Antibody targeting b7-h3, and chimeric antigen receptor comprising same

Abstract

The present invention relates to a novel antibody targeting B7-H3 or an antigen-binding fragment thereof, a chimeric antigen receptor (CAR) comprising same, an immune cell comprising the CAR, and uses thereof. The CAR-T cells according to the present invention have low toxicity and excellent antitumor activity, and thus can be effectively used in immune cell treatment for cancer treatment.

Description

[0001] Specification Title of the invention: Antibody targeting B7-H3, and chimeric antigen receptor comprising same Technical field The present invention relates to a novel antibody targeting B7-H3 or an antigen-binding fragment thereof, a chimeric antigen receptor (CAR) comprising the same, an immune cell comprising the CAR, and a use thereof. Background art Chimeric antigen receptor T cell therapy is a gene-transduced T cell in which a chimeric antigen receptor gene linking a recombinant antibody that recognizes a tumor antigen expressed on the surface of a cancer cell and a signal transduction region that induces T cell activation is introduced into the T cell. Currently, successful cases of CAR-T cell therapy are limited to CD19-positive acute leukemia, and its therapeutic efficiency is reported to be low in the case of solid tumors. One of the reasons is understood to be that solid tumors more firmly establish a tumor microenvironment with immunosuppressive properties. For example, in the case of CD19-positive hematological cancers, the efficacy of CAR-T cell therapy is known to be much lower in the case of lymphomas that form solid tumors (complete remission rate of 50%) than in leukemias where tumor cells mainly proliferate in the blood (complete remission rate of 70-80%). Therefore, there is an urgent need to develop CAR-T cells with improved performance that can overcome the immune resistance of tumors. Another factor in the low efficacy of CAR-T cell therapy in solid tumors is the heterogeneity of tumor antigens. The heterogeneity of tumor antigens refers to the concept that not all tumor cells in a single patient have the same tumor antigen, and the theory is that CAR-T cells targeting one tumor surface antigen will not be able to eliminate all tumor cells. As a countermeasure to this, it has been suggested that if antigens expressed simultaneously on tumor cells and surrounding normal cells, such as tumor stromal cells or tumor vascular cells, are targeted, it is possible to produce CAR-T cells that attack the entire tumor tissue by utilizing the homogeneous antigen expression of these normal cells.

