RU2778890C1 - Hla-dr-binding chimeric antigen receptor and car-t cell - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular, to an HLA-DR-binding antigen-binding molecule and to a composition containing T cells containing said molecule as a pharmaceutically effective ingredient.

EFFECT: invention is effective in treating cancer.

14 cl, 17 dwg, 2 tbl, 18 ex

Description

Technical field

[0001] The present invention relates to chimeric antigen receptors and CAR-T cells that bind HLA-DR.

State of the art

[0002] T cells engineered to express a chimeric antigen receptor (CAR) have a high therapeutic potential in the treatment of cancer (Grupp et al., 2013; Kochenderfer et al., 2010, 2015; Porter et al., 2011).

[0003] The clinical efficacy of these cells is a result of the hybrid structure of CAR, in which various signaling domains and antigen-binding domains with different avidity are artificially linked (Maus et al., 2014; van der Stegen et al., 2015).

[0004] CAR refers to synthetic molecules based on an extracellularly expressed antigen-binding domain that recognizes a target antigen, including a recognition site, a transmembrane domain (module), one or more costimulatory signaling domains, and a chimeric intracellular signal that carries an activation signal (Jensen and Riddell , 2015).

[0005] The binding of CAR-bearing T cells to tumor cell epitopes is independent of the major histocompatibility complex (MHC) and can induce apoptosis and tumor cell death through the mechanism of cytotoxic T cell action (Ramos and Dotti, 2011).

[0006] In recent years, CAR-Ts targeting CD19 (differentiation cluster 19) have shown significant results in the treatment of patients with relapsed/refractory acute lymphocytic leukemia (ALL) (Kochenderfer J.N. et al. (2010) Blood 116: 4099-4102 ; Porter D. L. et al. (2011) N. Engl. J. Med., 365: 725-733; Grupp S. A. et al. (2013) N. Engl. J. Med., 368: 1509-1518; Kochenderfer J. N. et al. (2015) J. Clin. Oncol., 33: 540-549 Brown C. E. et al. (2016) N. Engl. J. Med., 375: 2561-2569).

[0007] Although CD19 CAR-T cell therapy was effective in the treatment of relapsed/refractory B-cell non-Hodgkin's lymphoma, the objective response rate was increased from 20-30% to 79%, with complete remission in 30-50% cases. This indicator is 7 times higher than previous results (Crump et al., 2016; Locke et al., 2017).

[0008] For the cancer receptor (MVR) variant used herein, splenocytes were isolated from Balb/c mice multiple immunized with human B-cell lymphoma cells and hybridized with SP2/0 myeloma cells to generate pools of hybridomas and anti-MVR hybridomas were selected from a pool of hybridomas that specifically responded only to B-cell lymphoma and showed high reactivity (WO2016-094304).

[0009] CD19 is expressed in both normal and tumor cells, thus the CD19 antibody cannot accurately distinguish between normal cells and tumor cells, but the MVR antibody can accurately distinguish between normal and tumor cells, thus providing a high therapeutic effect and safety (Han et al., 2018).

[0010] There is a problem that CAR-T cells are not produced from certain high affinity HLA-DR type T cells. In order to improve this, antibodies having different binding affinities have been prepared in the present patent, and finally an antibody suitable as a therapeutic agent based on CAR-T cells has been selected.

[0011] The present invention is a patent designed to provide different binding affinities for an antigen by altering the sequence of a mouse anti-MVR antibody; it is expected that the antibody itself can be used as a therapeutic agent, or it can be used in therapeutics using the antibody (CAR-T cell therapeutics), and the like. In addition, a humanized antibody was prepared for the antibody of the present invention using a mouse anti-MVR antibody to minimize immunogenicity. In addition, by detecting antibodies having different binding affinities, CAR-T production can be expected to outperform parental antibody production when CAR-T is used, and can be applied to CAR-T cell-based therapeutics for blood cancer.

Detailed description of the invention

Technical problem to solve

[0012] It is an object of the present invention to provide an MVR that can be used in clinical practice and to provide CAR-T cells with a high therapeutic effect. In particular, the present invention relates to a co-stimulatory domain that can be introduced into various CAR-T cells as a co-stimulatory domain that plays a major role in its function in second-generation CAR-T cells. Furthermore, it is an object of the present invention to provide various antigen-binding domains that are capable of binding to antigens expressed on the surface of certain cancer cells and capable of forming CAR-T cells.

Solutions to a technical problem

[0013] 1. An antigen-binding molecule comprising a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1) containing the amino acid sequence represented by sequence No. 1, HCDR2 containing the amino acid sequence represented by sequence No. 2, and HCDR3 containing the amino acid the sequence represented by sequence #3; a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence represented by sequence No. 4; LCDR2 containing the amino acid sequence represented by sequence No. 5; and LCDR3 containing the amino acid sequence represented by sequence No. 6; where the antigen-binding molecule is a chimeric antigen receptor (CAR).

[0014] 2. An antigen-binding molecule according to claim 1, comprising a heavy chain variable region represented by sequence #7 and a light chain variable region represented by sequence #8.

[0015] 3. An antigen-binding molecule according to claim 1, comprising the amino acid sequence represented by sequence #9.

[0016] 4. An antigen-binding molecule according to claim 1, further comprising the amino acid sequence represented by sequence #13.

[0017] 5. An antigen-binding molecule according to claim 1, further comprising the amino acid sequence represented by sequence #14.

[0018] 6. An antigen-binding molecule according to claim 1, further comprising a transmembrane domain and an intracellular signaling domain for activating T cells.

[0019] 7. An antigen-binding molecule according to claim 6, wherein said transmembrane domain is selected from the group consisting of T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, and CD154, wherein said intracellular signaling domain is a CD3zeta signaling domain and a costimulatory signaling domain.

[0020] 8. An antigen binding molecule according to claim 7, wherein the costimulatory signaling domain is selected from the group consisting of CD28, OXO40, CD27, ICAM-1, CD278, and CD137.

[0021] 9. A nucleic acid molecule that encodes an antigen-binding molecule according to any one of claims 1-8.

[0022] 10. An expression vector containing the nucleic acid molecule according to claim 9.

[0023] 11. A cell containing a nucleic acid molecule according to claim 9.

[0024] 12. A cell according to claim 11 which is a T cell.

[0025] 13. The cell of claim 12, wherein the T cells are CD8+ T cells and/or CD4+ T cells.

[0026] 14. The cell according to claim 11, which is a chimeric antigen receptor (CAR-T) modified T cell.

[0027] 15. A pharmaceutical composition for the treatment of cancer, containing a cell according to claim 11 as a pharmaceutically effective ingredient.

[0028] 16. The pharmaceutical composition according to claim 15, wherein the cancer is esophageal adenocarcinoma, colorectal cancer, melanoma, ocular melanoma, small cell lung cancer, neuroblastoma, teratoma, fetal cancer, squamous cell carcinoma, head and neck squamous cell carcinoma, thymoma , lymphocytic leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, leukemia, acute myeloid leukemia, and the like.

[0029] 17. A method of treating cancer, comprising the step of administering to a patient suffering from cancer a pharmaceutical composition comprising a T cell containing a therapeutically effective amount of an antigen-binding molecule according to any one of claims 1-8.

[0030] 18. The method of treating cancer according to claim 17, wherein the pharmaceutical composition comprises CD8+ T cells, or comprises CD4+ T cells and CD8+ T cells.

[0031] 19. The method of treating cancer according to claim 18, wherein the ratio of CD4+ T cells to CD8+ T cells is generally 1:1.

[0032] 20. A method for producing a chimeric antigen receptor-modified (CAR-T) T cell for cancer treatment, comprising the step of transfecting the T cell with nucleic acids that encode an antigen-binding molecule comprising a heavy chain variable region comprising complementarity-determining region 1 of the heavy chain (HCDR1) containing the amino acid sequence represented by sequence No. 1, HCDR2 containing the amino acid sequence represented by sequence No. 2, and HCDR3 containing the amino acid sequence represented by sequence No. 3; a light chain variable region comprising the light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence represented by sequence No. 4, LCDR2 containing the amino acid sequence represented by sequence No. 5, and LCDR3 containing the amino acid sequence represented by sequence No. 6; where the antigen-binding molecule is a chimeric antigen receptor (CAR).

[0033] 21. A pharmaceutical composition for the treatment of cancer, comprising, as a pharmaceutically effective ingredient, T cells containing an antigen-binding molecule according to any one of claims 1 to 8.

[0034] 22. The pharmaceutical composition according to claim 21, wherein the pharmaceutical composition contains CD8+ T cells or contains CD4+ T cells and CD8+ T cells.

[0035] 23. The therapeutic pharmaceutical composition according to claim 18, wherein the ratio of CD4+ T cells to CD8+ T cells is generally 1:1.

Invention Effect

[0036] In the case of the MVR developed in the present invention, an increased level of HLA-DR can be selectively recognized in tumor cells, and when CAR-T cells are obtained using it, the MVR shows high efficiency in vitro and high efficiency in animals.

[0037] There is also a performance enhancing effect by adding five amino acids to the conventional 4-1BB co-stimulatory domain.

Brief description of the figures

[0038] FIG. 1 shows the corresponding amino acid sequence of a VH mouse anti-MVR antibody and 2 humanized antibodies produced (huMVR.L1H1, huMVR.L2H2).

[0039] FIG. 2 shows the corresponding amino acid sequence of the VL mouse anti-MVR antibody and the 2 humanized antibodies produced (huMVR.L1H1, huMVR.L2H2).

[0040] FIG. 3 shows the results of the avidity assay of muMVR and 2 humanized antibodies (huMVR.L1H1, huMVR.L2H2).

[0041] In FIG. 4 shows molecular modeling and prediction of affinity hotspots for the humanized huMVR.M2H2 antibody, which is MVR.

[0042] FIG. 5 shows the results of the avidity analysis of 15 types of mutants for which the affinity hot spot method was used.

[0043] FIG. 6a schematically shows the CD19 CAR, CD19CAR_euCD137, and huMVR.L2H2CAR_euCD137 constructs. CD19CAR used 214-255aa of domain 4-1 (CD137) in the costimulatory domain, and CD19CAR_euCD137 and huMVR.L2H2CAR_euCD137 used 209-255aa in domain 4-1 (CD137).

[0044] Figure 6b shows lentiviral vectors and corresponding genes showing the basic functions of CAR-T cells and the sequence of the CD19 CAR construct gene. In particular, a CD8 leader sequence is shown, including the EF1 alpha promoter, scFv huMVR.L2H2, the CDα hinge and human CD8 transmembrane domain, and the 4-1BB and CD3zeta signaling domains to increase the level of CAR expression. Also shown are the corresponding genes required to produce safe lentiviruses.

[0045] Figure 7 shows lentiviral vectors and corresponding genes showing the basic functions of CAR-T cells and the gene sequence of the CD19 CAR construct. In particular, the CD8 leader sequence is shown, including the EF1-alpha promoter, scFv MVR.L2H2, the CDα hinge and human CD8 transmembrane domain, and the 4-1BB and CD3zeta signaling domains to increase the level of CAR expression. Also shown are the corresponding genes required to produce safe lentiviruses.

[0046] Figure 8 shows a method for obtaining MVR CAR-T cells.

[0047] FIG. 9 shows experimental results confirming the apoptotic function of MVR CAR-T in vitro.

[0048] FIG. 10 shows the production level and efficacy results for two types of CD19 CAR-T cells in which CD19CAR and CD19CAR_euCD137 were introduced. A: 14 days after receiving 2 CD19 CAR-T cells, CD8 and CAR-T were stained and the proportion of CAR-T production was shown using FACS. B: Comparison of the cytotoxicity of the corresponding CAR-T following a luciferase assay 4 hours after interaction of 2 species of derived CAR-T with a target is shown.

[0049] FIG. 11 shows cell line HLA-DR expression and avidity of huMVR L2H2 scFv using FACS prior to evaluating the effect of huMVR CAR-T in an animal model.

[0050] FIG. 12 shows efficacy evaluation in an animal model using the CAR-T cell line after determination of the proportion of CAR-T cell production and cytotoxicity using the cell line shown in FIG. 9. A: Results of using an IVIS imaging device to evaluate the performance of CAR-T after reproducing a subcutaneous tumor animal model with subcutaneous injection of a luciferase-expressing tumor cell line into mice. B: Evaluation results and graphical depiction of photon values for each mouse after imaging.

