). After culturing at 30° C., the resulting colonies were cultivated in 2×YTAG medium at 30° C. overnight, and a part of the culture solution was used to prepare DNA by miniprep DNA kit (manufactured by QIAGEN) and sequenced. I went. At this stage, 19 clones were determined to be positive, that is, capable of binding to A24-WT1. Further, 50 μL of the culture solution was mixed with 1.5 ml of 2×YTAI (2×YTA medium containing 0.5 mM IPTG), incubated at 30° C. overnight, and then centrifuged to obtain a supernatant phage solution. This phage solution was used for the ELISA method.

(3) Acquisition of WT1 peptide/HLA-A24 complex-specific clone 500 ng neutravidin (manufactured by PIERCE) suspended in 50 μL of PBS was put in Maxisopose (manufactured by NUNC), and shaken overnight at 4° C. on a plate. Neutraavidin was immobilized. After the completion, the solution was discarded, 200 μL of 2% BSA/PBS was added, and the mixture was left standing overnight. A24-WT1 prepared in Example 1-(1) was placed in this plate so as to be 300 ng/50 μL PBS and shaken at 4° C. overnight. After completion, the plate was washed with PBS to give an antigen plate. As a control, the HLA-A24-restricted peptides of A24-hTERT, A24-SAGE, A24-Her2, A24-CMV, and A24-EBNA prepared in Example 1-(1) were used, and Example 1-(1) was used. Similarly, the biotinylated HLA-A24 complex was prepared to prepare an antigen-immobilized plate.
100 μL of the phage solution prepared in Example 1-(2) was placed in each antigen-immobilized plate, shaken at room temperature for 1 hour, washed with PBS, and then with 0.05% Tween20/PBS. 100 μL of 2000-fold diluted anti-cp3 rabbit antibody was added and shaken at room temperature for 1 hour. After washing the plate with PBS, 100 μL of HRP-labeled anti-rabbit IgG (manufactured by MBL) diluted 4000-fold with 0.05% Tween 20/PBS was added and shaken at room temperature for 1 hour. After the plate was washed with PBS, OPD (manufactured by WAKO) suspended in 0.01% H ₂ O ₂ , 0.1M Na ₂ PO ₄ , and 0.1M citric acid (pH 5.1) was reacted. After confirming the color development, the reaction was stopped with 2N sulfuric acid, and the absorbance was measured at a wavelength of 490 nm with SpectraMax M2 (manufactured by Molecular Devices).

  From the 19 phage clones obtained in Example 1-(2), a WT#213 clone that specifically binds to the WT1 peptide/HLA-A24 complex was obtained. Furthermore, the concentration of the WT#213 clone was changed to confirm the affinity with the WT1 peptide/HLA-A24 complex. The results are shown in FIGS. 1 and 2. The vertical axis of FIG. 2 represents the absorbance and the horizontal axis represents the dilution of the WT#213 clone. From FIG. 1 and FIG. 2, it was confirmed that the scFv presented by the WT#213 clone had very high binding specificity with the WT1 peptide/HLA-A24 complex and high affinity.

(4) Evaluation of WT#213 clone The Biacore (manufactured by GE Healthcare Bioscience) was used for the dissociation constant measurement of the scFv of the WT#213 clone and the WT1 peptide/HLA-A24 complex. The WT#213 clone was immobilized and the WT1 peptide/HLA-A24 complex was measured as an analyte. HBS buffer (0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% (v/v) Surfactant P20) was used as a running buffer. For the analysis of the data, the association rate constant (ka), the dissociation rate constant (kd) and the affinity (KD) were calculated using analysis software. As a result, the ka value was 3.01e ⁴ , the kd value was 0.117, and the KD value was 3.89 μM. The analysis graph is shown in FIG. From FIG. 3, it was confirmed that the scFv of the WT#213 clone had a high KD value and a smooth kd curve, and thus this scFv has a strong binding force to the antigen and is difficult to separate.