[0002] B7-H3 is an immune checkpoint receptor similar to PD-L1. It is known to mediate tumor immune resistance by suppressing the immune response of tumor cells and tumor stem cells by being highly expressed in tumor cells and tumor stem cells (CZ / k Cancer Res., 2016, 22(14), 3425-3431). In addition, it is known to promote tumor migration and invasion through signal transmission into tumor cells, and to promote tumor growth itself by mediating tumor angiogenesis and EMT (epithelial-mesenchymal transition). In particular, B7-H3 is highly expressed not only in tumor cells but also in vascular endothelial cells in tumor tissues, whereas B7-H3 expression is not observed in vascular cells of normal tissues (Cancer Ce / Z 2017, 31, 501-515). Therefore, since B7-H3 can simultaneously target tumor cells and intratumoral blood vessels, it can be considered as a CAR target that can overcome tumor antigen heterogeneity and effectively treat solid tumors. In the case of anti-B7-H3 scFv used in existing B7-H3 CAR-T cells, it specifically recognizes only human B7-H3 and has no binding affinity to mouse B7-H3. Therefore, although the efficacy of CAR-T cells against tumors can be confirmed in mouse in vivo efficacy tests, there was a disadvantage in that CAR-T cytotoxicity against B7-H3 expressed at low levels in normal tissues could not be evaluated (CZ / k Cancer Res. 2019; 25:2560—2574). For some B7-H3-targeting CAR-T cells, there was an example of observing both efficacy and toxicity using antibodies that simultaneously bind to human B7H3 and mouse B7H3 (cross-reactive), but in the case of this antibody, the affinity for mouse B7-H3 was significantly lower than that for human B7-H3, so there was a limitation in properly evaluating toxicity to normal tissues (Cancer Cell 2019; 35: 221-237).Accordingly, the present inventors developed novel anti-B7-H3 antibodies having a similar affinity for human B7-H3 and mouse B7-H3, and used them to produce anti-B7-H3 CARs, thereby developing anti-B7-H3 CAR-T cells with low toxicity and excellent anti-tumor activity. Detailed Description of the Invention Technical Problem An object of the present invention is to provide a novel antibody or an antigen-binding fragment thereof that specifically binds to B7-H3. Another object of the present invention is to provide a chimeric antigen receptor comprising an anti-B7-H3 antigen-binding domain comprising the antibody or an antigen-binding fragment thereof, and an immune cell comprising the same. Still another object of the present invention is to provide a nucleic acid encoding the chimeric antigen receptor, an expression vector comprising the nucleic acid, and a host cell comprising the expression vector. Still another object of the present invention is to provide a composition for treating cancer comprising the immune cell, a method for treating cancer using the immune cell, a use of the immune cell for treating cancer, and a use of the immune cell for producing a drug for treating cancer. Technical solution To achieve the above object, the present invention provides an antibody or an antigen binding fragment thereof that specifically binds to the B7-H3 antigen. As used herein, the term "antibody" refers to an antibody that specifically binds to B7-H3, which includes not only a complete antibody form but also an antigen binding fragment of an antibody molecule. As used herein, the term "antigen binding fragment" refers to a fragment that has an antigen binding function, and includes Fab, F(ab'), F(ab')2, chemically linked F(ab')2, and Fv, etc. Among antibody fragments, Fab (fragment antigen binding) has a structure having variable regions of light and heavy chains, constant region of light chain, and first constant region (CH1) of heavy chain, and has one antigen binding site.F(ab') differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced by disulfide bonding between the cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are disclosed in PCT International Patent Application Publication Nos. W0 88 / 10649, W0 88 / 106630, W0 88 / 07085, W0 88 / 07086, and WO 88 / 09344. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv (single-chain variable fragment, scFv) generally has a heavy chain variable region and a single chain variable region covalently linked via a peptide linker or directly linked at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv. Such antibody fragments can be obtained using a proteolytic enzyme (for example, a Fab can be obtained by restriction digestion of a whole antibody with papain, and an F(ab')2 fragment can be obtained by digestion with pepsin), or can be produced by genetic recombination technology. The antibody or antigen-binding fragment thereof of the present invention is specifically in the form of a scFv or a complete antibody. In addition, the heavy chain constant region can be selected from any one of the isotypes of gamma (x), mu (01), alpha (a), delta (6), or epsilon (e). Specifically, the heavy chain constant region is of the gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4) isotype, most specifically of the gamma 4 (IgG4) isotype. The light chain constant region can be of the kappa (K) or lambda (X) type, specifically of the kappa (K) type.Therefore, the specific antibody of the present invention is an scFv form or an IgG4 form having a kappa (K) light chain and a gamma 4 (IgG4) heavy chain, but is not necessarily limited thereto. As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, including a variable region domain VH of an antibody and three constant region domains CHI, CH2 and CH3 and a hinge, which comprise an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. In addition, as used herein, the term "light chain" refers to a full-length light chain and fragments thereof, including a variable region domain VL and a constant region domain CL of an antibody, which comprise an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, and a framework region (FR) exists between these CDRs to support the CDR loops. The CDR is a loop-shaped region involved in antigen recognition, and provides key contact residues for the binding of an antibody to an antigen or epitope, thereby determining the specificity of the antibody for the antigen. The term “Framework” or “FR” above refers to variable domain residues other than hypervariable region residues.The FR of a variable domain is typically composed of four FR domains FR1, FR2, FR3 and FR4. As used herein, the term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have a similar structure, each of which comprises four conserved framework regions and three hypervariable regions (HVRs) (Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind to a specific antigen can be isolated using the VH or VL domain from antibodies that bind to the antigen and screen a library of complementary VH or VL domains, respectively. As used herein, the term "specifically binding" or "specifically recognizing" means that an antibody or antigen-binding fragment thereof, or other construct such as scFv {{scFv}}, specifically interacts with an antigen or a corresponding substance to produce an immunological response. Specific binding is at least about 1 x IO'. 6 M or less (e.g. 9 x 10') 7 M, 8 x 10' 7 M, 7 x 10' 7 M, 6 x 10' 7 M, 5 x 10' 7 M, 4 x 10' 7 M, 3 x 10' 7 M, 2 x 10' 7 M, or 1 x 10' 7 M), specifically 1 x 10'7 M or less (e.g. 9 x 10') 8 M, 8 x 10' 8 M, 7 x 10' 8 M, 6 x 10' 8 M, 5 x 10' 8 M, 4 x 10' 8 M, 3 x 10' 8 M, 2 x 10' 8 M, or 1 x 10' 8 M), more specifically 1 x 10'

[0003] 8 M or less (e.g. 9 x 10') 9 M, 8 x 10' 9 M, 7 x 10' 9 M, 6 x 10' 9 M, 5 x 10'