[0051] FIG. 13 shows the proportion and absolute amount of CAR-T present in blood using FACS after blood sampling from the orbital sinus venosus in mice at 3-4 day intervals. A: Graph showing the total proportion of CD19 CAR-T in blood by FACS staining of CD8+/CAR+ cells. B: Graph showing the proportion of CD8 and CD4 CAR-T after confirming the total proportion of CAR-T. C: Graph showing the number of CAR-T cells present in blood using FACS counting beads during FACS staining.

[0052] FIG. 14 shows the results of evaluating the efficacy of CD8 huMVR CAR-T and CD4/CD8 huMVR CAR-T using an intraperitoneal tumor animal model. A: Results of IVIS imaging of the effects of CD8 huMVR CAR-T and CD4/CD8 huMVR CAR-T after reproducing animal models by injecting tumor cell lines expressing luciferase into the abdominal cavity of mice. B: Graphs showing the photon values of tumor cells in an animal model when injected into the abdominal cavity.

[0053] FIG. 15 shows results of MVR CAR-T performance evaluation using an animal model. A: IVIS imaging results showing the effect of MVR CAR-T after reproducing animal models by injecting mice with tumor cell lines expressing luciferase. B: Graphs showing the dimensions of tumors developed after subcutaneous inoculation as measured by automatic calipers.

[0054] FIG. 16 is a graph showing the viability of mice in each group based on FIG. fifteen.

[0055] FIG. 17 shows the proportion and amount of CAR-T present in blood using FACS after blood sampling from the orbital sinus venosus in mice at 3-4 day intervals after MVR CAR-T administration. A: Graph showing the proportion of hCD45+/CAR+ cells in the blood of mice. B: Graph showing the number of MVR CAR-T cells in the blood of mice using FACS counting pellets during FACS staining.

Best Mode for Carrying Out the Invention

[0056] The present invention relates to a cell expressing a chimeric antigen receptor, a pharmaceutical composition containing it, and a method of treating cancer using it.

[0057] As used herein, the term "chimeric antigen receptor (CAR)" refers to an antigen-binding domain that functions through a receptor exposed on the outside of a cell and recognizes a target molecule; including one or more hinge domains or spacer domains; transmembrane domains; one or more intracellular costimulatory signaling domains; and an intracellular stimulatory domain.

[0058] In the context of the present invention, the term "T cell" refers to lymphocytes derived from the thymus and plays an important role in cellular immunity. T cells include CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells, natural killer T cells, and the like. In one embodiment of the present invention, the T cells into which CAR is introduced are CD8+ T cells or CD8+ T cells and CD4+ T cells.

[0059] Within the scope of the present invention, the terms "antibody" and "antigen binding protein" can be used interchangeably, and "antigen binding protein" of the present invention encompasses not only the complete form of an antibody, but also functional fragments of an antibody molecule. A complete antibody is a structure having two full length light chains and two full length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. Functional fragments of antibody molecules refer to fragments having an antigen-binding function and include Fab, F(ab'), F(ab') ₂ , Fv, and the like. Of these antibody fragments, the Fab has one antigen-binding site with a structure including a light chain, a heavy chain variable region, a light chain constant region, and a first heavy chain constant region (CH1). Fab' differs from Fab in that it has a hinge region containing at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F(ab') ₂ antibodies are produced by the formation of disulfide bonds between cysteine residues in the Fab' hinge region.

[0060] Recombinant methods for creating Fv fragments with a minimum number of antibody fragments, in which Fv has only a heavy chain variable region and a light chain variable region, are disclosed in published international patent applications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. Double chain Fv (dsFv) are disulfide linked Fv fragments where the heavy chain variable region and the light chain variable region are linked, and the short chain Fv (scFv) is usually covalently linked to the heavy chain and light chain variable region via a peptide linker. Such antibody fragments can be prepared using proteolytic enzymes (eg, a complete antibody can be treated with papain to give a Fab, and pepsin digestion yields an F(ab') ₂ fragment).

[0061] As used herein, HLA-DR (human leukocyte antigen-antigen D linked) refers to a major histocompatibility complex class II molecule (Shackelford D.A. et al., (1982) Immunol. Rev., 66: 133-187). HLA-DR, which is a peptide of nine or more amino acids, and its ligand is a ligand for TCR. HLA-DR molecules are activated in response to signaling. In the event of infection, peptides (eg, staphylococcus enterotoxin I peptides) bind to the DR molecule and enter a number of multiple T-cell receptors found on T-helper cells. These cells bind to B cell surface antigens, which stimulate B cell proliferation.

[0062] The main function of HLA-DR is to present foreign peptide antigens that were not originally present in the immune system to induce or inhibit a helper T cell response to produce antibodies to that peptide antigen. HLA-DR is an α,β dimer and cell surface receptor; each subunit includes two extracellular domains, a membrane domain and a cytoplasmic tail. Both α- and β-chains are attached to the cell membrane. The N-terminal domain of the mature protein forms an alpha helix that constitutes the open portion of the linking group, and the C-terminal cytoplasmic region interacts with other chains to form beta-folds under the linking group across the cell membrane. Most of the contact sites of the peptide are located on the first 80 residues of each strand.

[0063] HLA-DR is expressed to a limited extent in antigen presenting cells such as dendritic cells, macrophages, monocytes, and B cells. Due to the fact that a large number of DR "antigens" on the cell surface often respond to stimuli, DR is also a marker of immune action due to the high expression of HLA-DR in cellular malignant neoplasms and a limited expression spectrum in normal cells; antibodies against HLA-DR have been developed and tested on B-cell malignancies in preclinical and clinical studies (Nagy Z.A. et al. (2002) Nat. Med., 8: 801-807; DeNardo G.L. et al. (2005) Clin. Cancer Res., 11: 7075s-7079s Ivanov A. et al (2009) J. Clin. Invest., 119: 2143-2159 Lin T. S. et al. (2009) Leuk Lymphoma, 50: 1958 -1963). Although serious toxicity was not established in phase I/II clinical trials, further studies were halted due to limited efficacy (Lin T.S. et al. (2009) Leuk. Lymphoma, 50: 1958-1963). Given the potential of CAR-T cells to enhance the therapeutic efficacy of monoclonal antibodies by incorporating antigen specificity into large-scale T-cell responses, it is believed that HLA-DR-targeted CAR-T cells may be useful therapeutic agents for the treatment of B-cell malignancies. .

[0064] In one embodiment of the present invention, the antigen binding protein comprises scFv and is in a form in which the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signaling domain are operably linked. For the transmembrane domain, the alpha, beta, or zeta chain of the T cell receptor or one or more of CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 can be listed. , CD137 or CD154, but not limited to. The intracellular signaling domain is basically the primary signaling domain of CD3zeta; one or more of CD28, OXO40, CD27, ICAM-1, ICOS (CD278), and 4-1BB (CD137) can be used as the co-stimulatory signaling domain, but is not limited thereto. In one embodiment of the present invention, the transmembrane domain is CD8 and the co-stimulatory signaling domain is 4-1BB.

[0065] The scFv of the present invention includes a light chain variable region (VL) and a heavy chain variable region (VH) and has a VL-VH or VH-VL structure, and the VL and VH can be linked directly or linked through a linker. When a linker is used, a linker known in the art can optionally be used, for example, (GGGGS) ₂ , (GGGGS) ₃ , (Gly) ₆ , (Gly) ₈ or (EAAAK) _n (where n is any integer from 1 to 3), without limitation. In one embodiment of the present invention, the antigen binding protein comprises a VL-linker-VH construct and the linker is (GGGGS) ₃ .

[0066] The scFv antigen-binding molecule of the present invention specifically binds to an antigen present on the surface of a cell; such an antigen is particularly a cell surface protein that is specifically expressed in target cells, such as tumor cells, or is overexpressed in tumor cells, and may be, for example, at least one selected from the group consisting of CD30, CD20, CD19, CD22 and CD138. In one embodiment of the present invention, the antigen is HLA-DR.

[0067] In one embodiment of the present invention, the scFv is huMVR, an anti-HLA-DR antibody. huMVR contains a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1) containing the amino acid sequence represented by sequence No. 1, HCDR2 containing the amino acid sequence represented by sequence No. 2, and HCDR3 containing the amino acid sequence represented by sequence No. 3; a light chain variable region comprising the light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence represented by sequence No. 4, LCDR2 containing the amino acid sequence represented by sequence No. 5, and LCDR3 containing the amino acid sequence represented by sequence No. 6; where the antigen-binding molecule is a chimeric antigen receptor (CAR). In one embodiment of the present invention, the scFv comprises a heavy chain variable region represented by sequence No. 7 and a light chain variable region represented by sequence No. 8; in yet another embodiment of the present invention, the scFv comprises the amino acid sequence represented by huMVR sequence #9.

[0068] The antigen-binding molecule of the present invention is a chimeric antigen receptor (CAR) having scFv as an antigen-binding site; in one embodiment of the present invention, the antigen-binding molecule further comprises a transmembrane domain at the C-terminus of the scFv and an intracellular signaling domain for T cell activation.

[0069] In yet another embodiment of the present invention, the CAR comprises an amino acid sequence represented by sequence #7 or sequence #8. In one embodiment of the present invention, the transmembrane domain is selected from the group consisting of T cell receptor alpha chain, T cell beta chain β-cell receptor, T-cell receptor zeta chain, CD28, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137 and CD154 and variants thereof, where the specified intracellular signaling domain is a signaling domain and costimulatory signaling domain CD3zeta; in one embodiment of the present invention, the costimulatory signaling domain is selected from the group consisting of CD28, OX40, CD27, ICAM-1, CD278, and CD137.

[0070] "Isolated polypeptide", "isolated peptide", or "isolated protein" refers to a polypeptide or protein that is substantially free of compounds with which it is normally associated in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates , lipids). "Isolated" does not mean the removal of artificial or synthetic mixtures with other compounds, the removal of impurities that do not interfere with biological activity, or the removal of impurities that may be present, such as by adding a stabilizer to an intermediate or formulation of a pharmaceutically acceptable preparation.

[0071] The term "variable region" refers to a portion of an antibody molecule that has many sequence variations when performing the function of specific binding to an antigen; CDR1, CDR2 and CDR3 are in the variable region. "Complementarity determining regions (CDRs)" are sites involved in antigen recognition; these sites are important in determining the specificity of an antibody for an antigen according to changes in the sequence of that site. Between the CDRs is a "frame region (FR)" in the correct orientation that supports the CDR loops; in particular, FR1, FR2, FR3 and FR4 are present.

[0072] In one embodiment of the present invention, another antigen-binding protein of the present invention may be a humanized antibody. In the present invention, the term "humanized antibody" generally refers to an antibody that is not immunogenic or has low immunogenicity in humans, as described above. Humanized antibodies are modified antibodies, and the amino acid sequence of an antibody can be rearranged to achieve the desired goal. These possible changes are numerous and can range from a change in one or more amino acids to a complete reconfiguration of the variable or constant region of an antibody. In general, while modification of the variable region is performed to increase avidity and affinity for antigen, modification of the constant region is performed to increase intracellular activity such as complement fixation, membrane interaction, and the function of other effective agents. The humanized antibodies provided in the present invention can be combined with all kinds of constant regions by recombinant techniques. The heavy chain constant region belongs to the gamma (γ), mu (μ), alpha (α), delta (δ) epsilon (ε) types and is subclassified into gamma 1 (γ1) gamma 2 (γ2) gamma 3 (γ 3) gamma 4 (γ4) alpha1 (α1), alpha2 (α2). Light chain constant regions are of kappa (κ) and lambda (λ) types (Coleman et al., Fundamental Immunology, 2nd ^Ed ., 1989, 55-73).

[0073] The term "fragment" as used in relation to polynucleotide or polypeptide sequences refers to a nucleic acid sequence or peptide sequence that is shorter than the aforementioned nucleic acid or protein and includes at least a portion that is identical to the nucleotide sequence or peptide sequence of the above nucleic acid or protein. Such nucleic acid fragments and polypeptide fragments of the present invention may, if necessary, be included in larger polynucleotides or polypeptides as their components. Such fragments may contain or consist of oligonucleotides or oligopeptides having continuous nucleotide or peptide sequences from the nucleic acid or protein of the present invention, at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000 residues or more.