Example 2 Evaluation of WT#213 scFv (1) Preparation of WT1 scFv tetramer The nucleotide sequence of VH of scFv of WT#213 clone is shown in SEQ ID NO:4, and the nucleotide sequence of VL is shown in SEQ ID NO:5. The amino acid sequence of VH is shown in SEQ ID NO:6, and the amino acid sequence of VL is shown in SEQ ID NO:7. The amino acid sequences of CDR1, CDR2 and CDR3 of VH are shown in SEQ ID NOs: 8, 9 and 10, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 11, 12 and 13, respectively.
Furthermore, a DNA fragment encoding scFv was prepared by PCR using the DNA extracted from the WT#213 clone as a template. As the primers, F primer (SEQ ID NO: 14) and R primer (SEQ ID NO: 15) were used. With reference to the bulletin of the American Academy of Sciences (Proc. Natl. Acad. Sci. USA), 2003, Vol. 100, No. 13, p. 7480-7485, the amplified DNA fragment was digested with the restriction enzyme site SalI-of pHisAvi. The resulting plasmid was inserted into AscI and designated as pWT#213scFvHisAvi. pWT#213scFvHisAvi expresses a fusion protein with a PelB leader, a His tag, a WT#213scFv and an Avi tag, and a fusion protein with a PelB leader and biotin ligase (BirA). Escherichia coli DH5α strain transformed with pWT#213scFvHisAvi was cultured overnight at 30° C. in 500 mL of 2×YTA medium (2×YTAIB medium) containing 0.5 mg/mL IPTG and 2 μM biotin. The culture solution was centrifuged (8000 rpm, 4° C. for 10 minutes), and 145.5 g of ammonium sulfate was added to the obtained supernatant to make it 50% saturated and completely dissolved. The precipitate collected by centrifugation (8000 rpm, 4° C. for 10 minutes) was suspended in PBS containing complete (Roche). The suspension was centrifuged (100,000 rpm, 4° C. for 30 minutes), the collected supernatant was filtered with a 0.45 μm filter (manufactured by Millipore), and the filtrate was then subjected to Ni-NTA agarose (manufactured by QIAGEN). ) The column was applied, and the column was washed with 0.1% Tween 20/PBS and PBS. The protein bound to the column was eluted with 50 mM citrate (pH 2.5) and then neutralized with 3 M Tris. Then, dialysis with PBS was performed and concentration with Amicon Ultra (Millipore) was performed.

13 μg of the protein obtained by the above operation was mixed with 4 μg of PE-labeled Streptavidin (manufactured by Prozyme), 0.05% NaN ₃ /PBS was added to make the total volume 400 mL, and the mixture was shielded from light and slowly at 4° C. WT #213 scFv tetramer was prepared by rotating and mixing overnight.

(2) Reactivity of WT1 peptide pulsed LCL and WT#213 scFv In Example 1-(1), a lymphoblastoid cell line (LCL), which is an HLA-A24-positive cell, was prepared in RPMI medium. The prepared WT1 peptide was pulsed for 24 hours. The pulsed LCL was washed with PBS, the WT#213 scFv tetramer prepared in Example 2-(1) was added, and the mixture was allowed to stand at 4°C for 1 hour. The LCL was washed with PBS, fixed with PBS containing 1% paraformaldehyde, and flow cytometry was performed. As a control, LCL not pulsed with peptide was similarly prepared. The results are shown in Fig. 4. From FIG. 4, it was confirmed that WT#213scFv shows reactivity to LCL pulsed with WT1 peptide.

(3) Reactivity of WT1 peptide pulse T2A24 and WT#213scFv T2A24, which is a HLA-A24 positive cell, was used to pulse WT1 peptide in the same manner as in Example 2-(2) to show the reactivity of WT#213scFv. confirmed. As a control, cells pulsed with CMV peptide were prepared. Results are shown in FIG. From FIG. 5, it was confirmed that WT#213scFv specifically reacts with the WT1 peptide.

(4) Reactivity of WT1-expressing tumor cells with WT#213scFv Using MEG-01, a human megakaryoblastic leukemia cell line that endogenously expresses WT1 in HLA-A24-positive cells, and using WT#213scFv. The reactivity with was confirmed in the same manner as in Example 2-(2). As a control, HSC-2, which is a human oral squamous cell carcinoma cell line that is HLA-A24 positive cells but does not express WT1 was used. The results are shown in Fig. 6. As a control, K562-A2 cells expressing HLA-A2 and WT1 were also used, and it was confirmed that WT#213scFv showed no reactivity. From the above, it was confirmed that WT#213scFv reacts with the WT1 peptide in an HLA-A24-restricted manner.

