JP2016204358A - WT1-derived peptide recognition antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody that specifically recognizes a WT1 peptide/HLA-A24 composite or an antigen-binding fragment thereof.SOLUTION: An anti-WT1 peptide/HLA-A24 composite antibody or an antigen-binding fragment thereof consists of: a heavy chain including VH-CDR1 made up of an amino acid sequence shown in sequence number 8 of a sequence table, VH-CDR2 made up of an amino acid sequence shown in sequence number 9 of the table, and VH-CDR3 made up of an amino acid sequence shown in sequence number 10 of the table; and a light chain including VL-CDR1 made up of an amino acid sequence shown in sequence number 11 of the sequence table, VL-CDR2 made up of an amino acid sequence shown in sequence number 12 of the table, and VL-CDR3 made up of an amino acid sequence shown in sequence number 13 of the table.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an antibody that specifically recognizes a WT1 peptide / HLA-A24 complex or an antigen-binding fragment thereof, a nucleic acid that encodes an antibody or an antigen-binding fragment thereof, a vector containing this nucleic acid, an antibody or an antigen-binding property 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, a cell expressing the chimeric antigen receptor, and a composition containing these.

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

  In patients with hematopoietic tumors and the like, it is known that a humoral immune response to WT1-derived proteins and a cellular immune response by CD8 positive T cells are naturally induced. A plurality of cytotoxic T lymphocyte (CTL) epitope peptides that are recognized by CD8 positive T cells from WT1 have been identified. For example, human leukocyte type antigen HLA-A0201-restricted 126-134 peptide, HLA-A2402-restricted 235-243 peptide, and the like have been reported. CTLs that recognize these peptides have been reported to damage HLA-restricted tumor cells that express the WT1 antigen. The expression of WT1 gene in normal cells excluding normal hematopoietic stem cells is extremely low, but normal hematopoietic stem cells are said to have the same expression as tumor cells. However, in human in vitro colony formation inhibition experiments using WT1 peptide-specific CTLs and in vivo experiments using WT1 peptide vaccine in mice, no damage to normal hematopoietic stem cells has been observed (Non-patent Document 2). .

  Currently, many tumor antigens expressed on the surface of various tumor cells have been found, and the expression intensity in each tumor cell and normal tissue has been reported. Treatments based on cellular immunity or humoral immunity targeting these tumor antigens are being studied, but there are a wide variety of tumors and tumor antigens, and selection and selection of appropriate tumor antigens for the treatment of each tumor Trial and error continues to find therapeutic strategies based on tumor antigens.

US Pat. No. 7,696,180

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

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

That is, if the present invention is outlined,
[1] Weight including VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing, VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10. And VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 11, VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 12, and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 13. An anti-WT1 peptide / HLA-A24 complex antibody consisting of 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 in 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 The antibody or 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 in the sequence listing;
[4] The antigen-binding fragment of an antibody is any one of [1] to [3], wherein the antigen-binding fragment is Fab, Fab ′, F (ab ′) ₂ , Fv, or single chain Fv (scFv). An antigen-binding fragment of
[5] An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of [1] to [4],
[6] The nucleic acid according to [5], comprising a base sequence encoding the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing and a base sequence encoding the amino acid sequence shown in SEQ ID NO: 7.
[7] A vector comprising 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 signaling protein is a CD3ζ chain,
[12] The chimeric antigen receptor according to [11], further comprising 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 comprising a nucleic acid encoding the chimeric antigen receptor according to [13],
[15] A cell that expresses 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.
About.

  According to the present invention, an antibody that specifically recognizes the WT1 peptide / HLA-A24 complex or an antigen-binding fragment thereof, a nucleic acid that encodes the antibody or an antigen-binding fragment thereof, a vector containing this nucleic acid, an antibody or an antigen-binding property thereof Provided are a method for producing a fragment, a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof, a nucleic acid encoding the chimeric antigen receptor, a vector comprising the nucleic acid, a cell expressing the chimeric antigen receptor, and a composition comprising these. Is done. An antibody or 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 tumor cells that express the WT1 peptide / HLA-A24 complex It is extremely useful for detection, treatment, and research and testing for it.

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. It is a figure which shows the coupling | bonding of the cell which presents a WT1 peptide / HLA-A24 complex, and the antibody fragment of this invention. It is a figure which shows the coupling | bonding of the cell which presents a WT1 peptide / HLA-A24 complex, and the antibody fragment of this invention. It is a figure which shows the coupling | bonding of the cell which presents a WT1 peptide / HLA-A24 complex, and the antibody fragment of this 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 this specification, “WT1” is a protein encoded by a gene isolated as one of the causative genes of 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 comprising two heavy chains (H chains) and two light chains (L chains) linked together by disulfide bonds. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain includes 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 regions of VH and VL can be further subdivided into regions having hypervariability called complementarity determining regions (CDRs) and regions conserved to some extent called framework regions (FR). In VH and VL, three CDRs and four FRs are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus. CDR3 is known to be important for antigen binding. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen.