[0074] A "variant" of a polypeptide or protein refers to any analog, fragment, derivative, or mutant derived from that polypeptide or protein and retaining at least one biological property of that polypeptide or protein. Various variants of such a polypeptide or protein may be found in nature. These variants may be allelic variants characterized by different nucleotide sequences of the structural gene encoding the protein, or may include differentiated splicing or post-translational modifications. A person skilled in the art can make variants containing one or more amino acid substitutions, deletions, additions, or substitutions. These variants may include: (a) a variant in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) a variant in which one or more amino acids are added to a polypeptide or protein, (c) a variant in which one or more amino acids contain a substituent, and (d) a variant in which the polypeptide or protein is fused to another polypeptide, such as serum albumin.

[0075] Conservative variants also refer to amino acid sequences having sequence changes that do not adversely affect the biological function of the protein. If the altered sequence disrupts or completely abolishes the biological function associated with the protein, then the substitution, insertion, or deletion is considered to adversely affect the protein. For example, the overall charge, structure, or hydrophobicity-hydrophilicity of a protein can be altered without adversely affecting biological activity. Accordingly, the amino acid sequence can be changed such that, for example, the peptide exhibits greater hydrophobicity or hydrophilicity without adversely affecting the biological activity of the protein. Methods for obtaining such variants, which include genetic (suppression, deletion, mutation, etc.), chemical and enzymatic methods, are known to specialists in this field.

[0076] In one embodiment, the present invention discloses a nucleic acid molecule encoding an antigen-binding molecule. The nucleic acid molecule may contain a nucleic acid sequence encoding the amino acid sequence represented by sequence No. 7 or a nucleic acid sequence encoding the amino acid sequence represented by sequence No. 7.

[0077] In yet another embodiment of the present invention, the nucleic acid molecule may contain a sequence for encoding an scFv represented by sequence No. 9. In another embodiment of the present invention, the nucleic acid molecule may contain a nucleic acid sequence encoding a CAR represented by sequence No. 15 or No. 16. In addition, in another embodiment of the present invention, the nucleic acid molecule may contain a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology for encoding a polypeptide that has the same amino acid sequence as the protein encoded by the above sequence, or that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology.

[0078] The terms "nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are used interchangeably in the present invention and refer to the single-stranded or double-stranded helix forms of the phosphate esters of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecules "") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecules"); or phosphorothioate or any phosphate ester analog such as a thioether. Of these, helical DNA-DNA, DNA-RNA and RNA-RNA are possible. The term "nucleic acid molecule" and, in particular, a DNA or RNA molecule refers only to the primary and secondary structures of the molecule and is not limited to any particular form of tertiary structure. Thus, the term includes among these molecules linear or cyclic DNA molecules (eg, restriction enzyme fragments), plasmids, supercoiled DNA, and double-stranded DNA found in chromosomes. When discussing the structure of a particular double-stranded DNA molecule in this specification, the structure may be described according to the general convention that the sequence is only presented in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand with the sequence corresponding to the mRNA) . "Recombinant DNA molecules" are DNA molecules that have undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.

[0079] As is known in the art, the term "percent identity" means the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. In addition, in this field, the term "identity" refers, as the case may be, to the degree of sequence match between polypeptide or polynucleotide sequences, which is determined by the degree of match between sections of the sequences. "Identity" and "similarity" can be readily determined by known methods, including but not limited to those described in Computational Molecular Biology ((Lesk A.M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993) Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994) Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987) and Sequence Analysis Primer (Gribskov M. and Devereux J., eds.) Stockton Press, New York (1991).

[0080] Preferred methods for determining identity are designed to provide optimal matching between test sequences. Methods for determining identity and similarity are at the heart of publicly available computer programs. Sequence alignment and percent identity calculations can be performed using sequence analysis software such as the Megaalign program from the LASERGENE bioinformatics software package (DNASTAR Inc., Madison, Wis.). Multiple sequence alignment can be performed using the Clustal alignment method with default parameters (gap penalty=10, gap extension penalty=10) (Higgins et al., CABIOS. 5: 151 (1989)). The default parameters for pair alignment using the Clustal method can be chosen from KTUPLE 1, gap penalty=3, window=5 and retained diagonals=5. As known in the art, "similarity" between 2 kinds of polypeptides is determined by comparing the amino acid sequence and conservative amino acid substitutions of the polypeptide with the sequence of the 2nd polypeptide. Identity or homology to these sequences in this application refers to the alignment of sequences and the introduction of gaps necessary to achieve the maximum percentage of homology, without considering any conservative substitutions as part of the sequence identity, and then is defined as the percentage of amino acid residues in a candidate sequence that is identical to the known peptides. An extension, deletion, or insertion in a peptide sequence at the N-terminus, C-terminus, or within should not be considered as affecting homology.

[0081] The term "homology" refers to the percentage identity between two polynucleotides or two polypeptide fragments. Correspondence between the sequences of one part and another part can be determined by methods known in the art. For example, homology can be determined by aligning known sequences and directly comparing the known sequence between two polypeptide molecules using available computer programs. Otherwise, homology can be determined by hybridizing polynucleotides under conditions that form a stable duplex between regions of the same type, followed by digestion with a single strand-specific nuclease and size determination of the cleaved fragments.

[0082] As used herein, all grammatical and spelling forms of the term "homology" refer to correspondence between proteins of "common evolutionary origin" (Reeck et al., Cell, 50: 667 (1987)), including proteins from a superfamily (e.g., the immunoglobulin superfamily ) and homologous proteins of different species (for example, myosin light chain, etc.). These proteins (and the genes that code for them) share sequence homology due to the high similarity of their sequences. However, in general usage and in this application, when modified by an adverb such as "very", the term "homologous" refers to sequence homology and does not indicate a common source of evolution.

[0083] Thus, the term "sequence similarity" in all grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences, which may or may not have a common evolutionary origin (Reeck et al., Cell, 50: 667 (1987 )). In one embodiment, when about 50% (e.g., at least about 75%, 90%, or 95%) of the nucleotides correspond to a DNA sequence of a certain length or more, then the two DNA sequences are "substantially homologous" or "substantially similar". ". Substantially homologous sequences can be identified by comparing sequences using available standard software with a sequence data bank or, for example, Southern hybridization experiments under stringent system-specific conditions. Determination of suitable hybridization conditions is within the technical ability of the art (see, for example, Sambrook et al. 1989).

[0084] As used herein, the term "substantially similar" refers to a nucleic acid fragment in which a change in one or more nitrogenous bases causes a change in one or more amino acids, but does not affect the functional properties of the protein that this DNA sequence encodes. "Substantially similar" also refers to a nucleic acid fragment in which a change in one or more nitrogenous bases does not affect the ability of the nucleic acid fragment to mediate changes in gene expression using antisense or cosuppression techniques. "Substantially similar" also refers to modifications of the nucleic acid fragments of the present invention, such as deletions or insertions of one or more nitrogenous bases, which do not significantly affect the functional properties of the resulting transcript. Therefore, it is to be understood that the present invention also includes the specific sequences shown. Each of the proposed modifications is known to those of ordinary skill in the art, for example, to determine the retention of biological activity of an encoded product.

[0085] In addition, those skilled in the art will appreciate that the substantially similar sequences encompassed by the present invention are defined by the ability to hybridize to the sequences described herein under stringent conditions (0.1x SSC, 0.1% SDS, 65 °C and 0.1× SSC, 0.1% SDS after washing with 2× SSC, 0.1% SDS). Substantially similar nucleic acid fragments of the present invention are nucleic acid fragments whose DNA sequences are at least about 70%, 80%, 90%, or 95% identical to the DNA sequences of the nucleic acid fragments described herein.

[0086] In one embodiment, the present invention relates to an expression vector that contains a nucleic acid molecule that encodes an antigen-binding protein of the present invention. The term "expression" refers to the biological production of a product encoded by a coding sequence. In most cases, a DNA sequence containing a coding sequence is transcribed to form a messenger RNA (mRNA). The messenger RNA is then translated to form a polypeptide product with the appropriate biological activity. In addition, the expression process may include an additional step of processing RNA transcription products (eg, splicing to remove introns) and/or post-translational processing of the polypeptide product.

[0087] In the context of the present invention, the term "expression vector" refers to a vector, plasmid or carrier designed to transform a host after expression of an inserted nucleic acid sequence.

[0088] Cloned genes, namely inserted nucleic acid sequences, are usually under the control of regulatory elements such as promoters, minimal promoters, enhancers, and the like. Numerous regulatory regions or promoters are well known to those skilled in the art that can be used to induce expression of nucleic acids in a desired host cell. Any promoter capable of substantially inducing the expression of these genes includes, but is not limited to, viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue-specific promoters, pathogenesis or disease-related promoters, developmental stage-specific promoters. , inducible promoters, light-controlled promoters, and the like; and includes, but is not limited to, promoters containing the early (SV40) SV40 promoter region and the Rous sarcoma virus (RSV) 3' long repeat (LTR); E1A adenovirus promoter (Ad) or major late promoter (MLP); immediate early promoters of human cytomegalovirus (HCMV); herpes simplex virus (HSV) thymidine kinase (TK) promoters; promoters of baculovirus IE1; elongation factor 1 alpha (EF1) promoters; glyceraldehyde-3-phosphate dehydrogenase (GSPDH) promoters, phosphoglycerate kinase (PGK) promoters; ubiquitin C (Ubc) promoters; albumin promoters; mouse metallothionein-L promoters and regulatory sequences for transcriptional regulatory regions; ubiquitous promoters (HPRT, vimentin, β-actin, tubulin, etc.); intermediate filaments (desmin, neurofibrils, keratin, GFAP, etc.), therapeutic gene promoters (MDR, CFTR or factor VIII forms, etc.), onset or disease-related promoters; and promoters that have been used in transgenic animals and exhibit tissue specificity, such as the regulatory regions of the elastase genes, which are active in pancreatic acinar cells (such as pancreatic acinar cells); regulatory regions of the insulin gene active in pancreatic beta cells; regulatory regions of the immunoglobulin gene active in lymphoid cells; mouse breast cancer virus regulatory regions active in testis, mammary gland, lymph nodes and macrophages; regulatory regions of albumin genes active in the liver; Apo AI and Apo AII regulatory regions, alpha-fetoprotein gene regulatory regions active in the liver; regulatory regions of the alpha-1-antitrypsin gene, active in the liver; regulatory regions of the beta-globin gene, active in bone marrow cells; regulatory regions of myelin basic protein, active in brain oligodendrocytes; myosin-2 light chain gene regulatory regions active in skeletal muscle and gonadotropin-releasing hormone active in the hypothalamus; pyruvate kinase promoters; villin promoters; intestinal fatty acid binding protein promoters; β-actin promoters in smooth muscle cells.

[0089] The term "vector" encompasses both non-viral and viral carriers for introducing nucleic acids into cells in vitro, ex vivo or in vivo.

[0090] The vector may be a replicon with another DNA fragment attached to amplify the attached fragment. The term "replicon" refers to any genetic element (eg, plasmid, phage, cosmid, chromosome, virus) that is capable of functioning as an autonomous unit of DNA replication in vivo, that is, replicating under its own control. Many vectors known in the art can be used to generate nucleic acids, incorporate reactive elements and promoters into genes, and the like. Preferred vectors include, for example, plasmids or modified viruses, including, for example, adenoviruses, retroviruses, adeno-associated viruses, herpes viruses, or plasmids such as PBR322 or pUC derivatives of the plasmid, or Bluescript vectors. For example, DNA fragments corresponding to reaction elements and promoters can be inserted into appropriate vectors by combining the appropriate DNA fragments with selected vectors having complementary sticky ends. If they are not present, then the ends of the DNA molecules can be enzymatically modified, or any site can be created by linking a nucleotide sequence (linker) to the ends of the DNA. Such vectors can be manipulated to contain a selectable marker gene for screening for cells that have incorporated the marker into the cell's genome. Such markers allow identification and/or screening of host cells expressing the protein encoded by the marker.

[0091] The vector provides the necessary regulatory sequences (eg, transcriptional and translational elements) to regulate expression of the fusion protein in the appropriate host cell. Regulatory sequences may include promoter regions, enhancer regions, transcription termination sites, ribosome binding sites, initiation codons, splicing signals, introns, polyadenylation signals, Shine/Dalgarno translation sequences, and Kozak consensus sequences. The regulatory sequence is selected based on the host cell in which the fusion protein will be produced. Suitable bacterial promoters include, but are not limited to, bacteriophage λpL or pR, T6, T7, T7/lacO, lac, recA, gal, trp, ara, hut, and trp-lac. Suitable eukaryotic promoters include, but are not limited to, PRBI, GAPDH, metallothionein, thymidine kinase, viral LTR, cytomegalovirus, SV40, or tissue-specific or tumor-specific promoters such as the α-fetoprotein, amylase, cathepsin E, M1 muscarinic receptor, or γ-glutamyl transferase promoter.