Example 3 Chimeric antigen receptor (CAR)
(1) Preparation of retrovirus plasmid expressing CAR having WT#213 scFv scFv against EGFR and human IgG-CL of pMS3-EGFR-LC-zG-CAR plasmid vector described in WO 2013/051718 pamphlet. The DNA encoding the (light chain constant region) domain was replaced with the DNA fragment encoding WT#213scFv prepared in Example 2-(1) to prepare a pMS3-WT#213-zG-CAR plasmid vector. This vector expresses CAR having a leader sequence, WT#213scFv, CD28 transmembrane region (TM), CD3 zeta chain intracellular domain, and GITR (glucocorticoid-induced tumor necrosis factor receptor) intracellular domain in this order from N terminus. . This CAR will be referred to as WT#213 CAR.

(2) Preparation of Retrovirus Solution Escherichia coli JM109 was transformed with the plasmid vector prepared in Example 3-(1) to obtain a transformant. The plasmid DNAs retained by these transformants were each purified using NucleoBond Xtra Midi Kit (manufactured by Machliner Gel) and subjected to the following operations as DNA for transfection.
293T cells were respectively transfected with each of the prepared DNAs for transfection and the pGP vector and pEeco vector contained in Retrovirus Packaging Kit Eco (manufactured by Takara Bio Inc.). This operation was performed according to the product protocol of the kit. A supernatant containing ecotropic virus was obtained from each of the obtained transduced cells and filtered with a 0.45 μm filter (Milex HV, manufactured by Millipore). Using this supernatant, PG13 cells (ATCC CRL-10686) were infected with ecotropic virus by a method using polybrene. The culture supernatant of the obtained cells was collected and filtered through a 0.45 μm filter to obtain a retrovirus solution for expressing WT#213 CAR.

(3) Expression of WT#213CAR Peripheral blood mononuclear cells (PBMC) separated from human peripheral blood obtained by obtaining informed consent were added to the retrovirus for WT#213CAR expression prepared in Example 3-(2). The solution was infected twice by a standard method using RetroNectin (registered trademark, manufactured by Takara Bio Inc.) to prepare WT#213 CAR expressing PBMC.
Cells (GMC) 14 days after virus infection and PBMC (NGMC) into which no vector was introduced as a control were stained with anti-human IgG Lambda antibody and Alexa Flour 488-labeled anti-Rabbit IgG antibody. Separately, PE (phycoerythrin: Becton Dickinson) labeled WT1-A24 tetramer was added. Using a flow cytometer, regarding the cells after staining, the proportion of cells that are positive for fluorescence labeling, that is, the proportion of cells that are positive for CAR and positive for CAR that binds to the WT1 peptide/HLA-A24 complex, It was measured. The result is shown in FIG. 7. As shown in FIG. 7, a high CAR positive rate was confirmed, and it was found that CAR binding to the WT1 peptide/HLA-A24 complex was expressed on the cell surface.

(4) Functional evaluation of WT#213 CAR expressing cells (cytokine production, marker expression)
The WT#213 CAR expressing cells prepared 14 days after virus infection prepared in Example 3-(3) were collected, and intracellular cytokines were stained with a 96-well plate as follows. In the medium containing the intracellular transport inhibitor Brefeldin A (manufactured by Sigma), the WT#213 CAR expressing cells (GMC) and the PBMC (NGMC) into which the vector was not introduced as a control were adjusted to 1.0×10 ⁶ cells/mL. The suspension suspended in 100 μL was added to each well of the plate. Further, 100 μL of 1.0×10 ⁶ cells/mL suspension of cells pulsed with WT1 peptide was added to the T2A24 cell line, and cocultured for 5 hours. As controls, the T2A24 cell line not pulsed with the WT1 peptide and the T2A24 cell line pulsed with the CMV peptide were used, and the same operation was performed. The co-cultured cells were stained with anti-Human CD8 antibody and anti-Human CD4 antibody, and then treated with IntraPrep Reagent (manufactured by Beckman Coulter, Inc.) and stained with anti-Human IFNγ antibody, anti-Human CD107a antibody and anti-Human Mip1b antibody. went. Using a flow cytometer, the ratio of each cytokine-producing cell in CD8-positive cells or CD4-positive cells was measured for the stained cells. The results are shown in FIG. As shown in FIG. 8, it was confirmed that WT#213CAR expressing cells (GMC) recognize the WT1 peptide and produce various cytokines and markers.