  As used herein, “HLA (Human Leukocyte Antigen)” means a human major histocompatibility complex (MHC), which is a cell surface molecule required for antigen presentation to immune cells. Refers to a complex of genes that encode. HLA can be classified into class I and class II. Class I HLA is composed of an 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 positive T cells. Class I HLA can be further classified into HLA-A, HLA-B, and HLA-C.

  The term “chimeric antigen receptor (CAR)” as used herein refers to a fusion protein comprising 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 called “chimeric receptor”, “T-body”, “chimeric immune receptor (CIR)”. An “extracellular domain that binds to an antigen” refers to any oligo or polypeptide that can bind to an antigen, and an “intracellular domain” refers to a signal that results in the activation or inhibition of a biological process in the cell. By any oligo or polypeptide known to function as a transducing domain.

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

Hereinafter, the present invention will be specifically described.
(1) The antibody of the present invention or an antigen-binding fragment thereof and the nucleic acid encoding them The anti-WT1 peptide / HLA-A24 complex antibody of the present invention is an HLA-A24-restricted WT1 235-243 peptide (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 an 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 WT1 peptide or HLA-A24 present alone. Furthermore, the antibody of the present invention does not bind to a complex other than WT1 peptide and HLA-A24, and does not bind to a complex of HLA other than WT1 peptide and 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 shown in SEQ ID NO: 8 in the sequence listing, VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10. A VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 11, VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 12, and VL- consisting of the amino acid sequence shown in SEQ ID NO: 13 It consists of a light chain containing CDR3.

  As one aspect of the present invention, the antibody of the present invention comprises a heavy chain comprising a heavy chain variable region (VH) consisting of the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing and a light chain variable consisting of the amino acid sequence shown in SEQ ID NO: 7. It consists of a light chain containing a region (VL). The base sequence encoding the amino acid sequence of VH is shown in SEQ ID NO: 4, and the base 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. The antibodies thus obtained are also encompassed by the present invention.

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

In one aspect of the invention, the invention includes a combination of antigen binding fragments. Examples of such molecules include Fab ₃ , Diabody, Triabody, Tetrabody, Minibody, Bis-scFv, (scFv) ₂ -Fc, and intact-IgG. Holliger et al., Nature Biotechnology, Vol. 23, No. 9, p. . 1126-36 (2005).

  The antibody or antigen-binding fragment of the present invention can 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 the FR region. Such modifications include, for example, site-specific mutagenesis methods (point mutagenesis and cassette mutagenesis, etc.), gene homologous recombination methods, primer extension methods, PCR methods, and the like, which are well known to those skilled in the art. It can be easily implemented by combining the methods.

  The present invention further includes a nucleic acid encoding the antibody or antigen-binding fragment of the present invention. As one embodiment 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 the host cell (optimum codon) without changing the encoded amino acid sequence. By changing to the optimal codon in this way, 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 using methods known to those skilled in the art, for example, various means such as genetic engineering techniques or chemical synthesis. As genetic engineering techniques, for example, a replicable cloning vector or expression vector containing the nucleic acid of the present invention is prepared, 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. Thus, the nucleic acid can be expressed, and the resulting polypeptide can be recovered and purified. When the nucleic acid of the present invention consists of a plurality of nucleic acid molecules, a combination (set) of a plurality of vectors containing each nucleic acid molecule is introduced into the host cell, or a single vector containing a plurality of nucleic acids is introduced into the host cell. May be. In producing a polypeptide, a peptide tag can be linked to the target polypeptide and purified using the peptide tag.

  A vector that can be used in the present invention is operably linked to appropriate control sequences so as to express the nucleic acid of the present invention in an appropriate 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 and translation. Control sequences and the like are included. Furthermore, various sequences known to those skilled in the art, for example, restriction enzyme cleavage sites, marker genes (selection genes) such as drug resistance genes, signal sequences, leader sequences and the like may be used as necessary. These various sequences or sites can be appropriately selected and used by those skilled in the art depending on conditions such as the type of polypeptide to be expressed, the host cell used, the culture medium, and the like.