[0004] 9 M, 4 x 10' 9 M, 3 x 10' 9 M, 2 x 10' 9 M, or 1 x 10' 9The equilibrium dissociation constant (KD) of two molecules can be characterized by a KD of 1 M (e.g., a smaller KD indicates a tighter binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As used herein, the terms “homology” and “identity” refer to the degree to which two given base sequences are related, which can be expressed as a percentage. The terms homology and identity are often used interchangeably. Whether any two sequences have homology or identity can be determined using a well-known computer algorithm such as the “FASTA” program using default parameters, for example, as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Additionally, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLAST?, BLASTN, FASTA (Atschul, [S.] [F„ ] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ed.,] Academic Press, San Diego, 1994,and [CARILLO ETA / . ] (1988) SIAM J Applied Math 48:1073). For example, sequence homology or identity can be determined using BLAST of the National Center for Biotechnology Information database, or ClustalW. The antibodies or antigen-binding fragments thereof of the present invention include full-length or intact polyclonal or monoclonal antibodies, as well as antigen-binding fragments thereof (e.g., Fab, F(ab'), F(ab')2, Fab3, Fv and variants thereof), fusion proteins comprising one or more antibody portions, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single-chain variable fragments (scFv), scFv-Fc, bispecific antibodies, multispecific antibodies, other modified configurations of immunoglobulin molecules comprising an antigen recognition site of the required specificity, glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Specific examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engager), T andAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX— bodies (peptide modified antibodies),Duobodies and triomAbs are included. The list of such antibodies and antigen-binding fragments thereof is not limited to the above. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention may be selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a full-length antibody, an Fv, an scFv, a Fab, F(ab'), F(ab')2, and a nanobody comprising the heavy chain variable region and the light chain variable region described above, but is not limited thereto. Specifically, the antibody or antigen-binding fragment thereof may be a humanized antibody. The term "humanized antibody" as used herein refers to a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, F(ab'), F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin of a non-human (e.g., murine) antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, residues from the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may comprise residues that are not found in either the recipient antibody or in the imported CDR or framework sequences. Such modifications are made to further improve and optimize antibody performance. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin,All or substantially all of the FR region has the sequence of the FR region of a human immunoglobulin. The humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc region) sequence, or substantially a human immunoglobulin constant region sequence. The framework region of the isolated antibody or of the antigen-binding portion thereof comprises one or more back mutations, wherein a human germline framework amino acid is replaced with a native non-human amino acid at a corresponding position. The framework region of the isolated antibody or of the antigen-binding portion thereof also comprises one or more conservative substitutions. As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the essential properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-direct mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the amino acid residue in question (e.g., has similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. Such families include amino acids having alkaline side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with P-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g.,(tyrosine, phenylalanine, tryptophan, histidine). Therefore, the amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12 (10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference). In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to the present invention comprises a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 13, 22, 31, 40 and 49, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 14, 23, 32, 41, 50 and 135, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, A heavy chain CDR2 comprising an amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 24, 33, 42, 51 and 136, a portion thereof or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,A heavy chain variable region (VH) comprising a heavy chain CDR3 comprising an amino acid sequence having 98% or 99% identity thereto; and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 16, 34, 43 and 52, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 17, 26, 35, 44 and 53, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, A light chain variable region (VL) comprising a light chain CDR2 comprising an amino acid sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain CDR3 comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or a portion thereof, selected from the group consisting of SEQ ID NOs: 9, 18, 27, 36, 45 and 54. In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to the present invention comprises:

[0005] (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 33; A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 136;

[0006] (ii) a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 36; A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 45; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 54. In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to the present invention comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; orA heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 24; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27; A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 33; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 36; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 45;A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 54; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 136; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 138, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 139. In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to the present invention;

[0007] ( i ) Sequence numbers 2, 11, 20, 29, 38, 47, 105, 114, 123, 132, 141, 150, 159, 168,

[0008] A heavy chain variable region comprising an amino acid sequence selected from the group consisting of 177, 186 and 195, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or

[0009] ( ii ) Sequence numbers 3, 12, 21, 30, 39, 48, 106, 115, 124, 133, 142, 151, 160, 169,

[0010] A light chain variable region comprising an amino acid sequence selected from the group consisting of 178, 187 and 196, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In another embodiment, the antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to the present invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 105; and SEQ ID NO:

[0011] A light chain variable region comprising an amino acid sequence of SEQ ID NO: 106; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 114; and

[0012] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 115; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 123; and

[0013] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 124; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 132; and

[0014] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 133; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141; and

[0015] A light chain variable region comprising an amino acid sequence of SEQ ID NO: 142; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 150; and

[0016] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 151; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 159; and