(5) Functional evaluation of WT#213 CAR expressing cells (cytotoxic activity)
The WT#213 CAR expressing cells (GMC) prepared 14 days after virus infection prepared in Example 3-(3) were collected, and the cytotoxic activity was measured by a Calcein release assay on a 96-well plate. Calsein-AM (manufactured by Dojindo Co., Ltd.) was incorporated into T2A24 cell line, K562-A2 cell line, and MEG-01 cell line, so as to be 1.0×10 ⁵ cells/mL. For the T2A24 cell line, WT1 peptide pulsed cells, CMV peptide pulsed cells and non-pulsed cells were prepared. 100 μL was added to each well of each cell, and the WT#213 CAR-expressing cells and PBMC (NGMC) into which no vector had been introduced as a control were suspended, and 100 μL was added so that the ET ratio was 20, 10, 5, 2.5. did. In place of PBMC, a well was prepared in which 100 μL of 0.1% Triton X-100 was added as a medium and Low control as a medium. After preparing the cells and controls, the 96-well plate was incubated for 4 hours in a 37° C. CO ₂ incubator equilibrated with 5.0% CO ₂ gas. Then, with respect to 100 μL of the supernatant, the fluorescence intensity was measured at λ _ex =490 nm and λ _em =515 nm to measure the amount of released Calsein. The result of calculating the cytotoxic activity (Lysis) by the following formula is shown in FIG.

  Cytotoxic activity (%)=100×(measured value of each well-measured value of Low control)/(measured value of High control-measured value of Low control)

  As shown in FIG. 9, in the T2A24 cell line pulsed with WT1 peptide, high cytotoxic activity by PBMC into which WT#213CAR was introduced was observed. It was confirmed that WT#213CAR has high specificity since it does not show reactivity with the cell line pulsed with the CMV peptide and the cell line not pulsed with the peptide. In addition, the cytotoxic activity of the K562-A2 cell line and the MEG-01 cell line that both endogenously express WT1 was confirmed against the HLA-A24-positive MEG-01 cell line. Moreover, no cytotoxic activity specific to the HLA-A24 negative K562-A2 cell line was confirmed, which indicates that WT#213 CAR reacts with the WT1 peptide in a HLA-A24-restricted manner.

Example 4 Evaluation Using Tumor-Bearing Animals (1) Preparation of WT#213 CAR-expressing T Cell Group PBMC 5.3×10 ⁵ cells separated from human peripheral blood collected by obtaining informed consent was 0.2%. Of human serum albumin (HSA), 600 IU/mL IL-2, and 0.6% plasma in GT-T503 medium (manufactured by Takara Bio Inc.), and suspended in 5 μg/mL OKT-3 (manufactured by eBioscience). ) And 25 μg/mL of RetroNectin (manufactured by Takara Bio Inc.) were seeded on a solidified plate and cultured in a 37° C. CO ₂ incubator equilibrated with 5.0% CO ₂ gas for 4 days to expand T cells. I went. The T cells were infected twice with the WT#213 CAR expressing retrovirus solution prepared in Example 3-(2) by a standard method. The infected T cells were further cultured for 4 days in GT-T503 medium containing 0.2% human serum albumin (HSA), 600 IU/mL IL-2 and 0.6% plasma to express WT#213 CAR. A T cell population was prepared. The expression of CAR in the T cell population 8 days after the start of culture was measured by the same method as in Example 3-(3). As a result, it was confirmed that 87.7% of the lymphocytes contained in the T cell group were CD8-positive cells, and 94% of them expressed CAR. Further, 7.8% of the lymphocytes contained in the T cell group were CD4 positive cells, and 98% of them were expressing CAR, which confirmed the high expression rate of WT#213 CAR.

(2) Effect of WT#213CAR-expressing T cells in tumor-bearing mice 8 to 10-week-old NOG mice (NOD/Shi-scid, IL-2γ KO) (Institute for Experimental Animals, Central Research Institute) Irradiation was performed. On the next day, 2.5×10 ⁶ cells of WT1-positive and HLA-A24-negative K562 cell lines were subcutaneously injected into the left back of each mouse, and WT1-positive and HLA-A24-positive K562-A24 cell lines were injected into the right back. 5×10 ⁶ cells were injected subcutaneously. At the same time, 1.0×10 ⁷ cells of WT#213 CAR-expressing T cells prepared in Example 4-(1) were intravenously injected. In addition, as controls, a mouse injected with T cells not infected with a virus solution and a mouse injected with PBS instead of T cells were prepared. Five mice were used for each group. Tumor diameter and body weight were measured every 2-3 days after injection. The tumor diameter was calculated by measuring the maximum diameter and the minimum diameter of the tumor and multiplying them by the value.