  As the vector that can be used in the present invention, any of a vector that integrates into the host genome, a vector that does not integrate, and an episomal vector that exists in the cytoplasm and replicates autonomously can be used. For example, retrovirus vectors (including oncoretrovirus vectors, lentivirus vectors, pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors or Sendai virus vectors, Epstein-Bar Viral vectors such as viral (EBV) vectors and HSV vectors can be used. As the above-mentioned virus vector, those lacking replication ability are suitable so that the virus cannot self-replicate in infected cells. Non-viral vectors can also be used in combination with condensing agents such as liposomes and cationic lipids. Furthermore, the nucleic acid of the present invention can be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation, or 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.

  An antibody or antigen-binding fragment of the invention expressed in a host cell can be purified from host cell culture media, host cell extracts and / or lysates. The purification method can be performed by appropriately combining arbitrary methods known to those skilled in the art. For example, centrifugation, hydroxyapatite chromatography, gel electrophoresis, dialysis, fractionation on ion exchange columns, 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 or the like), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and affinity chromatography. Affinity chromatography is one of the preferred purification techniques with high efficiency utilizing the affinity with a peptide tag possessed by a polypeptide.

(2) Composition of the present invention The present invention includes 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 a complex of WT1 peptide and HLA-A24. In particular, WT1 has been shown to be 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, as a detection composition or a diagnostic composition.

  As a detection method, conventional general detection methods such as enzyme-linked immunosorbent assay (ELISA), fluorescent antibody assay, radioimmunoassay, radioimmunoprecipitation, 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, a tissue or cell at a disease site, blood, serum, plasma, lymph, urine or other body fluid. The antibody of the present invention is brought into contact with these samples, or samples that have been pre-purified, homogenized, centrifuged, or diluted as necessary, in an appropriate buffer, and the complex of the antigen and the antibody is mixed with the above-mentioned complex. This is detected by the detection method. 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, fluorescamine, dansyl chloride, derivatives thereof, etc.), tag peptides Etc.

The antibodies or antigen-binding fragments of the invention can be used to deliver a medicament 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 cytokines (IL2, TNF, IFN-γ, etc.) and proteins such as receptors thereof, cytotoxins (lysine, diphtheria, gelonin, etc.), radionuclides ( ⁹⁰ Y, ¹³¹ I, ²²⁵ Ac, ²¹³ Bi, ²²³ Ra, and ²²⁷ Th), cells (T cells, NK cells, etc.) or low molecular 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, site and size of the subject to be administered, various conditions of the patient, administration route, etc. it can. A typical single dose or daily dose depends on the above conditions and, if possible, first in vitro and then, for example, using assays known in the art for tumor cell survival or growth. In addition, appropriate dose ranges can be determined with appropriate animal models that can extrapolate dose ranges for human patients.

  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 according to various conditions such as the type of active ingredient, pharmaceutical form, administration method / purpose, and pathological condition of the administration target. Various pharmaceutically acceptable components (for example, carriers, excipients, buffers, stabilizers, etc.) can be added as appropriate. The pharmaceutical composition of the present invention is a tablet, solution, powder, gel, spray, or microcapsule, colloidal distribution system (liposome, microemulsion, etc.), macroemulsion, etc., depending on the above various conditions. Various drug forms are possible.

  Examples of administration methods include intravenous, intraperitoneal, intracerebral, intraspinal, intramuscular, intraocular, intraarterial, in particular intrabiliary or intralesional injection or injection, and sustained release system formulations. It is done. The pharmaceutical compositions of the invention can be administered continuously by infusion or by bulk injection.

  The composition of the present invention includes hematopoietic tumors such as chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), stomach 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, mesothelioma. It is particularly useful for treatment, diagnosis and detection of WT1-positive, HLA-A24-positive tumors and cancers.

(3) Chimeric antigen receptor of the present invention The present invention includes a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment of the present invention described in (1) above and an 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 a cell expressing CAR. The CAR of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes an antibody or antigen-binding fragment of the invention. When the extracellular domain and the complex specifically bind to each other, a signal is transmitted into the cell via the intracellular domain of CAR, thereby stimulating the cell expressing CAR. Stimulated cells produce cytokines and the like, can exert cytotoxicity against target cells expressing the complex, or can induce cytotoxicity of other immune cells.

  Signal that is a molecule capable of transmitting a signal into the cell when the extracellular domain existing in the same molecule binds (interacts) with the WT1 peptide / HLA-A24 complex as the intracellular domain of CAR. 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 a primary cytoplasmic signaling sequence derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Further, for example, intracellular domains containing secondary cytoplasmic signal (costimulatory signal) transduction sequences derived from CD2, CD4, CD5, CD8α, CD8β, CD28, CD137, CD134, ICOS, GITR and CD154 are exemplified. Furthermore, the intracellular domain containing the tertiary cytoplasmic signal transduction sequence derived from IL-2 receptor and IL-21 receptor is illustrated.