[0017] A light chain variable region comprising an amino acid sequence of SEQ ID NO: 160; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 168; and

[0018] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 169; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 177; and

[0019] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 178; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 186; and

[0020] A light chain variable region comprising an amino acid sequence of SEQ ID NO: 187; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 195; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 196. In another embodiment, the antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to the present invention may comprise, but is not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 10, 19, 28, 37, 46, 104, 113, 122, 131, 140, 149, 158, 167, 176, 185 and 194, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In addition, the present invention provides a nucleic acid encoding an antibody or an antigen-binding fragment thereof that specifically binds to the B7-H3, an expression vector comprising the nucleic acid, or a host cell comprising the expression vector. The antibody or the antigen-binding fragment thereof that specifically binds to the B7-H3 is as described above. The expression vector comprising the nucleic acid encoding the antibody is not particularly limited thereto, but may be a vector capable of replicating and / or expressing the nucleic acid in a eukaryotic or prokaryotic cell, including a mammalian cell (e.g., human, monkey, rabbit, rat, hamster, mouse cell, etc.), a plant cell, a yeast cell, an insect cell, or a bacterial cell (e.g., Escherichia coli, etc.), and specifically, may be a vector that is operably linked to an appropriate promoter so that the nucleic acid can be expressed in the host cell, and includes at least one selectable marker. For example, the nucleic acid may be introduced into a phage, a plasmid, a cosmid, a mini-chromosome, a virus, or a retrovirus vector.An expression vector comprising a nucleic acid encoding the antibody may be an expression vector comprising a nucleic acid encoding a heavy chain or a light chain of the antibody, respectively, or an expression vector comprising both nucleic acids encoding the heavy chain or the light chain. The host cell comprising the expression vector may be a cultured cell, for example, a mammalian cultured cell such as CHO (Chinese hamster ovary cells), BHK, NSO, SP2 / 0, YB2 / 0 derived from a rodent (rat, mouse, guinea pig, or hamster); or a human tissue or hybridoma cell, yeast cell, and insect cell, and a cell contained in a transgenic animal or cultured tissue, without limitation. In addition, the present invention provides (a) an anti-]37-H3 antigen binding domain comprising an antibody or an antigen binding fragment thereof that specifically binds to the B7-H3.