Tumor size ^(mm 2) = maximum diameter (mm) × minimum diameter (mm)

  FIG. 10 shows the changes over time in the tumor diameter and body weight of the K562 cell line and the K562-A24 cell line in each mouse. In the figure, GMC is a mouse injected with WT#213 CAR-expressing T cells intravenously, NGMC is a control mouse injected with T cells not infected with the virus solution, and PBS is a control mouse injected with PBS instead of T cells. Shown respectively. The horizontal axis shows the number of days, and the vertical axis shows the product of tumor diameters. The suppression of tumor growth of the K562-A24 cell line was confirmed only in mice infused with WT#213 CAR expressing T cells. This indicates that WT#213 CAR suppresses tumor growth with the WT1 peptide restricted to HLA-A24. In addition, no difference was observed in the weight of each mouse, confirming the high safety of WT#213CAR.

  According to the present invention, an antibody or an antigen-binding fragment thereof that specifically recognizes the WT1 peptide/HLA-A24 complex, a nucleic acid encoding the antibody or an antigen-binding fragment thereof, a vector containing this nucleic acid, an antibody or an antigen-binding thereof A method for producing a fragment, a chimeric antigen receptor containing an antibody or an antigen-binding fragment thereof, a nucleic acid encoding the chimeric antigen receptor, a vector containing this nucleic acid, a cell expressing the chimeric antigen receptor, and a composition containing these are provided. To be done. An antibody or an antigen-binding fragment thereof that specifically recognizes the WT1 peptide/HLA-A24 complex can be used in the fields of cell medicine and gene therapy, and is a tumor cell expressing the WT1 peptide/HLA-A24 complex. It is extremely useful for the detection, treatment, research and testing therefor.

SEQ ID NO:1: WT1 peptide sequence
SEQ ID NO:2: Extracellular domain of HLA-A24 heavy chain amino acid sequence
SEQ ID NO:3: beta 2M amino acid sequence
SEQ ID NO:4: VH nucleic acid sequence
SEQ ID NO:5: VL nucleic acid sequence
SEQ ID NO:6: VH amino acid sequence
SEQ ID NO:7: VL amino acid sequence
SEQ ID NO:8: VH-CDR1 amino acid sequence
SEQ ID NO:9: VH-CDR2 amino acid sequence
SEQ ID NO:10: VH-CDR3 amino acid sequence
SEQ ID NO:11: VL-CDR1 amino acid sequence
SEQ ID NO:12: VL-CDR2 amino acid sequence
SEQ ID NO:13: VL-CDR3 amino acid sequence
SEQ ID NO:14: F primer sequence
SEQ ID NO:15: R primer sequence

Claims (16)

  A heavy chain containing VH-CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8, VH-CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9, and VH-CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10, and From a light chain containing VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 11 of the sequence listing, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 12 and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 13. An anti-WT1 peptide/HLA-A24 complex antibody or an antigen-binding fragment thereof.   A heavy chain containing a heavy chain variable region (VH) consisting of the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing, and a light chain containing a light chain variable region (VL) consisting of the amino acid sequence shown in SEQ ID NO: 7. Item 1. The antibody or the antigen-binding fragment thereof according to Item 1.   The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the WT1 peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. The antigen-binding fragment according to any one of claims 1 to 3, wherein the antigen-binding fragment of the antibody is Fab, Fab', F(ab') ₂ , Fv, or single-chain Fv(scFv). . An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.   The nucleic acid according to claim 5, comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO:6 and a base sequence encoding the amino acid sequence represented by SEQ ID NO:7 in the sequence listing.   A vector comprising the nucleic acid according to claim 5 or 6.   A method for producing an anti-WT1 peptide/HLA-A24 complex antibody or an antigen-binding fragment thereof, comprising the step of expressing the nucleic acid according to claim 5 or 6 or the vector according to claim 7.   A composition comprising the antibody according to any one of claims 1 to 4 or an antigen-binding fragment thereof.   A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and an intracellular domain of a signal transduction protein.   The chimeric antigen receptor according to claim 10, wherein the signal transduction protein is CD3ζ chain.   The chimeric antigen receptor according to claim 11, which further comprises an intracellular domain of CD28.   A nucleic acid encoding the chimeric antigen receptor according to claim 10.   A vector comprising a nucleic acid encoding the chimeric antigen receptor according to claim 13.   A cell expressing the chimeric antigen receptor according to any one of claims 10 to 12.   A pharmaceutical composition comprising the cell according to claim 15 as an active ingredient.

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