[0092] Additional vectors include lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to nucleic acids, the vector may also contain one or more regulatory regions and/or selectable markers suitable for selection, evaluation and monitoring of nucleic acid delivery outcomes (delivery to a specific tissue, duration of expression, etc.).

[0093] Vectors can be introduced into the desired host cell by a method known in the art, such as injection, transfection, electroporation, microinjection, transduction, cell fusion, lipofection, calcium phosphate precipitation (Graham F. L. et al., Virology, 52: 456 (1973); and Chen and Okayama, Mol. Cell. Biol., 7: 2745-2752 (1987)), liposome mediated method (Wong T. K. et al., Gene, 10:87 (1980); Nicolau and Sene , Biochim. Biophys. Acta, 721: 185-190 (1982), and Nicolau et al., Methods Enzymol., 149: 157-176 (1987)), treatment with DEAE-dextran (Gopal, Mol. Cell Biol., 5 : 1188-1190 (1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) using gene species or carrier DNA vectors (see e.g. Wu et al., J. Biol. Chem. 267: 963 (1992); Wu et al., J. Biol. Chem. 263: 14621 (1988); and Hartmut et al., Canadian Patent Application No. 2012311).

[0094] Viral vectors have been used in a wide range of applications to transfer genes into cells as well as live animals. Viral vectors that can be used include, but are not limited to, adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, herpes simplex virus, lentivirus, baculovirus, Sendai virus, measles virus, simian 40 virus, and Epstein virus vectors. Barra. Non-viral vectors include plasmids, lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to nucleic acids, the vector may contain one or more regulatory regions and/or selectable markers suitable for screening, evaluating and monitoring the results of delivery of nucleic acids (delivery to tissue, maintenance of expression, etc.).

[0095] The polynucleotides of the present invention can be administered in vivo by lipofection. In recent decades, the use of liposomes for in vitro encapsulation and transfection of nucleic acids has increased. Synthetic cationic lipids designed to limit the difficulties and risks faced by liposome-mediated transfection can be used to prepare liposomes for in vivo gene transfection (Feigner et al., Proc. Natl. Acad. Sci. USA, 84: 7413 (1987) ); Mackey et al., Proc. Natl. Acad. sci. USA 85: 8027 (1988); and Ulmer et al., Science, 259:1745 (1993)). The use of cationic lipids can promote the encapsulation of negatively charged nucleic acids, and can also promote fusion with negatively charged cell membranes (Feigner et al., Science, 337: 387 (1989)). Particularly useful lipid compounds and nucleic acid delivery compositions are described in WO95/18863, WO96/17823 and US Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into certain tissues in vivo has several practical advantages. Molecular targeting of liposomes to specific cells is one advantage. Clearly, direct transfection into certain cell types will be particularly desirable in tissues with cellular heterogeneity such as the pancreas, liver, kidney and brain. Lipids can be chemically linked to other targeting molecules (Mackey et al., 1988). Target peptides such as hormones or neurotransmitters and proteins such as antibodies or non-peptide molecules can be chemically linked to liposomes.

[0096] As used herein, the term "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by a cell. When exogenous or heterologous RNA or DNA is introduced into a cell, the cell is "transfected" with such RNA or DNA. When a textured RNA or DNA type causes a phenotypic change, the cell is "transformed" by exogenous or heterologous RNA or DNA. The RNA or DNA causing this transformation can be inserted (covalently) into chromosomal DNA to become part of the cell's genome.

[0097] As used herein, the term "transformation" refers to the delivery of nucleic acid fragments to a host organism resulting in genetically stable inheritance. Host organisms containing transformed nucleic acid fragments are referred to as "transgenic", "recombinant", or "transformed" organisms.

[0098] As used herein, the term "recombinant vector" refers to a gene construct that is an expression vector capable of expressing a target protein in a suitable host cell and includes the necessary regulatory elements that are operably linked to express the inserted gene. .

[0099] Other molecules such as cationic oligopeptides (eg WO95/21931), peptides derived from DNA binding proteins (eg WO96/25508), or cationic polymers (eg WO95/21931) can also be used to provide transfection of nucleic acids in vivo.

[0100] You can also enter the vector in vivo as a naked DNA plasmid (see US patents No. 5693622, 5589466 and 5580859). Receptor-mediated DNA delivery can also be used (Curiel et al., Hum. Gene Ther., 3: 147 (1992); and Wu et al., J. Biol. Chem., 262: 4429 (1987)).

[0101] In one embodiment, the present invention discloses a cell that contains a nucleic acid molecule that encodes an antigen-binding protein of the present invention. In one embodiment of the present invention, the cell is a T cell; in another embodiment, the T cell is a CD8+ T cell and/or a CD4+ T cell; and in yet another embodiment, the cell is a chimeric antigen receptor (CAR-T) T cell.

[0102] In one embodiment, the present invention relates to a pharmaceutical composition for the treatment of cancer, containing as a pharmaceutically effective ingredient a cell expressing an antigen binding protein.

[0103] In the framework of the present invention, the term "anticancer" includes "prevention" and "treatment"; "prevention" means any action in which cancer is inhibited or retarded by the administration of a composition of the present invention, and "treatment" means any action which attenuates or favorably alters the symptoms of cancer by the administration of an antibody of the present invention. Prevention may be complete, for example, the complete disappearance of symptoms in the subject. Prevention can also be partial, for example, if the subject's symptoms are less severe than they would be without the application of the present invention.

[0104] The "composition" disclosed in the present invention refers to the combination of cytotoxic T cells of the present invention as an active ingredient and inactive ingredients such as natural or artificial carriers, labels or detectors, active ingredients such as adjuvants, diluents, binding agents, stabilizers, buffers, salts, lipophilic solvents and preservatives, and includes a pharmaceutically acceptable carrier. The carrier may also include pharmaceutical excipients and additional proteins, peptides, amino acids, lipids, and carbohydrates (e.g., monosaccharides; disaccharides; trisaccharides; tetrasaccharides; oligosaccharides; alditol, aldonic acid, polysaccharides, sugar derivatives such as an esterified sugar or a sugar polymer or the like. ), alone or in combination, in an amount of from 1 to 99.99 wt.% or vol.%. Protein excipients include, but are not limited to, human serum albumin, recombinant human albumin, gelatin, casein, and the like.

[0105] Typical amino acid components that can act as a buffer include, for example, alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and etc., but not limited to. Carbohydrate excipients also include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides such as lactose, sucrose, trehalose, cellobiose; polysaccharides such as raffinose, maltodextrin, dextran and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, sorbitol and myoinositol; not limited to this.

[0106] A person skilled in the art can formulate the pharmaceutical composition of the present invention using methods known in the art. For example, if necessary, it can be used parenterally in the form of an injectable sterile solution or suspension with water or other pharmaceutically acceptable liquid. For example, it can be suitably combined with pharmaceutically acceptable carriers or media, in particular sterile water or saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, carrier, preservative, binder, and the like; the composition can be prepared by mixing to obtain a single dosage form according to the requirements of conventional pharmaceutical practice. The amount of active ingredient used in the formulation is such that a suitable dosage can be provided within the indicated range.

[0107] In addition, sterile injectable compositions can be formulated in accordance with conventional formulation practice using liquid excipients such as distilled water for injection. As an aqueous solution for injection, for example, combinations of saline; isotonic solutions containing glucose or other auxiliary agents, eg D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable dissolution aids, eg alcohols, eg ethanol, and polyalcohols, eg propylene glycol, polyethylene glycol; and nonionic surfactants such as polysorbate 80™, HCO-50. Oily liquids include, for example, sesame oil and soybean oil, and can be used in combination with benzyl benzoate and benzyl alcohol as a solubilizer.

[0108] Injectable formulations can, for example, be administered by intravenous injection, intra-arterial injection, selective intra-arterial injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intraventricular injection, intracranial injection, intramedullary injection, and the like, however, they are preferably administered by intravenous injection.

[0109] The composition of the present invention contains a pharmaceutically effective amount of T cells. An effective amount can be readily determined by those skilled in the art based on the disclosure provided herein.

[0110] Typically, a pharmaceutically effective amount is determined by first administering at a low concentration of the active ingredient, and then gradually increasing the concentration until the desired effect is achieved in the subject without any side effects (e.g., cancer-related symptoms are alleviated or eliminated). ). Methods for determining suitable dosages or intervals between administrations for administering compositions of the present invention are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th ^Edition , McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, ^20th and 21st ^Editions , Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005).

[0111] The route of administration of the composition of the present invention can be determined based on various factors in the subject, such as the type of cancer, age, body weight, gender, health status, disease severity, route of administration, and other self-administered drugs. Therefore, although the route of administration may vary greatly, it can be determined according to the method commonly used.

[0112] The amount of the composition of the present invention to be administered to a subject may be determined based on a variety of factors, such as the route of administration, the subject's health status, body weight, and physician's advice; all of these factors are within the skill of the art.

[0113] The pharmaceutical composition of the present invention may contain about 1×10 ⁶ cells/ml or more, about 2×10 ⁶ cells/ml or more, about 3×10 ⁶ cells/ml or more, about 4×10 ⁶ cells/ ml or more, about 5×10 ⁶ cells/ml or more, about 6×10 ⁶ cells/ml or more, about 7×10 ⁶ cells/ml or more, about 8×10 ⁶ cells/ml or more, about 9 ×10 ⁶ cells/ml or more, 1×10 ⁷ cells/ml or more, about 2×10 ⁷ cells/ml or more, 3×10 ⁷ cells/ml or more, about 4×10 ⁷ cells/ml or more , about 5×10 ⁷ cells/ml or more, about 6×10 ⁷ cells/ml or more, about 7×10 ⁷ cells/ml or more, about 8×10 ⁷ cells/ml or more, about 9×10 ⁷ cells/ml or more, about 1x10 ⁸ cells/ml or more, about 2x10 ⁸ cells/ml or more, about 3x10 ⁸ cells/ml or more, about 4x10 ⁸ cells/ml or more, about 5×10 ⁸ cells/ml or more, about 6×10 ⁸ cells/ml or more, about 7×10 ⁸ cells/ml or more, about 8×10 ⁸ cells/ml or more, or about 9×10 ⁸ cells/ml or more CAR-T cells, but one skilled in the art can adjust the titer of CAR-T cells within a range where similar effects can be obtained. Factors such as preparation methods, routes of administration, age of the patient, body weight, gender, morbidity, diet, administration time, route of administration, rate of elimination, and sensitivity to response may influence prescribing in different ways.

[0114] The pharmaceutical composition can also be combined with buffers, such as phosphate buffers or sodium acetate buffers; analgesics, such as procaine hydrochloride; stabilizers such as benzyl alcohol, phenols and antioxidants. The prepared solution for injection is usually poured into suitable ampoules.

[0115] Suspensions and emulsions may contain as carriers, for example, natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection together with the active compound contain pharmaceutically acceptable carriers such as sterile water, olive oil, ethyl oleate, glycols, for example propylene glycol, and, if necessary, appropriate amounts of lidocaine hydrochloride.

[0116] The pharmaceutical composition of the present invention can be administered to a subject, for example, by intravenous injection (bolus injection) or continuous infusion. For example, the pharmaceutical composition of the present invention can be administered at least 1 time, at least 2 times, at least 3 times, at least 4 times, or at least 5 times, continuously or at intervals intervals of at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 8 hours, at least at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least , 7 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 3 months, at least 6 months, or at least , intervals are determined by clinical judgment. Injectable preparations can be formulated as an ampoule or as a single dosage form in a multi-dose container. However, a person skilled in the art will understand that the dosage of the pharmaceutical composition of the present invention may vary depending on various factors such as age, weight, height, sex of the subject, general health and previous treatment history.