[0021] (b) extracellular domain,

[0022] (c) a transmembrane domain, and

[0023] (d) Provides a chimeric antigen receptor (CAR) specific for B7-H3, comprising an intracellular domain. As used herein, the term "antigen binding domain" means a domain that directly binds to an antigen, such as an antibody or an antigen binding fragment thereof. As used herein, the term "extracellular domain (EC)" means a domain that is exposed to the outside of a cell and connects a transmembrane domain and an antigen binding domain. As used herein, the term "transmembrane domain (TM)" means a domain that connects an extracellular domain and an intracellular domain and is located in a cell membrane. In this specification, the term "intracellular domain" is also called a cytoplasmic domain, and means a domain located inside the cell membrane of a cell, i.e., in the cytoplasm. In this specification, the term "intracellular signaling domain" is a part located inside the cell membrane of an immune cell, i.e., in the intracellular domain, and means a site that transmits a signal within a cell when an antigen binding domain linked to an extracellular domain binds to a target antigen. In the present invention, the antigen binding domain includes an antibody or an antigen binding fragment thereof that specifically binds to B7-H3, and the antibody or antigen binding fragment thereof that specifically binds to B7-H3 is as described above: In the present invention, the N-terminal portion of the antigen binding domain may additionally include a signal peptide (signal peptide: SP). In the present invention, the signal peptide may be derived from a molecule selected from the group consisting of CD8a, GM-CSF receptor a, Ig-kappa, and IgG1 heavy chain. However, it is not limited to this.Specifically, a CD8a signal peptide may be used as the signal peptide, and the CD8a signal peptide may include an amino acid sequence of SEQ ID NO: 55. In the present invention, the extracellular domain may be an extracellular domain derived from IgG1, IgG2, IgG4, IgD, CD8 or CD28, but is not limited thereto. In one embodiment, the extracellular domain may be an extracellular domain derived from CD8. It may include all or a part of the extracellular domain derived from CD8, and specifically, the CD8 may be human CD8, but is not limited thereto. The CD8-derived extracellular domain may comprise a human CD8-derived extracellular domain, and may specifically include, but is not limited to, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In the present invention, the transmembrane domain may be a transmembrane domain derived from a protein selected from the group consisting of T cell receptor (TCR) a chain, TCRP chain, TCRx chain, TCR5 chain, CD3 zeta (e), CD3 epsilon (e), CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, OX40 (CD134), 4-lBB (CD137), ICOS, and CD154, or a transmembrane domain derived from killer immunoglobulin-like receptor (KIR), but is not limited thereto. In one embodiment, the transmembrane domain may be a CD8-derived transmembrane domain. The CD8-derived transmembrane domain may comprise all or part of a CD8-derived transmembrane domain, and specifically, the CD8 may be, but is not limited to, human CD8.The CD8-derived transmembrane domain may comprise a human CD8-derived transmembrane domain, and specifically, may comprise an amino acid sequence of SEQ ID NO: 59, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, but is not limited thereto. In the present invention, the chimeric antibody receptor may further comprise a hinge domain. The hinge domain may be composed of any oligopeptide or polypeptide, and may comprise 1 to 100 amino acid residues, specifically 10 to 70 amino acid residues. In the present invention, the intracellular domain may comprise an intracellular signaling domain and / or a costimulatory domain. The intracellular signaling domain may be one or more intracellular signaling domains selected from the group consisting of CD3 zeta (x), CD3 gamma (x), CD3 delta (6), CD3 epsilon (e), FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b and CD66d, but is not limited thereto, and specifically may be CD3 zeta (&). In one embodiment, the intracellular signaling domain of CD3 zeta (&) according to the present invention may have an amino acid sequence including the amino acid sequence of SEQ ID NO: 61, but is not limited thereto. In addition, the intracellular domain according to the present invention may additionally include a costimulatory domain, but is not limited thereto.The co-stimulatory domain according to the present invention may be one or more co-stimulatory domains selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-lBB (CD137), OX40 (CD134), ICOS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C and B7-H3, but is not limited thereto. Specifically, the co-stimulatory domain may be a 4-1BB co-stimulatory domain. In one embodiment, the intracellular domain according to the present invention may include a CD3 zeta (&) intracellular signaling domain and a 4-1BB co-stimulatory domain, but is not limited thereto. Specifically, the intracellular domain according to the present invention may include a CD3 zeta (&) intracellular signaling domain comprising an amino acid sequence of SEQ ID NO: 61 and a costimulatory domain of 4-1BB comprising an amino acid sequence of SEQ ID NO: 60. In particular, the chimeric antigen receptor according to the present invention may include one or more intracellular signaling domains and one or more costimulatory domains. When the chimeric antigen receptor according to the present invention includes one or more intracellular signaling domains and one or more costimulatory domains, the one or more costimulatory domains and the one or more intracellular signaling domains may be serially linked to each other. In this case, each domain may be directly linked, or may be selectively linked via an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acid residues, and specifically, a glycine-serine continuous sequence may be exemplified as such a linker sequence. In the present invention, the chimeric antigen receptor may further include a T cell immune function promoting factor, and the T cell immune function promoting factor may include IL-.

[0024] Examples thereof include, but are not limited to, interleukin 7, IL-12, IL-15, IL— 18, IL— 21 or CCL19. With respect to factors promoting immune function of T cells, see WO 2016 / 056228 A. In the present invention, the chimeric antigen receptor may further comprise an interleukin receptor chain comprising a JAK binding motif and a STAT 3 / 5 association motif, such as, but not limited to, IL-2RP. In this regard, see WO 2016 / 127257 A.

[0025] The first-generation CAR included an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and used only CD3 as the signaling domain, but had a minimal therapeutic effect on cancer and a short duration of action. This first-generation CAR is specifically described in U.S. Patent No. 6,319,494, which is incorporated herein by reference. To improve responsiveness to immune cells, a second-generation CAR was manufactured that combined a costimulatory domain (CD28 or CD137 / 4-1BB) and CD3, and the number of CAR-containing immune cells remaining in the body significantly increased compared to the first-generation CAR. While the second-generation CAR used one costimulatory domain, the third-generation CAR used two or more costimulatory domains. To achieve expansion and persistence of immune cells containing CARs in vivo, the co-stimulatory domain can be combined with 4-IBB, CD28, or 0X40. Second generation CARs are specifically described in U.S. Patent Nos. 7,741,465, 7,446,190, or 9,212,229, and third generation CARs are specifically described in U.S. Patent No. 8,822,647, which are incorporated herein by reference.