  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 T cell receptor, The transmembrane domain of CD134, CD137, ICOS, CD154, GITR can be used. Alternatively, an artificially designed arrangement may be used.

  The present invention includes a nucleic acid encoding the CAR of the present invention. A nucleic acid encoding CAR can be linked to another nucleic acid so that it is expressed under the control of a suitable promoter. As the promoter, any of those that constitutively promote expression and those that are induced by drugs or the like (for example, tetracycline or doxorubicin) can be used. For example, phosphoglycerate kinase (PGK) promoter, Xist promoter, β-actin promoter, RNA polymerase II promoter and other mammalian promoters, SV40 early promoter, cytomegalovirus promoter, herpes simplex virus thymidine kinase promoter, various retroviruses Virus-derived promoters such as the LTR promoter can be used. In addition, in order to achieve efficient transcription of the nucleic acid, a promoter or other regulatory element cooperating with the transcription start site, for example, a nucleic acid containing an enhancer sequence or terminator sequence may be linked. Furthermore, a gene that can serve as a marker for confirming nucleic acid expression (for example, a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein) 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. Furthermore, 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 introducing a nucleic acid encoding the CAR of the present invention into the cell. A cell that expresses CAR is activated by binding to the WT1 peptide / HLA-A24 complex via CAR and transmitting a signal into the cell. Cell activation differs depending on the type of host cell and intracellular domain, but can be confirmed using, for example, cytokine release, improvement of cell proliferation rate, changes in cell surface molecules, and the like as indicators. For example, the release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells results in the destruction of tumor cells that express the complex. In addition, other immune cells such as B cells, dendritic cells, NK cells, macrophages and the like are stimulated by cytokine release and changes in cell surface molecules. Therefore, the CAR-expressing cells of the present invention are useful in adoptive immunotherapy, particularly adoptive immunotherapy for WT1-positive, HLA-A24-positive tumors or cancer.

  The step of introducing a nucleic acid encoding CAR into a cell is performed ex vivo or in vivo. As a cell into which a nucleic acid is introduced, a mammal, for example, a cell derived from a human or a cell derived from a non-human mammal such as monkey, mouse, rat, pig, cow or dog 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 cells [dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes or NK cells, hematopoietic cells (neutrophils, basophils, monocytes)], hematopoietic stem cells, cord blood mononuclear cells, fibers 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 cells. T cells include CD8 positive T cells or CD4 positive T cells, regulatory T cells, cytotoxic T cells, and tumor infiltrating lymphocytes. Cell populations containing T cells and T cell progenitor cells include PBMC. The cells used in the present invention may be either those collected from a living body, those obtained by culturing them, or those established as a cell line. When it is desired to transplant a cell into which a nucleic acid is introduced or a cell differentiated from the cell, it is preferable to introduce the nucleic acid into a cell collected from the living body itself or the same kind of living body.

The cells expressing the CAR of the present invention may be cultured and / or stimulated using an appropriate medium and / or stimulating 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 and the like, and a medium containing IL-2 is preferably used. Although there is no limitation in particular as a density | concentration in the culture medium of IL-2, For example, it is 0.01-1 * 10 < ⁵ > U / mL suitably, More preferably, it is 1-1 * 10 < ⁴ > U / mL. Examples of suitable proteins include CD3 ligand, CD28 ligand, and anti-IL-4 antibody. In addition, a lymphocyte stimulating factor such as lectin can also be added. Further, serum or plasma may be added to the medium. The amount added to these media is not particularly limited, but is exemplified to be more than 0 to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be decreased in stages and used. The origin of serum or plasma may be either self (meaning that the origin is the same as the cell being cultured) or non-self (meaning that the origin is different from the cell being cultured), but preferably Self-derived ones are used from the viewpoint of safety.

The cell culture equipment used for cell culture 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. As the bag, a CO ₂ gas permeable bag for cell culture can be used. Moreover, when manufacturing a large cell population industrially, a large culture tank can be used. Moreover, although culture | cultivation can be implemented by any of an open system and a closed system, it is preferable to culture | cultivate by a closed system from a viewpoint of the safety | security of the cell population obtained suitably.

The present invention includes a pharmaceutical composition containing a cell expressing CAR as an active ingredient. The pharmaceutical composition of the present invention is used parenterally administered to a subject. Parenteral administration methods include methods such as intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. In order to enhance the antitumor action, it can also be administered to meningiomas or nearby tissues, such as subcutaneously. The dose is appropriately selected according to the state, weight, age, etc. of the subject, but usually, the number of cells is about 10 ⁷ to 10 ⁹ cells per time for a subject weighing 60 kg, 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), a medium and the like in order to stably maintain the cells. Although it does not specifically limit as a culture medium, Media, such as RPMI, AIM-V, and X-VIVO10, are generally mentioned.