[0117] In the framework of the present invention, the term "cancer" refers to any of the numerous diseases or disorders caused by abnormal uncontrolled cell growth. Cells that can cause cancer are called cancer cells and they have unique typological characteristics such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation. Often the cancer cells may be in the form of a tumor, but such cells may be present individually in mammals or may be non-tumor cells such as leukemic cells. Cancer can be diagnosed clinically or radiologically with tumor detection; by testing cells from tumors or other biological samples obtained by biopsy; detection of blood cancer markers such as CA125, PAP, PSA, CEA, AFP, HCG, CA 19-9, CA 15-3, CA 27-29, LDH and NSE; or detection of cancer marker genotypes such as TP53 and ATM. However, a negative result of the above method does not necessarily mean a diagnosis of no cancer: for example, a subject who has been found to be completely cured of cancer may still have cancer; which is confirmed in the form of a relapse.

[0118] For the purposes of the present invention, the term "about" can be understood within a range commonly used in the art, such as within 2 standard deviations from the mean. "About" can mean between 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4 %, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the indicated value.

[0119] In the framework of the present invention, the term "administration" means the introduction of a certain substance to a patient; administration can be by any suitable route, so that the route of administration of the composition containing the antibody of the present invention can be administered by any general route, provided that it can reach the target tissue. Administration may be by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, or rectal administration, without limitation. However, in the case of oral administration, since protein is degradable, it is desirable to formulate the oral composition in such a way as to coat the active agent or protect it from degradation in the stomach.

[0120] In addition, the pharmaceutical composition can be administered using any device from which the active substance is able to migrate to the target cell.

[0121] In one embodiment of the present invention, the cancer may be a solid cancer or cancer of the blood. More specifically, in one embodiment of the present invention, cancers such as adenocarcinoma of the esophagus, colorectal cancer, melanoma, melanoma of the eye, small cell lung cancer, neuroblastoma, teratoma, fetal cancer, squamous cell carcinoma, squamous cell carcinoma can be treated with the pharmaceutical composition of the present invention. head and neck, thymoma, lymphocytic leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, leukemia, acute myeloid leukemia, and the like.

[0122] Another embodiment of the present invention discloses a method for treating cancer by administering a pharmaceutical composition to a patient suffering from cancer. The pharmaceutical composition includes CD8+ T cells, or includes CD4+ T cells and CD8+ T cells, or includes both CD4+ T cells and CD8+ T cells; the ratio of CD4+ T cells to CD8+ T cells, depending on the number of cells, is generally 1:1.

[0123] In one embodiment, the present invention discloses a method for producing a chimeric antigen receptor (CAR-T) modified T cell for cancer treatment, comprising the step of transfecting the T cell with nucleic acids that encode an antigen-binding molecule comprising a heavy chain variable region comprising heavy chain complementarity determining region (HCDR1) containing the amino acid sequence represented by Sequence No. 1, HCDR2 containing the amino acid sequence represented by Sequence No. 2, and HCDR3 containing the amino acid sequence represented by Sequence No. 3; a light chain variable region comprising the light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence represented by sequence No. 4, LCDR2 containing the amino acid sequence represented by sequence No. 5, and LCDR3 containing the amino acid sequence represented by sequence No. 6; where the antigen-binding molecule is a chimeric antigen receptor (CAR).

[0124] In the framework of the present invention, the term "construct" usually refers to a composition that is not found in nature. The constructs can be obtained by synthetic methods (eg, preparation and expression of recombinant DNA) or by methods of chemical synthesis of nucleic acids or amino acids. Designs can also be created by adding or linking one substance to another so that the result is a form that does not exist in nature.

Experimental Example 1 Humanization of Mouse Anti-MVR Antibodies

[0125] The preparation of a humanized antibody from a mouse anti-MVR antibody (WO2015-133817 A1) was developed in two versions, low avidity and high avidity, respectively, for use in avidity optimization.

1.1 Humanization of the heavy chain variable region

[0126] To select a human antibody VH framework for making humanized antibodies, VH frameworks were selected using Blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins') with sequences similar to mouse anti-MVR antibodies (https://www.ncbi.nlm.nih.gov/protein/AAV40168.1).

[0127] Based on this, 3 VH CDRs were determined according to the Kabat numbering system, and the combination of the selected human antibody framework region and the CDR of the identified anti-MVR antibody was used to sequence the low avidity VH variant (huMVR.H1, sequence #10).

[0128] In addition, a high avidity VH variant was generated by backmutating VH27, VH29, VH48, VH67, VH71, VH73, VH78 from huMVR.H1 (huMVR.H2, sequence #7) (FIG. 1)

1.2 Humanization of the light chain variable region

[0129] In order to select the human antibody VL framework for the production of humanized antibodies, the trastuzumab VL framework (US Patent Application 5,821,047 A, sequence #25) was selected, which is known to be highly stable. Based on this, 3 VL CDRs were determined according to the Kabat numbering system, and a selected human antibody framework region and a specific anti-MVR antibody CDR were combined, and a low avidity VL variant from "hu4D5 framework-CDR combination" was obtained to introduce the human consensus sequence. anti-MVR" mutation K to R in VL24, I to L in VL48, S to R in VL53, T to S in VL56, R to G in VL66, F to Y in VL71, Q to G in VL100 (huMVR.L1 , sequence no. 11). In addition, individually VL49, VL69 and VL71 were backmutated from "hu4D5 framework region-CDR anti-MVR combination" and a high avidity VL variant was generated by an R to G mutation in VL66 (huMVR.L2, sequence no. 8) ( Fig. 2)

[0130] The engineered humanized antibody gene, which is huMVR.L1H1 (huMVR.L1 and huMVR.H1) (sequence No. 10, sequence No. 11, sequence No. 12), huMVR.L2H2 (huMVR.L2 and huMVR.H2) (sequence No. 7, sequence No. 8, sequence No. 9) was obtained with 2 antibodies. In particular, two humanized antibodies in the form of scFv VL-(G4S) ₃ -VH were prepared in pOptivec plasmid (Invitrogen), which is an animal cell expression vector, and the 6× His tag was conjugated to the C-terminus to obtain the gene. Plasmids into which the gene was inserted were expressed as scFv using the Expi293 expression system (Invitrogen) and purified using an AktaPure (GE healthcare) and HisTrap (GE healthcare) purifier column.

Experimental Example 2: Avidity Assay for Blood Cancer Lines

[0131] B-cell lymphoma lymphoblastoid cell lines derived from PBMC were prepared in FACS sample tubes (Falcon, catalog number 352052) at 1 x 10 ⁵ cells per sample and then treated with muMVR scFv, huMVR. binding) and huMVR.L2H2 scFv (high binding) at 0.5 µg/mL, 0.1 µg/mL, 0.05 µg/mL generated using Expi293F cells (Invitrogen, A14527) and each sample was incubated at 4°C for 15 min. Thereafter, 3 ml wash buffer (0.5% FBS, 0.1% sodium azide in PBS) was added to each sample tube. Each sample was centrifuged at 2000 rpm for 5 min, the supernatant was removed and phycoerythrin-conjugated anti-His antibody (BioLegend, catalog number 362603) was added at a 1:200 dilution to reach a total volume of 200 µl. All samples were incubated at 4°C for 15 min.

[0132] Thereafter, 3 ml wash buffer (0.5% FBS, 0.1% sodium azide in PBS) was added to each sample tube, each sample was centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. After diluting 4% paraformaldehyde (Biosessang, P2031) to 1%, cells were immobilized by adding 200 µl to each sample; immobilized samples were analyzed using a FACS Celesta flow cytometer (BD Biosciences).

[133] Two species (huMVR.L1H1, huMVR.L2H2) humanized antibody scFv bound to target antigens expressed in cells in a dose-dependent manner and huMVR.L2H2 exhibited similar avidity to mouse MVR scFv at an antibody concentration of 1 μg/ ml, while huMVR.L1H1 showed very low avidity (Fig. 3)

Experimental Example 3: Predicting Affinity Hot Spots to Optimize Humanized Antibody Activity

[0134] To generate mutants having different binding avidity between two humanized antibodies, affinity hotspots were predicted based on the mouse anti-MVR antibody. Molecular modeling was performed using the Swiss Model (https://swissmodel.expasy.org/) (Figure 4) based on the avidity analysis result of two humanized antibodies and two affinity hot spots in the heavy chain variable region framework (VH27, VH71) and affinity hotspots (VL91, VL92) in the light chain variable region CDRs (Table 1). 15 scFv type single mutants were prepared for the 4 affinity hotspots using huMVR.L2H2 as template, expressed and purified in the same manner as described above.

Table 1 "Hot Spots" Mutant amino acids VH_27F Y, L, G VH_71K H, L, V VL_91Y F, S, V, W, A VL_91W F, Y, L, A

Experimental Example 4: Avidity Analysis of 15 Mutants Used to Mutate Affinity Hot Spots

[0135] B-cell lymphoma lymphoblastoid cell lines derived from PBMC were generated in FACS sample tubes (Falcon, catalog number 352052) at 1 x 10 ⁵ cells per sample and then treated with muMVR scFv or huMVR.L2H2 derived from using Expi293F cells (Invitrogen, A14527) or 15 mutant species at a concentration of 0.1 μg/ml, and each sample was incubated at 4°C for 15 min.

[0136] Thereafter, 3 ml of wash buffer (0.5% FBS, 0.1% sodium azide in PBS) was added to each sample tube. Each sample was centrifuged at 2000 rpm for 5 min, the supernatant was removed and phycoerythrin-conjugated anti-His antibody (BioLegend, catalog number 362603) was added at a 1:200 dilution to reach a total volume of 200 µl. All samples were incubated at 4°C for 15 min. Thereafter, 3 ml of wash buffer (0.5% FBS, 0.1% sodium azide in PBS) was added to each sample tube, each sample was centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded.

[0137] After diluting 4% paraformaldehyde (Biosessang, P2031) to 1%, cells were immobilized by adding 200 μl to each sample, and immobilized samples were analyzed using a FACS Celesta flow cytometer (BD Biosciences).

[138] 15 species of mutant scFv huMVR.L2H2 (sequence no. 9) exhibited different avidity to LCL cells, and among them huMVR.L2H2.F27L, huMVR.L2H2.K71H, huMVR.L2H2.Y91F, huMVR.L2H.Y91W had successively lower affinity than muMVR and huMVR.L2H2 (sequence #9) (FIG. 5).

Experimental Example 5: Construction of CAR_euCD137

[0139] In the present invention, to improve the immunological efficacy in the cytoplasmic signaling domain 4-1BB, euCD137 (4-lBB aa209-255, sequence No. 14), to which 5 amino acids (4-1BB aa 209-213) were added in the cytoplasmic domain 4 -1BB 214-255aa (sequence #3) was used in CAR-T as a costimulatory signaling factor to generate a new CAR expression vector (CAR_euCD137) (Kwon et al., (1989) Cellular Immunology, 121: 414-422, Kwon et al., (1998) Biochemical and Biophysical Research Communication 242:613-620). The complete construct contains the anti-CD19 or anti-MVR huMVR.L2H2 binding domain, which is a scFv including the EFI-alpha promoter, human CD8 hinge and transmembrane domain, and an intracellular signaling domain. In particular, the intracellular signaling domain consists of a stimulatory domain and a costimulatory signaling domain. For the transmembrane domain, the alpha, beta, or zeta chain of the T cell receptor or one or more of CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154, but not limited to. Of these, CD8 was used in the present invention. The intracellular signaling domains are primarily costimulatory signaling domains in the primary signaling domain of CD3zeta selected from CD28, OX40, CD27, ICAM-1, ICOS (CD278), 4-1BB (CD 137) and 4-1BB 5aa (euCD137). In the present invention, 4-1BB, a costimulatory signaling domain, was used, to which 5 consecutive amino acids were added to which CD3zeta was bound. The resulting CAR gene fragment was finally conjugated to BamH I and Sal I digested lentiviral ELPS expression vectors. In addition, cloning was performed using the BamH I/Nhe I restriction enzyme to replace only a portion of the scFv (FIG. 6).

[0140] In addition, a chimeric antigen receptor (huMVR CAR) containing 2 types of cytoplasmic signaling domains (4-1BB, CD3ζ) for T cell activation was prepared using huMVR.L2H2, a humanized anti-MVR antibody (huMVR) designed for minimizing immunogenicity based on mouse anti-MVR antibodies, which are scFvs suitable for therapeutic CAR-T development. Specifically, in the present invention, in order to increase immunological efficacy to the 4-1BB cytoplasmic signaling domain, the construct (MVR CAR_euCD137) to which 5 consecutive amino acids of RFSVV were added contains the EF1-alpha promoter and contains only the huMVR scFv binding domain. L2H2 anti-MVR, and also the CD8 hinge region and the transmembrane domain, and also euCD137 and the intracellular signaling domain.