[0026] The fourth generation CARs may further comprise an additional gene encoding a cytokine, such as IL-12 or IL-15, to enable additional expression of the cytokine-based immunoprotein, and the fifth generation CARs may further comprise an interleukin receptor chain, such as IL-2RP, for immune cell enhancement. The fourth generation CAR is specifically described in U.S. Pat. No. 10,316,102, and the fifth generation CAR is specifically described in U.S. Pat. No. 10,336,810, which are incorporated herein by reference. In one embodiment, the chimeric antigen receptor according to the present invention may comprise, but is not limited to, an anti-B7-H3 antigen binding domain comprising an anti-B7-H3 scFv*, a CD8-derived extracellular domain, a CD8-derived transmembrane domain, a 4-1BB costimulatory domain, and a CD3 & intracellular signaling domain. Specifically, the chimeric antigen receptor according to the present invention may include, but is not limited to, an anti-B7-H3 antigen binding domain comprising an anti-B7-H3 scFv; a CD8-derived extracellular domain comprising an amino acid sequence of SEQ ID NO: 58; a CD8-derived transmembrane domain comprising an amino acid sequence of SEQ ID NO: 59; a 4-1BB costimulatory domain comprising an amino acid sequence of SEQ ID NO: 60; and a CD3 zeta (&) intracellular signaling domain comprising an amino acid sequence of SEQ ID NO: 61. For example, a chimeric antigen receptor according to the present invention may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 63, 70, 77, 84, 91, 204, 211 and 218, a portion thereof or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In another aspect, the present invention relates to a nucleic acid encoding the chimeric antigen receptor.The nucleic acid (polynucleotide) encoding the chimeric antigen receptor according to the present invention can be modified by codon optimization, which is due to the degeneracy of codons, and it will be well understood by those skilled in the art that there are many nucleotide sequences encoding polypeptides or variant fragments thereof. Some of these polynucleotides (nucleic acids) have minimal homology to the nucleotide sequences of any naturally occurring gene. In particular, polynucleotides (nucleic acids) that are variable due to differences in codon usage, for example, polynucleotides (nucleic acids) optimized for codon selection in humans, primates and / or mammals, can be used. In another aspect, the present invention relates to an expression vector comprising the nucleic acid and a host cell (e.g., a virus) comprising the expression vector. The term “vector” in the present invention means a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to a vector nucleic acid molecule, for example, inserted into the vector nucleic acid molecule. The vector may comprise sequences directing autonomous replication in a cell, or may comprise sequences sufficient to enable integration into host cell DNA. The vector includes, but is not limited to, DNA vectors, RNA vectors, plasmids, phagemids, phage derivatives, viruses, and cosmids. The viral vector may be selected from the group consisting of, but is not limited to, lentiviral vectors, adenoviral vectors, herpesvirus vectors, and retroviral vectors. In the present invention, the nucleic acid or the vector is transformed or transfected into a host cell, for example, a virus producing cell (packaging cell line).Any of a variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells to “transform” or “transfect” them can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. In the present invention, a virus produced from a virus producing cell is “transduced” or “transduced” into an immune cell. The nucleic acid of the virus “transduced” or “transduced” into the cell is used to produce a chimeric antigen receptor protein, either integrated or not integrated into the genome of the cell. In another aspect, the present invention relates to an immune cell expressing the chimeric antigen receptor on its surface. In the present invention, the immune cell may be, but is not limited to, a T cell, an NK cell, an NKT cell, or a macrophage, and may be specifically a T cell. The immune cell expressing the chimeric antigen receptor according to the present invention may be a CAR-T cell, a CAR-NK cell (Chimeric Antigen Receptor Natural Killer Cell), a CAR-NKT cell (Chimeric Antigen Receptor Natural killer T Cell), or a CAR-macrophage (Chimeric Antigen Receptor Macrophage). In the present invention, the T cell may be selected from the group consisting of a CD4 positive T cell; a CD8 positive cytotoxic T lymphocyte (CTL); a gamma-delta T cell; a tumor infiltrating lymphocyte (TIL); and a T cell isolated from a peripheral blood mononuclear cell (PBMC).In another aspect, the present invention relates to a composition for treating cancer comprising an immune cell expressing the chimeric antigen receptor. In the present invention, the immune cell may be, but is not limited to, a T cell, an NK cell, an NKT cell, or a macrophage, and specifically may be a T cell. In the present invention, “cancer” and “tumor” are used interchangeably and refer to or mean a physiological condition of a mammal that is typically characterized by unregulated cell growth / proliferation. Cancers that can be treated with the CAR of the present invention include not only vascularized tumors but also tumors that are not vascularized or are not substantially vascularized yet. The cancer may include a non-solid tumor (e.g., a hematological tumor, such as leukemia and lymphoma) or may include a solid tumor. Types of cancers that can be treated with the CARs of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included. Hematologic cancers are cancers of the blood or bone marrow. Examples of blood (or hematopoietic) cancers include leukemias, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, and myeloblastic, prolymphocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic lymphocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. Solid tumors are abnormal masses of tissue that usually do not contain capsules or areas of liquid.Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, squamous cell carcinoma, rhabdomyosarcoma, rectal carcinoma, lymphoid malignancies, colon cancer, stomach cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, laryngopharyngeal cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, And CNS tumors (e.g., gliomas (e.g., brainstem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases). The therapeutic composition of the present invention is a composition for preventing or treating cancer, and the term “prevention” of the present invention means any act of inhibiting or delaying the progression of cancer by administering the composition of the present invention, and “treatment” means inhibiting the development of cancer, alleviating or eliminating symptoms. A pharmaceutical composition comprising an immune cell expressing a chimeric antigen receptor according to the present invention may additionally include a pharmaceutically acceptable excipient.Examples of such excipients include, but are not limited to, surfactants, specifically nonionic surfactants of the polysorbate series; buffers such as neutral buffered saline, human salt buffered saline; sugars or sugar alcohols such as glucose, mannose, sucrose, dextran, mannitol; amino acids or proteins or polypeptides such as glycine, histidine; antioxidants; chelating agents such as EDTA or glutathione; penetrants; adjuvants; and preservatives. The compositions of the present invention can be formulated using methods known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal other than a human. The formulations can be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powders. In another aspect, the present invention relates to a method for treating cancer, comprising administering to a subject an immune cell expressing the chimeric antigen receptor. The present invention also relates to the use of the immune cell for treating cancer. The present invention also relates to the use of the immune cell for the manufacture of a medicament for treating cancer. The subject may be a mammal having a tumor, and specifically, may be a human, but is not limited thereto. The immune cell expressing the chimeric antigen receptor according to the present invention or a composition comprising the same may be administered by, but is not limited to, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc.The dosage of the active ingredient can be appropriately selected depending on various factors such as the route of administration, the patient's age, sex, weight, and the severity of the disease, and the therapeutic composition according to the present invention can be administered in parallel with a known compound that has the effect of preventing, improving, or treating cancer symptoms. Effect of the invention The existing in vivo efficacy model of CAR-T cells is a model in which human CAR-T cells are administered to immunodeficient mice inoculated with human tumor cells, and immunodeficient mice have been used to prevent the mouse's immune cells from eliminating human tumor cells and human CAR-T cells through an immune response. However, this model is very different from the situation in which CAR-T cells are actually administered to patients, as CAR-T cells are administered after artificially removing immune cells in the mouse body. In addition, most human CAR-T cells only recognize human target antigens of human tumor cells inoculated into mice, and in many cases do not recognize mouse target antigens of mouse tissues, so it is often impossible to evaluate the toxicity of human CAR-T cells to normal tissues in vivo. In the present invention, novel anti-B7-H3 antibodies having similar affinities for human B7-H3 and mouse B7-H3 were developed, and CARs were produced using the same, thereby confirming that anti-B7-H3 mouse CAR-T cells have excellent anti-tumor effects in vivo in normal mice without immune cell deficiency, while exhibiting low toxicity to normal tissues. Human CAR-T cells were produced using the same antibodies, and were confirmed to exhibit effective in vitro anti-tumor effects. In addition, humanized anti-B7-H3 antibodies were developed, and human CAR-T cells were produced using the same, and effective in vitro anti-tumor effects were confirmed, thereby developing anti-B7-H3 CAR-T cells with excellent anti-tumor activity and low toxicity.Since B7-H3 is a target that