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.
Example 1 Screening of WT1 peptide / HLA-A24 complex-specific scFv (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, an extracellular fragment (SEQ ID NO: 2) of HLA-A24 heavy chain having a biotinylation site at the C-terminus 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 extracellular fragment of HLA-A24 heavy chain in 200 mL solution (100 mM Tris-HCl (pH 8), 400 mM L-Arginine-HCl, 2 mM Na2EDTA, 5 mM red. after mixing in glutathione, 0.5M oxid.glutathione, 0.1 mM PMSF, 0.2 mg pepstatin, 0.2 mg leupeptin, dialysis with distilled water, Labscale TFF system (Millipore) and Amicon Ultra 10k (Millipore) Concentration). The concentrate was subjected to fractionation using AKTA (manufactured by GE) to obtain a WT1 peptide / HLA-A24 complex. Similar to WT1 peptide, hTERT (human telomerase reverse transcriptase), SAGE (meat antigen), Her2 (human epidermal growth factor receptor type 2), CMV (cytomegalovirus), EBNA (Epstein Barr virus nuclear antigen) ) -Derived HLA-A24-restricted peptide and HLA-A24 complex. Next, the WT1 peptide / HLA-A24 complex was biotinylated with biotin ligase (manufactured by AVIDITY). The biotinylated product was purified again with AKTA, and this was designated as A24-WT1.

(2) Screening of scFv 20 μg of A24-WT1 prepared in Example 1- (1) was mixed with 200 mg of magnetic beads bound with streptavidin and reacted overnight at 4 ° C. in 0.05% Tween / PBS solution. After that, 3 μL of 2 mM biotin / PBS was added to this and reacted for 1 hour. This was washed twice with 0.05% Tween / PBS solution and then suspended in 400 μL of 0.05% Tween / PBS solution to obtain 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 and mixed by rotation at room temperature for 1 hour, and then mixed with 100 μL of the antigen bead solution and mixed by rotation for 1 hour.

  Thereafter, the magnetic beads trapped by a magnetic 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 E. coli DH12S and 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 cultured at 37 ° C. for 1 hour, and then 2 × YT (2 × Y) containing 50 μM kanamycin and 200 μM ampicillin. YTAK) 500 mL was added and cultured overnight at 30 ° C. The supernatant obtained by centrifuging this was mixed with 100 mL of 20% PEG # 600, 2.5 M NaCl solution, and centrifuged to collect the precipitate. This was suspended in 10 mL PBS and then sterilized by filter. The operation from the incubation with the antigen bead solution to the filter sterilization was repeated 4 times, and after the fourth time, the recovered E. coli was treated with LBGA plate (200 μg / mL ampicillin, normal agar medium containing 0.1% glucose (Nissui) Made)). Culturing at 30 ° C., the obtained colonies were cultured overnight in 2 × YTAG medium at 30 ° C., and DNA was prepared with a miniprep DNA kit (manufactured by QIAGEN) using a part of the culture solution and sequenced. Went. At this stage, 19 clones were judged 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), cultured at 30 ° C. overnight, and then centrifuged to obtain a supernatant phage solution. This phage solution was used in 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 in Maxisop loose (manufactured by NUNC) and shaken overnight at 4 ° C. Neutravidin was immobilized. After completion, the liquid was discarded, 200 μL of 2% BSA / PBS was added, and the mixture was allowed to stand overnight. Into this plate, A24-WT1 prepared in Example 1- (1) was placed at 300 ng / 50 μL PBS and shaken at 4 ° C. overnight. After completion, the plate was washed with PBS to obtain an antigen plate. As a control, the HLA-A24-restricted peptide of A24-hTERT, A24-SAGE, A24-Her2, A24-CMV, A24-EBNA prepared in Example 1- (1) was used, and Example 1- (1) In the same manner as above, a biotinylated HLA-A24 complex was prepared to prepare an antigen-immobilized plate.
100 μL of the phage solution prepared in Example 1- (2) was put in each antigen-immobilized plate, shaken at room temperature for 1 hour, washed with PBS, and then 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 times with 0.05% Tween20 / 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 , 0.1M citric acid (pH 5.1) was reacted. When the color was confirmed, the sample was stopped with 2N sulfuric acid, and the absorbance was measured at a wavelength of 490 nm using SpectraMax M2 (manufactured by Molecular Devices).