Experimental Example 6 Production of Recombinant huMVR Lentivirus

[0141] The 293T cell culture used to obtain recombinant lentiviruses contains a medium containing 10% FBS (Millipore, TMS-013-BKR) and 1 μl P/S (Gibco, 15140-122) in high glucose DMEM medium ( Welgene, LM001-05). 293T cells were incubated in DMEM containing 10% FBS for 24 h before transduction in a 5% CO ₂ incubator at 37°C. The next day for transfection, the transfection reagent and four lentiviral plasmids were mixed in an appropriate ratio and incubated for 48 hours. The supernatant containing the lentivirus was then collected and centrifuged at 400×g for 10 minutes. In addition, the supernatant was filtered through a 0.45 µm syringe filter using a 50 ml syringe. The resulting supernatant was mixed in a 3:1 ratio with the lentivirus enrichment kit reagent (Clontech, 631231) and reacted at 4°C for 24-48 hours. Then centrifugation was performed for 2 hours at 4°C and 4000 rpm to obtaining a virus, which was resuspended in 0.5 ml of RPMI medium (Welgene, LM001-01) not containing FBS, to obtain lentivirus.

6.1: Quantification of infected lentiviral particles using flag conjugated to N-terminal chimeric antigen

[0142] Transducing units (TU/ml) were determined by assaying the number of lentivirus particles actually capable of transduction using Jurkat cells. On the first day, Jurkat cells were seeded in 96-well plates at the rate of 1×10 ⁵ cells/100 μl per well. On the second day, serial dilutions of lentivirus were prepared in 1/3 increments in 96-well plates and lentiviral transduction was performed on already seeded Jurkat cells. At this time, polybrene (Millipore) was added to RPMI medium (10% FBS and 1x P/S) to further enhance lentivirus transduction. After centrifugation at 1200×g and 25° C. for 2 h, the cells were incubated for 3 h in a 5% CO ₂ incubator at 37° C. and only 100 μl of RPMI medium per well was added. On day 5, flag lentivirus introduced into the cell was stained with anti-Flag-DYKDDDDK (Biolegend, catalog number 637310) to determine the percentage of transduced cells using a flow cytometer. Using this, the titer was calculated as described in Follenzi and Naldini, 2002 (Follenzi and Naldini, 2002) and the result was 1.4×10 ¹⁰ IU/ml.

Experimental Example 7 Thawing and T Cell Activity

[0143] Frozen peripheral blood mononuclear cells (PBMC) were thawed for 5 minutes in a 37°C constant temperature bath and suspended in RPMI1640 medium. Then centrifugation was performed at 1500 rpm for 5 min and the supernatant was removed. T cells were isolated using CD4 microbeads (Miltenyi Biotec, 130-045-101) and CD8 microbeads (Miltenyi Biotec, 130-045-201). For the method of isolating CD4, CD8 T cells, the protocol with CD4, CD8 microbeads (DS_130-045-101, DS_130-045-201) was used.

[0144] After adjusting the density of isolated cells to 1×10 ⁶ cells/ml, CAR-T medium (basal medium IL Optimizer CTS + 26 ml CTS optimizer + 50 ml CTS immune Cell SR mixture + 10 ml penicillin-streptomycin + 10 ml GlutaMAX-1), and IL-2 was added at a concentration of 20 IU/ml. In addition, 10 μl of T-cell TransAct activator (Miltenyi Biotec, 130-111-160) per 1×10 ⁶ cells was added, placed in a T75 flask or 24-well plate and incubated in a thermostat (37°C, 5% CO ₂ ) within 2 days (Fig. 8)

Experimental Example 8: CAR Transduction and Incubation of CAR-T Cells

[0145] T cells activated for 2 days were added at the rate of 2×10 ⁶ cells per 1 well of a 24-well plate, and lentivirus containing the CAR gene was added at a MOI of 1-3. Protamine sulfate (product number ADR301) was added and suspended to a final concentration of 10 μg/ml. A 24-well plate was centrifuged at 400×g and 32°C for 2 h and after suspending the cells in 20 ml of CAR-T medium (IL-2 200 IU/ml), they were placed in a T75 flask and incubated in a thermostat (37°C , 5% CO ₂ ) for 2 days. After 2 days, the number of cells was counted, the cell density was adjusted to 0.5×10 ⁶ cells/ml, and the cells were added to the CAR-T medium and IL-2 was added at the rate of 200 IU/ml for the total incubation broth. If the cell density was below 0.5×10 ⁶ cells/ml, then 200 IU/ml of IL-2 was added relative to the total amount of culture fluid without the addition of CAR-T medium. Thereafter, cell counts were performed every 2 days and CAR-T medium (IL-2 200 IU/ml) was added at a standard cell density of 0.5×10 ⁶ cells/ml. CAR-T cells were harvested between 7 and 14 days after the start of incubation (FIG. 7).

Experimental Example 9 Cytotoxicity Assay

[0146] Cytotoxicity of CAR-T cells to target cells was determined using a luciferase-based assay. Target cells were infected with EBV carrying the luciferase gene to obtain target cells carrying the luciferase gene. Co-cultivation with effector cells was carried out at an effector-target ratio (E/T) for 24 h at 37°C. After co-culture and subsequent treatment with CytoTox-Glo Cytotoxicity reagent (promega, G9290), RLU values for live target cells were measured using a Fluroskan FL microplate luminometer (Thermo).

[0147] More specifically, samples were prepared in triplicate in a 96-well flat-bottomed white plate (COSTAR, 3917) with effector cells at 4.5×10 ⁵ cells/50 μl, at an E/T ratio of 3:1 in calculated on 1.5×10 ⁴ target cells and diluted 1/3 to obtain ratios of E/T 3:1, 1:1, 0.3:1 and 0.1:1. In each well, effector cells along with 1.5×10 ⁴ target cells (50 μl) were co-cultured in an incubator (37°C, 5% CO ₂ ) for 18-24 h, and then 100 μl of CytoTox-Glo Cytotoxicity was added. (Promega, G9290), blocked the light, and after 5 min the luminescence value was measured using a Fluroskan FL microplate luminometer (Thermo).

[0148] As a result, huMVR CAR-Ts were found to exhibit 95.4% cytotoxic activity at an E/T ratio of 3:1 (FIG. 8).

Experimental Example 10: Cell Line Incubation

[0149] The CBKLCL-Luc and KHJ LCL-Luc (hereinafter LCL-Luc) cell lines, from the National Cancer Institute, were incubated in RPMI1640 medium (Welgene, catalog number LM011-01) with 20% fetal bovine serum (FBS, Millipore, catalog number TMS-013-KBR) and 1% penicillin/streptomycin mixture (Gibco, catalog number 15140-122).

Experimental Example 11: Determination of two types of CD19CAR-T cells in which CD19CAR and CD19CAR_euCD137 have been introduced

[0150] To improve immunological efficacy, CD19CAR (sequence No. 17, sequence No. 12 in US Patent Application 8906682) and CD19CAR_euCD137 (sequence No. 18), in which the 4-1BB domain in CD19CAR was replaced by euCD137, were obtained separately and cultured with CD19CAR T cells to assess the newly established efficacy of the CAR expression vector (CAR_euCD137), with euCD137 (4-1BBaa209-255, sequence no. 14) as a costimulatory signaling factor.

[0151] Cell staining for FACS was performed to determine the proportion of production of two types of CAR-T cells after 14 days of incubation. For each CAR-T cell type, from 2×10 ⁵ cells were harvested into FACS tubes (FALCON, catalog number 352052), then 2 ml of FACS buffer was added and centrifuged for 5 min at 2000 rpm using a centrifuge ( Thermo, ST16). After removal of the supernatant, 0.5 µl/tube anti-CD8 APC (SKI, Biolegend, cat. no. 344722), 0.5 µl/tube anti-CD4 BV650 (RPA-T4, Biolegend, cat. no. 300536) and 0.125 µl /anti-flag tube (L5, Biolegend, catalog number 637310) and stained at room temperature for 30 minutes. After adding 2 ml of FACS buffer and centrifuging at 2000 rpm for 5 minutes, this procedure was repeated one more time, 1 µl/tube of 7-AAD (Biolegend, cat. no. 420404) was added to stain for living/dead cells, and the mixture was left for 5 min at room temperature, and then analyzed using FACS (BD, FACSCelesta).

[0152] Prior to testing in a subcutaneous tumor animal model of the effect of CD19 CAR-T using the CAR construct (CD19CAR_euCD137, sequence #18), in which euCD137 (amino acids 209-255, sequence #14) was added to 4-1BB (amino acids 214- 255, sequence #13) containing five amino acids, and a conventional construct (CD19CAR, sequence #17, sequence #12 in US Patent Application 8,906,682), the proportion of CAR-T cells was determined using the method described in Experimental Example 2. Analysis of the relative number of CD19 CAR-T cells produced using FACS staining confirmed that in the case of an improved CD19 CAR-T cell construct (5aa CD19 CAR-T), the cell ratio was CD4+/CAR+ 29.4%, CD8+/CAR+ 50.8 % and overall CAR-T 80.2%. It was found that in the unimproved CAR-T cell construct (CD19 CAR-T), the cell ratio was 42.7% for CD4+/CAR+, 29.3% for CD8+/CAR+ and 72.0% for CAR-T as a whole. .

[0153] Therefore, 5aa CD19 CAR-T cells were shown to have 8.2% higher CAR expression than CD19 CAR-T cells, and CD8+/CAR+ cells were 21.5%, or about 2 times more (Fig. 9-A).

Experimental Example 12 Cytotoxicity Evaluation of Derived CD19CAR-T Cells

[0154] To assess the cytotoxicity of two types of CAR-T cells cultured for 14 days, the ratio of CAR-T (E): LCL (T) was adjusted to 30:1, 10:1, 3:1 and 1:1 in 96-well white plates (Corning, catalog number 3917). First, CAR-T cells were placed in wells at 6×10 ⁵ cells/50 μl, 2×10 ⁵ cells/50 μl, 9×10 ⁴ cells/50 μl and 2×10 ⁴ cells/50 μl, respectively. Then the target cell line, namely the cell line CBK LCL-Luc, was added to 2×10 ⁴ cells/50 μl in a CO ₂ incubator at 37°C (Mammert, INCO153med) and allowed to interact for 4 h. After 4 h 100 μl of Bright-Glo™ (Promega, catalog number E2620) was added to each well and relative light units (RLU) were measured 5 minutes later by ELISA (Thermo, Fluorescan FL).

[0155] There was no difference in cytotoxicity between CARTs injected with conventional 4-1BB and CAR-T cells injected with euCD137 containing five amino acids added to the 4-1BB domain.

[0156] In this experiment, when two CAR-T cell lines and a CBK LCL-Luc cell line were incubated together at a ratio of 30:1, the cytotoxicity was approximately 80% after 4 hours, and when the cells were incubated at a ratio of 10: 1, the cytotoxicity was about 50%. Since the corresponding number of CAR-T cells incubated with tumor cells decreased 3-fold, the cytotoxicity was reduced by about 3-fold; furthermore, when 5 amino acids were added to the 4-1BB domain in vitro, it was shown that there was no negative effect on cytotoxicity (Fig. 9B)

Experimental Example 13: HLADR Expression Evaluation and HuMVR L2H2 scFv Avidity Analysis in Cell Lines

[0157] FACS analysis was performed using a cultured cell line LCL-Luc, to assess the expression of HLADR in the cell line and analyze the avidity of the developed scFv huMVR L2H2. After transferring 2×10 ⁵ LCL-Luc cells, 2 ml of FACS buffer was added to the FACS tube and centrifuged at 2000 rpm for 5 minutes using a centrifuge. After the supernatant was removed, anti-HLADR APC-H7 (G46-6, BD Pharmigen™, catalog number 561358) was added at 0.5 μl/vial and Inventor's MVR L2H2 at 1.0 μg/vial and stained at room temperature for 30 min. 2 ml of FACS buffer was added for washing, followed by centrifugation at 2000 rpm for 5 minutes, and the supernatant was discarded. This procedure was repeated once more. For huMVR L2H2 scFv secondary staining, 1 μl/tube of anti-His PE (Biolegend, cat. no. 362603) was added and stained for 30 minutes at room temperature. Then, the washing procedure using FACS buffer was repeated twice, and 100 μl of FACS buffer was added, and then analysis was performed using FACS.