  A WT # 213 clone that specifically binds to the WT1 peptide / HLA-A24 complex was obtained from 19 phage clones obtained in Example 1- (2). Further, the affinity of the WT1 peptide / HLA-A24 complex was confirmed by changing the concentration of the WT # 213 clone. The results are shown in FIGS. In addition, the vertical axis | shaft of FIG. 2 shows a light absorbency, and a horizontal axis | shaft shows the dilution of a WT # 213 clone. From FIG. 1 and FIG. 2, it was confirmed that the scFv presented by the WT # 213 clone has a very high binding specificity to the WT1 peptide / HLA-A24 complex and a high affinity.

(4) Evaluation of WT # 213 clone Biacore (manufactured by GE Healthcare Bioscience) was used to measure the dissociation constant of the WT # 213 clone scFv 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. An 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 analysis of the data, the association rate constant (ka), dissociation rate constant (kd), and 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. An analysis graph is shown in FIG. FIG. 3 shows that the scFv of the WT # 213 clone has a high KD value and a gentle kd curve. Thus, it was confirmed that this scFv has a strong binding force to the antigen and is difficult to leave.

Example 2 Evaluation of WT # 213scFv (1) Preparation of WT1scFv tetramer The base sequence of VH of scFv of WT # 213 clone is shown in SEQ ID NO: 4, and the base sequence of VL is shown in SEQ ID NO: 5. Further, 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 DNA extracted from the WT # 213 clone as a template. As the primer, F primer (SEQ ID NO: 14) and R primer (SEQ ID NO: 15) were used. With reference to the American Academy of Sciences (Proc. Natl. Acad. Sci. USA), 2003, Vol. 100, No. 13, pp. 7480-7485, the amplified DNA fragment was restricted to the restriction enzyme site SalI- of pHisAvi. The resulting plasmid was inserted into Ascl and the resulting plasmid was designated as pWT # 213scFvHisAvi. pWT # 213scFvHisAvi expresses a fusion protein with PelB leader, His tag, WT # 213scFv and Avi tag, and a fusion protein with PelB leader and biotin ligase (BirA). E. 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. To the supernatant obtained by centrifuging the culture (8000 rpm, 4 ° C. for 10 minutes), 145.5 g of ammonium sulfate was added to 50% saturation and completely dissolved. This was centrifuged (8000 rpm, 4 ° C. for 10 minutes), and the collected precipitate was suspended in PBS containing complete (Roche). After centrifuging the suspension (100,000 rpm, 4 ° C. for 30 minutes), the collected supernatant was filtered with a 0.45 μm filter (Millipore), and then the filtrate was Ni-NTA agarose (QIAGEN). ) It was applied to the column, and the column was further washed with 0.1% Tween20 / PBS and PBS. The protein bound to the column was eluted with 50 mM citrate (pH 2.5), and then neutralized with 3M Tris. Subsequently, dialysis with PBS was performed, and concentration with Amicon Ultra (manufactured by 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 so that the total volume became 400 mL, and lightly shielded at 4 ° C. WT # 213scFv tetramer was prepared by rotary mixing overnight.

(2) Reactivity of WT1 peptide pulse LCL and WT # 213scFv In Example 1- (1), a lymphoblastoid cell line (hereinafter referred to as LCL) which is an HLA-A24 positive cell in RPMI medium. Pulsed with the prepared WT1 peptide for 24 hours. The pulsed LCL was washed with PBS, WT # 213scFv tetramer prepared in Example 2- (1) was added, and the mixture was allowed to stand at 4 ° C. for 1 hour. LCL was washed with PBS, fixed with PBS containing 1% paraformaldehyde, and flow cytometry was performed. LCL without pulsed peptide was prepared in the same manner as a control. The results are shown in FIG. From FIG. 4, it was confirmed that WT # 213scFv was reactive to LCL pulsed with WT1 peptide.

(3) Reactivity of WT1 peptide pulse T2A24 and WT # 213scFv Using T2A24 which is an HLA-A24 positive cell, WT1 peptide was pulsed in the same manner as in Example 2- (2), and the reactivity of WT # 213scFv was determined. confirmed. As a control, cells pulsed with CMV peptide were prepared. The 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 and WT # 213scFv Using MEG-01, a human megakaryoblastic leukemia cell line that endogenously expresses WT1 in HLA-A24-positive cells, WT # 213scFv 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. As a control, K562-A2 cells expressing HLA-A2 and WT1 were also used, and it was confirmed that no reactivity was observed with WT # 213scFv. From the above, it was confirmed that WT # 213scFv reacts with WT1 peptide in an HLA-A24-restricted manner.

Example 3 Chimeric Antigen Receptor (CAR)
(1) Preparation of retroviral plasmid expressing CAR having WT # 213scFv scFv and human IgG-CL for EGFR of pMS3-EGFR-LC-zG-CAR plasmid vector described in WO2013 / 051718 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 a CAR having a leader sequence, WT # 213scFv, CD28 transmembrane region (TM), CD3ζ chain intracellular domain, GITR (glucocorticoid-induced tumor necrosis factor receptor) intracellular domain in this order from the N-terminus. . This CAR is WT # 213CAR.

(2) Preparation of retrovirus solution Escherichia coli JM109 was transformed with the plasmid vector prepared in Example 3- (1) to obtain a transformant. Plasmid DNAs retained by these transformants were purified using NucleoBond Xtra Midi Kit (manufactured by Mach Liner Gel) and subjected to the following operations as transfection DNA.
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.) were each transfected into 293T cells. This operation was performed according to the product protocol of the kit. A supernatant containing ecotropic virus was obtained from each of the transduced cells obtained, and filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore). Using this supernatant, ecotropic virus was infected to PG13 cells (ATCC CRL-10686) 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 # 213CAR.

(3) Expression of WT # 213CAR A retrovirus for WT # 213CAR expression produced in Example 3- (2) on peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood collected with informed consent. The solution was infected twice by a standard method using RetroNectin (registered trademark, manufactured by Takara Bio Inc.) to prepare WT # 213CAR-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 Floor 488-labeled anti-Rabbit IgG antibody. Separately, PE (phycoerythrin: Becton Dickinson) labeled WT1-A24 tetramer was added. Using a flow cytometer, the percentage of cells that are positive for fluorescent labeling, that is, the percentage of cells that are positive for CAR and positive for CAR that binds to the WT1 peptide / HLA-A24 complex, is used for the stained cells. It was measured. The result is shown in FIG. 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 # 213CAR expressing cells (cytokine production, marker expression)
WT # 213CAR-expressing cells 14 days after virus infection prepared in Example 3- (3) were collected and stained for intracellular cytokines on a 96-well plate as follows. In a medium containing the intracellular transport inhibitor Brefeldin A (manufactured by Sigma), the above WT # 213CAR-expressing cells (GMC) and PBMC (NGMC) into which no vector was introduced as a control were adjusted to 1.0 × 10 ⁶ cells / mL. The suspension suspended in was added at 100 μL per well of the plate. Further, 100 μL of a 1.0 × 10 ⁶ cells / mL suspension of cells pulsed with WT1 peptide was added to the T2A24 cell line, and co-cultured for 5 hours. As a control, a T2A24 cell line not pulsed with WT1 peptide and a T2A24 cell line pulsed with CMV peptide were used, and the same operation was performed. Each of the co-cultured cells was stained with anti-Human CD8 antibody and anti-Human CD4 antibody, then treated with IntraPrep Reagent (manufactured by Beckman Coulter), 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 cells after staining. The results are shown in FIG. As shown in FIG. 8, it was confirmed that WT # 213CAR expressing cells (GMC) recognize WT1 peptide and produce various cytokines and markers.

(5) Functional evaluation of WT # 213CAR expressing cells (cytotoxic activity)
WT # 213 CAR-expressing cells (GMC) 14 days after virus infection prepared in Example 3- (3) were collected, and cytotoxic activity was measured by Calsein release assay on a 96-well plate. The T2A24 cell line, K562-A2 cell line, and MEG-01 cell line into which Calsein-AM (manufactured by Dojin Chemical Co., Ltd.) was incorporated were suspended at 1.0 × 10 ⁵ cells / mL. For the T2A24 cell line, cells pulsed with WT1 peptide, cells pulsed with CMV peptide, and cells not pulsed were prepared. Add 100 μL per well of each cell. Suspend the above WT # 213CAR expressing cells and PBMC (NGMC) into which no vector was introduced as a control, and add 100 μL so that the ET ratio is 20, 10, 5, 2.5 did. Instead of PBMC, a well was prepared in which a medium was added as Low control, and 100 μL of 0.1% Triton X-100 was added as High control. After preparing cells and controls, 96-well plates were incubated for 4 hours in a 37 ° C. CO ₂ incubator equilibrated with 5.0% CO ₂ gas. Next, with respect to 100 μL of the supernatant, the fluorescence intensity was measured at λ _ex = 490 nm and λ _em = 515 nm, and the amount of released calsein was measured. The results of calculating the cytotoxic activity (Lysis) by the following formula are 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 the WT1 peptide, high cytotoxic activity by PBMC introduced with WT # 213CAR was observed. Since WT # 213CAR does not show reactivity with a cell line pulsed with CMV peptide and a cell line not pulsed with peptide, it was confirmed that WT # 213CAR has high specificity. Moreover, the cytotoxic activity with respect to the HLA-A24 positive MEG-01 cell line was confirmed about K562-A2 cell line and MEG-01 cell line which are expressing both WT1 endogenously. In addition, cytotoxic activity specific to the HLA-A24 negative K562-A2 cell line was not confirmed. This indicates that WT # 213CAR reacts with WT1 peptide in an HLA-A24-restricted manner.