[0158] FACS staining and analysis was performed to assess the expression of HLA DR, which is a target for huMVR L2H2, and the avidity of huMVR L2H2 scFv in tumor cells.

[0159] The results of the evaluation of HLA DR expression in the LCL-Luc cell line showed that 100% of HLA DR is expressed. When avidity analysis was performed using huMVR L2H2 scFv, at least 97% binding to expressed HLA DR was shown (Fig. 10)

Experimental Example 14: Reproduction of Subcutaneous Tumor Animal Models and Evaluation of CAR-T with Automated Calipers and IVIS Imaging

[0160] NSG mice (NOD-scidIL2rγµL1) (The Jackson Laboratory) were used as experimental animals and kept under constant conditions in an animal nursery. The temperature was 23±2°C with a 12-hour light/dark cycle and a humidity of 50±10%; food and water were given ad libitum. In efficacy experiments using CD19CAR-T (5aaCD19CAR-T) with five amino acids added to the 4-1BB domain, CBK LCL-Luc cell lines were prepared at 2×10 ⁶ cells/100 μl DPBS/head and injected subcutaneously 6 -week-old female mice to reproduce the subcutaneous tumor animal model. When tumors reached 50 to 100 mm ³ as measured with an automatic caliper (Youngbio, TM900), improved CD19 CAR-T cell construct and non-improved CD19 CAR-T cell construct were injected at 2×10 ⁶ cells/100 μl DPBS/head (dose 1) and 6×10 ⁶ cells/100 μl DPBS/head (dose 2) once through the tail vein to evaluate efficacy. In the following experiment, the efficacy of huMVR CAR-T cells was evaluated in an animal model using a construct (huMVR CAR_euCD137, Fig. 6C) that was engineered to express huMVR in a CAR construct containing five amino acids in the 4-1BB domain. Subcutaneous tumor animal models were induced by subcutaneous injection of LCL-Luc cells into 6 week old female mice at 4×10 ⁶ cells/100 μl DPBS/head. To assess efficacy, when tumors reached the range of 50-100 mm ³ , a single dose of MVR CAR-T was administered at a rate of 1×10 ⁶ cells/100 μl DPBS/head, 2×10 ⁶ cells/100 μl DPBS/head (dose 1 ) and 5×10 ⁶ cells/100 μl DPBS/head (dose 3) via the tail vein. In all animal experiments, the size and viability of the tumors were periodically determined.

[0161] More specifically, tumor size and photon values were determined using automatic calipers and an IVIS imaging device (PerkinElmer, Luna III) at 3- and 4-day intervals after CAR-T administration. In the case of using the TM900, the size of the tumors was determined after placing the equipment at the location of the tumors. When imaging and determining photon values using the IVIS imaging device, mice were first injected intraperitoneally with 150 mg/kg XenoLight™ D-luciferin (PerkinElmer, catalog number 122799). After 15 minutes, inhalation anesthesia with isoflurane was performed, and after 5 minutes IVIS was used to visualize the presence of tumor cells. After imaging, normalization was performed, and then luciferase values (photon values) were determined and plotted.

[0162] More specifically, after reproducing a subcutaneous tumor animal model using the CBK LCL-Luc cell line, the effects of CD19 CAR-T and 5aa CD19 CAR-T cells were compared using IVIS imaging. As shown in FIG. 11A, the effect was observed within 1 week after administration of 2 kinds of CAR-T cells.

[0163] In the experimental group in which CD19 CAR-T with CD19CAR_euCD137 was administered at 2×10 ⁶ cells/100 μl DPBS/head and 6×10 ⁶ cells/100 μl DPBS/head, tumor cells were observed by IVIS imaging through 1 week after injection. In addition, in the unimproved CD19 CAR-T construct group, when 6×10 ⁶ cells/100 μl DPBS/head were injected, tumor cells were rarely observed by imaging within 1 week after administration. However, tumor cells were identified in the experimental group administered unimproved CD19 CAR-T after 1 week.

[0164] 1 week after the administration of CAR-T, as shown in the images, the value of luciferase was determined in each animal after the imaging process; luciferase values were only confirmed in the group injected with CD19 CAR-T at 2×10 ⁶ cells/100 μl DPBS/head. When observed 3 weeks or more thereafter, tumors continued to grow in mice not injected with CAR-T cells, and no tumor cells were detected in the three experimental groups in which tumor cells were eliminated initially. However, after 10 days, luciferase levels decreased in the group treated with 2×10 ⁶ cells/100 μl DPBS/head for CD19 CAR-T, but the tumor cells did not completely disappear after 3 weeks. When administered with the improved 5aa CD19 CAR-T construct at 2×10 ⁶ cells/100 μl DPBS/head, experimental imaging of tumor cells revealed that the efficacy was similar to that in the group treated with CD19 CAR-T at 6×10 ⁶ cells/100 µl DPBS/head. The results showed that there was no difference in cytotoxicity between 5aa CD 19 CAR-T and CD 19 CAR-T in vitro, but the efficacy was shown to be 5-fold higher for 5aa CD19 CAR-T in animal models (FIG. 11).

Experimental Example 15: Determination of the proportion of CD19 CAR-T with 5 added amino acids in the 4-1BB domain in an in vivo animal model

[0165] After the administration of CAR-T cells in a subcutaneous tumor animal model to evaluate the effectiveness of the improved CAR-T cell construct, the presence of CAR-T was determined by blood analysis of mice.

[0166] More specifically, blood sampling from the orbital venous sinus of mice was performed at 3- and 4-day intervals after CAR-T administration. During each blood draw, 70 μl of blood was collected and 60 μl of blood was used to determine the proportion of CAR-T cells and count cells. 60 µl of blood was placed in a 5 ml FACS tube and live/dead cell staining was performed using Zombie Aqua BV510 (Biolegend, catalog number 423101). After reaching a concentration of 0.1 µl/100 µl DPBS/tube, staining was performed at room temperature for 10 min. Counting beads (Molecularprobes, cat. no. C36950), anti-CD45 FITC (HI30, Biolegend, cat. no. 304006), anti-CD8 BV786 (SK-1, Biolegend, cat. no. 344740), anti-CD4 BV650 and anti- -flag PE and stained at room temperature for 30 min. Each antibody was mixed at the rate of 0.5 μl/100 μl of FACS buffer per tube and 25 μl of counting beads were added. After 30 minutes, 2 ml of erythrocyte lysis buffer IX (Biolegend, catalog number 422401) was added and reacted at room temperature for 5 minutes. After centrifugation for 5 minutes at 2000 rpm using a centrifuge, all supernatant was removed.

[0167] To this tube was added 2 ml of FACS wash buffer and centrifuged for 5 minutes at 2000 rpm. This procedure was repeated once more, and then 50 μl of FACS buffer was added and analyzed by FACS.

[0168] One week after the administration of CAR-T cells in the group with the introduction of 5aa CD19 CAR-T, approximately 20% of 5aa CD19 CAR-T cells were in the blood as in groups with 2×10 ⁶ cells/100 μl DPBS/head , and in groups with 6×10 ⁶ cells/100 µl DPBS/head; but in the CD19 CAR-T injected group, only 5% of CD19 CAR-T was confirmed with 6×10 ⁶ cells/100 μl DPBS/head. After 3 days, the number and proportion of CAR-T cells in the mouse reached a maximum and then decreased. Within one week in 3 experimental groups in which CAR-T cells were detected (5aa CD19 CAR-T; 2×10 ⁶ cells/100 μl DPBS/head and 6×10 ⁶ cells/100 μl DPBS/head, CD19 CAR-T; 6×10 ⁶ cells/100 μl DPBS/head), tumor cells died rapidly, probably due to the fact that they could contact a relatively large number of CAR-T cells before they proliferated in the mouse. However, in the experimental group, mice which were injected with CD19CAR-T at 2×10 ⁶ cells/100 μl DPBS/head, the proportion and number of CAR-T cells reached a maximum after 2 weeks, and the proportion of CAR-T was approximately 25% while that the tumor cells proliferated relatively well. The improved 5aa construct CD 19 CAR-T was more stable in number than CD19 CAR-T after the proportion of CAR-T first increased and then decreased. However, in the case of CD19 CAR-T, the proportion of CAR-T cells increased and decreased at later times, and therefore, it took longer for tumors to disappear in mice. As a result, as shown by the data of this experiment, the group injected with 5aa CD19 CAR-T at 2×10 ⁶ cells/100 μl DPBS/head showed similar levels of CAR-T and effects in mice with the group injected with CD19 CAR- T based on 6×10 ⁶ cells/100 μl DPBS/head, indicating that 5aa CD19 CAR-T has a higher effect (Fig. 12).

Experimental Example 16: Reproduction of an intraperitoneal tumor animal model and evaluating the efficacy of CD8 and CD4/CD8 huMVR CAR-T (CAR_euCD137) in an intraperitoneal tumor animal model

[0169] The experimental animals used to reproduce the intraperitoneal tumor animal model were the same as those used in Experimental Example 14 and were kept under similar conditions. CD8 huMVR CAR-T and CD4/CD8 huMVR CAR-T were constructed using the improved CAR construct (CAR_euCD137) and efficacy was evaluated in an intraperitoneal tumor animal model. To reproduce an intraperitoneal tumor model in animals, LCL-Luc cells expressing luciferase were injected into the abdominal cavity at 2×10 ⁶ cells/100 μl DPBS/head, and after nine days the animals were divided into five groups according to photon values. After separation into appropriate groups, CD8 MVR CAR-T was injected into the tail vein at a dose of 0.5×10 ⁶ cells/100 μl DPBS/head (dose 1), 2.0×10 ⁶ cells/100 μl DPBS/head (dose 2) and 10×10 ⁶ cells/100 μl of DPBS per head (dose 3), or 0.5×10 ⁶ CD4 and CD8 huMVR CAR-T cells, respectively, were mixed and injected once. In all experiments on animals periodically determined the size and viability of the tumors (Fig.13). The specific experimental method was the same as described in Example 14.

[0170] After isolation of CD4 and CD8 from PBMC derived from healthy adult blood, huMVR scFv was expressed in a modified CAR construct and evaluated for efficacy in an intraperitoneal tumor animal model using huMVR CAR-T.

The performance of huMVR CAR-T was assessed by imaging and determining the photon values shown by luciferase-expressing tumor cells.

[0171] After being assessed 2 times a week using the IVIS imaging device, 27 days after the administration of CAR-T to the experimental groups, in the control group of mice that had tumors and CAR-T was not injected, all animals died. However, all mice that developed tumors and were injected with huMVR CAR-T survived for at least 27 days. In the group of mice injected with CD8 huMVR CAR-T at a low dose (0.5×10 ⁶ cells/100 μl DPBS/head), the tumors tended to decrease in size, and then increase again after 10 days; in the group injected with CD8 huMVR CAR-T at a medium dose (2.0×10 ⁶ cells/100 μl DPBS/head), there was a trend towards a slow decrease in photon values over 27 days. However, when CD8 huMVR CAR-T was administered at a high dose (10.0×10 ⁶ cells/head), all tumors were found to have disappeared after 13 days. In addition, when CD4 and CD8 MVR CAR-T were administered together at low doses, tumors were shown to disappear in most mice (3 out of 5 mice) by about day 27, although they were not as effective as CD8 huMVR. CAR-T at a high dose. Therefore, the results of this trial showed that huMVR CAR-T has an effect at a medium dose or higher, and when used together with CD4, and not only CD8, the effect occurs even when used at a lower dose of CAR-T (Fig. 13 ).

Experimental Example 17 Evaluation of the Efficacy of CD4/8 huMVR CAR-T in Subcutaneous Tumor Animal Models

[0172] In Experimental Example 13, the production of huMVR CAR-T using CD4 and CD8 T cells together in constructing MVR CAR-T was shown to be more efficient in animal experiments. Based on the above example, CD4/CD8 huMVR CAR-T was prepared and administered at low, medium and high doses, and the efficacy of CAR-T was evaluated using a subcutaneous tumor animal model.

[0173] When tumor growth was assessed using an automatic caliper, in the high dose group (5.0×10 ⁶ cells/100 μl/head; dose 3), tumor growth slowed down by day 10, tumor growth was reversed by day 14 and most of the tumors were gone by day 17. As shown in FIG. ^14A , tumors were difficult to detect ^on day 21 images. μl/head; dose 1), tumor growth slowed from day 17 after CAR-T administration, and tumors decreased from day 21. However, as a result of using the imaging device, there were some mice in which tumor cells remained in the body for more than 4 weeks , and although the effect was seen at the medium and low dose, the effect was delayed compared to the high dose. When tumor cells were visualized with IVIS imaging, although the tumor model was replicated by subcutaneous inoculation, it was found over time that the tumors had spread to the lymph nodes. However, when MVR CAR-T was administered, all metastatic tumor cells died.