Example 4 Evaluation Using Cancer-bearing Animals (1) Preparation of WT # 213 CAR-expressing T cell group PBMC 5.3 × 10 ⁵ cells isolated from human peripheral blood collected with informed consent were 0.2%. Human serum albumin (HSA), 600 IU / mL IL-2, suspended in GT-T503 medium (manufactured by Takara Bio Inc.) containing 0.6% plasma, and 5 μg / mL OKT-3 (manufactured by eBioscience) ) And 25 μg / mL of RetroNectin (manufactured by Takara Bio Inc.) 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. Went. The T cells were infected twice with the standard WT # 213 CAR expression retrovirus solution prepared in Example 3- (2). Infected T cells were further cultured in GT-T503 medium containing 0.2% human serum albumin (HSA), 600 IU / mL IL-2, 0.6% plasma for 4 days to express WT # 213 CAR A T cell population was prepared. The expression of CAR in the T cell group on day 8 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 the cells expressed CAR. Further, 7.8% of lymphocytes contained in the T cell group were CD4 positive cells, 98% of which expressed CAR, and a high expression rate of WT # 213CAR was confirmed.

(2) Effects of WT # 213CAR expressing T cells in tumor-bearing mice 2.5Gy of 8-10 week old NOG mice (NOD / Shi-scid, IL-2γ KO) Irradiation was performed. On the next day, WT1-positive, HLA-A24-negative K562 cell line 2.5 × 10 ⁶ cells were subcutaneously injected into the left back of each mouse, and WT1-positive, HLA-A24-positive K562-A24 cell line was injected into the right back. 5 × 10 ⁶ cells were injected subcutaneously. At the same time, WT # 213 CAR-expressing T cells 1.0 × 10 ⁷ cells prepared in Example 4- (1) were intravenously injected. Further, as a control, 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 and minimum tumor diameters and multiplying them.

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

  FIG. 10 shows changes over time in the tumor diameter and body weight of the K562 cell line and K562-A24 cell line in each mouse. In the figure, GMC is a mouse injected intravenously with WT # 213CAR-expressing T cells, NGMC is a control mouse injected with T cells not infected with viral fluid, and PBS is a control mouse injected with PBS instead of T cells. Each is shown. The horizontal axis indicates the number of days, and the vertical axis indicates the product of the tumor diameter. Only in mice infused with WT # 213CAR-expressing T cells, suppression of tumor growth of the K562-A24 cell line was confirmed. This indicates that WT # 213CAR inhibits tumor growth in a HLA-A24-restricted manner with the WT1 peptide. Moreover, the difference in the weight of each mouse | mouth was not seen, and the high safety | security of WT # 213CAR was confirmed.

  According to the present invention, an antibody that specifically recognizes the WT1 peptide / HLA-A24 complex or an antigen-binding fragment thereof, a nucleic acid that encodes the antibody or an antigen-binding fragment thereof, a vector containing this nucleic acid, an antibody or an antigen-binding property thereof Provided are a method for producing a fragment, a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof, a nucleic acid encoding the chimeric antigen receptor, a vector comprising the nucleic acid, a cell expressing the chimeric antigen receptor, and a composition comprising these. Is done. An antibody or 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 tumor cells that express the WT1 peptide / HLA-A24 complex It is extremely useful for detection, treatment, and research and testing for it.

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 comprising VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 8 of the sequence listing, VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9 and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10, and From a light chain comprising VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing, VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 12 and VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 13. An anti-WT1 peptide / HLA-A24 complex antibody or an antigen-binding fragment thereof.   A heavy chain comprising a heavy chain variable region (VH) consisting of the amino acid sequence shown in SEQ ID NO: 6 in 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 4. The antibody or 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 an 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 shown in SEQ ID NO: 6 of the sequence listing and a base sequence encoding the amino acid sequence shown in SEQ ID NO: 7.   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 a step of expressing the nucleic acid according to claim 5 or 6 or the vector according to claim 7.   A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.   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 a CD3ζ chain.   The chimeric antigen receptor according to claim 11, further comprising an intracellular domain of CD28.   A nucleic acid encoding the chimeric antigen receptor according to any one of claims 10 to 12.   A vector comprising a nucleic acid encoding the chimeric antigen receptor of 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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