[0174] (Fig. 15) In addition, when animals were viable in this experiment by administering huMVR CAR-T, in the control group, all mice not treated with CAR-T died at day 18, but all mice survived at the group treated with MVR CAR-T (FIG. 16).

Experimental Example 18: In Vivo Identification of CD4/8 MVR CAR-T in Animal Models

[0175] The blood obtained from the mouse used in Experimental Example 17 was analyzed to determine the proportion and amount of CAR-T present in the body of the animal. Blood analysis showed that the proportion of CAR-T was low in mice administered CAR-T after 3 days, and the amount of CAR-T increased rapidly from 7% to 31% in the high dose group after 10 days. This was the point at which tumor size and photon values decreased in the high dose MVR group. In addition, for medium and low doses of huMVR CAR-T, the proportion of CAR-T increased from day 17 post-administration, and tumor size and photon values decreased from day 21 (FIG. 17).

table 2 Sequence number Information one heavy chain CDR1 huMVR.H2 2 huMVR.H2 heavy chain CDR2 3 huMVR.H2 heavy chain CDR3 four CDR1 light chain huMVR.L2 5 CDR2 light chain huMVR.L2 6 CDR3 light chain huMVR.L2 7 huMVR.H2 heavy chain variable region eight Light chain variable region huMVR.L2 9 scFv huMVRL2H2 ten huMVR.H1 heavy chain variable region eleven Light chain variable region huMVR.L1 12 scFv huMVRL1H1 13 4-IBB signaling domain fourteen euCD137 fifteen MVR.L2H2 CAR 16 MVR.L2H2 CAR_euCD137 17 CD19CAR eighteen CD19 CAR_euCD137 19 EF1a promoter twenty CD8-α leader sequence 21 Flag 22 CD19scFv 23 CD8/hinge/transmembrane sequence 24 CD3z 25 Groundhog/PRE 26 R/Region

[0176] For example, for the purposes of constructing the claims, it is not intended that the claims below be construed in any way narrower than its literal language, and thus it is not intended that exemplary embodiments from the description be in the claims. . Accordingly, it is to be understood that the present invention has been described by way of illustration and not by way of limitation of the scope of the claims. Accordingly, the present invention is only limited by the following claims. All publications, issued patents, patent applications, books and journal articles cited in this application are hereby incorporated by reference in their entirety.

Free text sequence listing

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Sequence list

<110> Eutilex Co., Ltd.

<120> CHIMERIC ANTIGEN RECEPTOR THAT BINDS HLA-DR AND

CAR-T-CELL

<130> 47683-0032002

<140> 16/715.462

<141> 2019-12-16

<150> 62/717.267

<151> 2018-08-10

<150> 62/867.503

<151> 2019-06-27

<150> PCT/KR2019/010244

<151> 2019-08-12

<160> 30

<170>PatentIn version 3.5

<210> 1

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<210> 1

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.H2 heavy chain CDR1

<400> 1

Arg Tyr Ser Val His

fifteen

<210> 2

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.H2 heavy chain CDR2

<400> 2

Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 3

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.H2 heavy chain CDR3

<400> 3

Asn Glu Gly Asp Thr Thr Ala Gly Thr Trp Phe Ala Tyr

1 5 10

<210> 4

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.L2 light chain CDR1

<400> 4

Lys Ala Ser Asp His Ile Asn Asn Trp Leu Ala

1 5 10

<210> 5

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.L2 light chain CDR2

<400> 5

Gly Ala Thr Ser Leu Glu Thr

fifteen

<210> 6

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.L2 light chain CDR3

<400> 6

Gln Gln Tyr Trp Ser Thr Pro Phe Thr

fifteen

<210> 7

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.H2 heavy chain variable region

<400> 7

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr

20 25 30

Ser Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Glu Gly Asp Thr Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.L2 light chain variable region

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

<211> 243

<212> PROTEIN

<213> Artificial sequence

<220>

<223> scFv huMVRL2H2

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr Ser Val His Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Gly

165 170 175

Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Glu Gly Asp Thr

210 215 220

Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210> 10

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.H1 heavy chain variable region

<400> 10

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Arg Tyr

20 25 30

Ser Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Glu Gly Asp Thr Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> huMVR.L1 light chain variable region

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Tyr Gly Ala Thr Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 12

<211> 243

<212> PROTEIN

<213> Artificial sequence

<220>

<223> scFv huMVRL1H1

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Tyr Gly Ala Thr Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Gly Ser Ile Ser Arg Tyr Ser Val His Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Met Ile Trp Gly Gly

165 170 175

Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Glu Gly Asp Thr

210 215 220

Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210> 13

<211> 42

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Signal domain 4-1BB

<400> 13

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 14

<211> 47

<212> PROTEIN

<213> Artificial sequence

<220>

<223>euCD137

<400> 14

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40 45

<210> 15

<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> sequence CAR MVR.L2H2

<400> 15

gatattcaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60

atcacctgca aggccagtga ccacatcaac aactggctgg cctggtatca acagaaacca 120

ggaaaagctc cgaaactact gatcagcggc gccacctctc tggaaaccgg agtcccttct 180

cgcttctctg gttccggatc tgggaaggat tacactctga ccatcagcag tctgcagccg 240

gaagacttcg caacttatta ctgtcagcag tactggtcca cccccttcac cttcggacag 300

360

ggatctcagg tgcagctgca ggagtcggggc ccaggactgg tgaagccttc ggagaccctg 420

tccctcacct gcactgtctc tggtttctcc ctgagtcggt actctgtgca ttggatccgg 480

cagcccccag ggaagggact ggagtggctg gggatgatct ggggaggcgg cagcaccgac 540

tacaacagcg ccctgaagtc ccgactgacc atatcaaagg acaactccaa gaaccaggtg 600

tccttgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaaat 660

gagggcgata ccaccgccgg cacttggttt gcctattgggg gccagggaac cctggtcacc 720

gtctcctcaa cccgacgcc agcgccgcga ccaccaacac cggcgcccac catcgctagc 780

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 960

1020

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1080

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1200

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260

1320

gatggccttt accagggtct cagtacagcc accaggaca cctacgacgc ccttcacatg 1380

caggccctgc cccctcgcta a 1401

<210> 16

<211> 1416

<212> DNA

<213> Artificial sequence

<220>

<223> sequence CAR_euCD137 MVR.L2H2

<400> 16

gatattcaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60

atcacctgca aggccagtga ccacatcaac aactggctgg cctggtatca acagaaacca 120

ggaaaagctc cgaaactact gatcagcggc gccacctctc tggaaaccgg agtcccttct 180

cgcttctctg gttccggatc tgggaaggat tacactctga ccatcagcag tctgcagccg 240

gaagacttcg caacttatta ctgtcagcag tactggtcca cccccttcac cttcggacag 300

360

ggatctcagg tgcagctgca ggagtcggggc ccaggactgg tgaagccttc ggagaccctg 420

tccctcacct gcactgtctc tggtttctcc ctgagtcggt actctgtgca ttggatccgg 480

cagcccccag ggaagggact ggagtggctg gggatgatct ggggaggcgg cagcaccgac 540

tacaacagcg ccctgaagtc ccgactgacc atatcaaagg acaactccaa gaaccaggtg 600

tccttgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaaat 660

gagggcgata ccaccgccgg cacttggttt gcctattgggg gccagggaac cctggtcacc 720

gtctcctcaa cccgacgcc agcgccgcga ccaccaacac cggcgcccac catcgctagc 780

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900

gtccttctcc tgtcactggt tatcaccctt tactgccgtt tctctgttgt taaacggggc 960

agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1020

gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1080

gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1140

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1200

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1260

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1320

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1380

gacgcccttc acatgcaggc cctgccccct cgctaa 1416

<210> 17

<211> 1398

<212> DNA

<213> Artificial sequence

<220>

<223> CAR CD19 sequence

<400> 17

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgctagccag 780

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020

1080

1140

cgaagagagg agtacgatgt ttttggacaag agacgtggcc gggaccctga gatggggggga 1200

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380

gccctgcccc ctcgctaa 1398

<210> 18

<211> 1420

<212> DNA

<213> Artificial sequence

<220>

<223> sequence CAR_euCD137CD19

<400> 18

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgctagccag 780

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900

cttctcctgt cactggttat caccctttac tgccgtttct ctgttgttaa acggggcaga 960

1020

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1080

aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1140

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1200

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1260

cagaaagata agatggcggga ggcctacagt gagattggga tgaaaggcga gcgccgggagg 1320

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1380

gcccttcaca tgcaggccct gccccctcgc taagtcgaca 1420

<210> 19

<211> 1184

<212> DNA

<213> Artificial sequence

<220>

<223> EF1a promoter

<400> 19

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacgggg ttatggccct tgcgtgcctt 300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360

ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540

tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840

ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900

ttccgtcctc agccgtcgct tcatgtgact ccactgagta ccgggcgccg tccaggcacc 960

tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140

gtga 1184

<210> 20

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> CD8-a leader sequence

<400> 20

ggatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60

gccaggccg 69

<210> 21

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Flag

<400> 21

gactacaagg acgacgatga caag 24

<210> 22

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223>CD19scFv

<400> 22

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctca 726

<210> 23

<211> 207

<212> DNA

<213> Artificial sequence

<220>

<223> CD8/Hinge/Transmembrane sequence

<400> 23

60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 24

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223>CD3z

<400> 24

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339

<210> 25

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> Woodchuck/PRE

<400> 25

atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 60

cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 120

tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 180

ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg 240

gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta 300

ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 360

tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 420

cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480

atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 540

gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct g 591

<210> 26

<211> 98

<212> DNA

<213> Artificial sequence

<220>

<223> R/area

<400> 26

gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60

ctgcttaagc ctcaataaag cttgccttga gtgcttca 98

<210> 27

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 27

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

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr

20 25 30

Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Glu Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Asn Glu Gly Asp Thr Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 28

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Asn Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Met Gly Ile Ala Ala Ala Gly Tyr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 29

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 29

Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly

1 5 10 15

Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 30

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<---

Claims (14)

1. An antigen-binding molecule that binds HLA-DR, containing the variable region of the heavy chain c SEQ ID NO: 7, and the variable region of the light chain c SEQ ID NO: 8, a transmembrane domain and an intracellular signaling domain for activating T cells; where the antigen-binding molecule is a chimeric antigen receptor (CAR). 2. An antigen-binding molecule according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 9. 3. The antigen-binding molecule of claim 1, wherein said transmembrane domain is selected from the group consisting of T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28, CD45, CD4, CD5 , CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, and CD154, wherein said intracellular signaling domain is a CD3zeta signaling domain and a costimulatory signaling domain. 4. The antigen binding molecule of claim 3, wherein the co-stimulatory signaling domain is selected from the group consisting of CD28, OXO40, CD27, ICAM-1, CD278, and CD137. 5. An antigen-binding molecule according to claim 3 or 4, wherein the costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 13. 6. An antigen-binding molecule according to claim 3 or 4, wherein the costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 14. 7. A method of treating cancer, comprising the step of administering to a patient suffering from cancer a pharmaceutical composition containing a T cell containing a therapeutically effective amount of an antigen-binding molecule according to claim 1. 8. The method according to claim 7, where the pharmaceutical composition contains CD8+ T cells or contains CD4+ T cells and CD8+ T cells. 9. The method of claim 9 wherein the pharmaceutical composition comprises CD4+ T cells and CD8+ T cells. 10. The method of claim 9 wherein the ratio of CD4+ T cells to CD8+ T cells is generally 1:1. 11. A therapeutic pharmaceutical composition for the treatment of cancer, containing, as a pharmaceutically effective ingredient, T cells containing an antigen-binding molecule according to claim 1 in an effective amount. 12. The therapeutic pharmaceutical composition according to claim 11, wherein the pharmaceutical composition contains CD8+ T cells or contains CD4+ T cells and CD8+ T cells. 13. The therapeutic pharmaceutical composition according to claim 12, wherein the pharmaceutical composition contains CD4+ T cells and CD8+ T cells. 14. The therapeutic pharmaceutical composition according to claim 13, wherein the ratio of CD4+ T cells to CD8+ T cells is generally 1:1.

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