can overcome tumor antigen heterogeneity, the anti-B7-H3 CAR-T cell according to the present invention can be applied to the treatment of refractory solid tumors. Brief description of the drawings Figures 1 and 2 show the results of evaluating the cross-reactivity of B7H3 antibody clones to human B7H3 and mouse B7H3 proteins. Figure 1 is a graph showing the results of ELISA screening analyzing the affinity of discovered scFv antibody clones to human B7H3 (h4Ig, h2Ig) and mouse B7H3 (m2Ig) (h4Ig, h2Ig: two isoforms of human B7H3 protein). Figure 2 shows the results of ELISA affinity analysis of some purified B7-H3 target scFv proteins. Figure 3 is a graph showing the results of evaluating the binding affinity of B7-H3 scFv antibody clones to four types of human cell lines. Figure 4 is a graph showing the results of evaluating the binding affinity of B7-H3 scFv antibody clones to six mouse cell lines. Figure 5 is a schematic diagram showing the structure of mouse B7-H3 CAR. (LTR, Long terminal repeat; mCD8a, mouse CD8 alpha chain; EC, extracellular domain; TM, transmembrane domain; cyt, intracellular domain) Figure 6 is a graph showing the CAR expression rate in mouse B7-H3 CAR T cells (the numbers in the graph indicate the proportion (%) of CAR positive cells). Figure 7 is a graph showing the results of evaluating the IFN- x production of mouse B7-H3 CAR-T cells. Figure 8 is a graph showing the cytotoxicity of mouse B7-H3 CAR-T cells against target tumor cells (E0771). Figure 9 is a graph showing the change in tumor size and body weight after administration of mouse B7-H3 CAR-T cells.Figure 10 is a schematic diagram showing the structure of a retroviral vector expressing a CTLA4-CD28 chimeric (CTC28) gene and / or an inducible IL-12 (iIL-12) gene together with a B7-H3 CAR gene (PGK, PGK promoter; 6xNFAT promoter (six tandem NFAT binding elements: six tandem NFAT binding elements); P2A, P2A peptide; m, mouse; IL-12f, two subunits of IL12 (. P4O and p35) are linked by a single chain (IL12). Figure 11 is a graph showing the expression rate of CAR and CTC28 of enhanced B7-H3 CAR-T cells. Figure 12 is a graph showing the production of IL-12 of enhanced B7-H3 CAR-T cells. (a) CAR-T cells not co-cultured with tumor cells, (b) CAR-T cells co-cultured with tumor cells. Figure 13 is a graph showing the production rate of IFN- x of enhanced B7-H3 CAR-T cells. Figure 14 is a graph showing the cytotoxicity of enhanced B7-H3 CAR-T cells against target cells (breast cancer cells). Figure 15 is a graph showing the tumor (breast cancer) size (a) and body weight change (b) after administration of enhanced B7-H3 CAR-T cells to small-sized tumors. Figure 16 is a graph showing the tumor (breast cancer) size (a) and body weight change (b) after administration of B7-H3 CAR-T cells with enhanced efficacy to advanced tumors (n / 5: n = number of animals in which the tumor was completely eradicated out of a total of 5 animals). Figure 17 is a graph showing the tumor (breast cancer) size and body weight change after administration of #12-CTC28-iIL12 CAR-T cells {#25— CTC28— iIL12 CAR-T cells}. Figure 18 is a graph showing the tumor (breast cancer) size and body weight change after administration of #12-CTC28-iIL12 CAR-T cells {#54— CTC28— iIL12 CAR-T cells}. Figure 19 is a graph showing the in vivo efficacy of #25-CTC28-iIL12 CAR-T cells against liver cancer cells. Figure 20 is a schematic diagram showing the structure of the human CAR gene, i.e., the hB7H3 CAR (hB7H3-BBz) gene, produced using the anti-B7-H3 antibody scFv> (h, human). Figure 21 is a graph showing the CAR expression rate in human B7-H3 CAR-T cells. Figure 22 is a graph showing the cytotoxicity of human B7-H3 CAR-T cells against target tumor cells (MDA-MB-231).Figure 23 is a graph showing the IFN- x production rate of human B7-H3 CAR-T cells. Figure 24 is a schematic diagram showing the structure of a lentiviral vector co-loaded with human B7H3 CAR, inducible IL-12, and CTLA4-CD28 chimeric (CTC28) genes (h, human; PGK, PGK promoter; 6xNFAT, six tandem NFAT binding element promoter; P2A, P2A peptide). Inducible IL-12 is denoted as iIL12 or simply IL12. Figure 25 is a graph showing the CAR and CTC28 expression rates of enhanced human B7-H3 CAR-T cells (UT, untransduced; ChlgY, FTTC-labeled anti-chicken IgY Fab; CTLA4-PE, PE-labeled anti-human CTLA4 antibody). Figure 26 is a graph showing the results of tumoricidal activity evaluation of enhanced human B7-H3 CAR-T cells (Unt, untransduced). (untransduced). Figure 27 is a graph showing the results of evaluating the IL-12 secretion ability of enhanced human B7-H3 CAR-T cells. Figure 28 is a graph showing the results of evaluating the IFN- x secretion ability of enhanced human B7-H3 CAR-T cells. Figure 29 is the results of analyzing the expression of CAR and hCTC28 in enhanced human B7-H3 CAR-T cells (CD4-positive and CD8-positive CAR-T cells) (ChlgY, FITC-labeled anti-chicken IgY Fab; CTLA4-PE, PE-labeled anti-human CTLA4 antibody). For convenience, it is abbreviated as CTC28-IL12# C12. Figure 30 is a graph showing the results of evaluating the tumor killing ability of enhanced human B7-H3 CAR-T cells (CD4-positive and CD8-positive CAR-T cells). Figure 31 is a graph showing the results of analyzing the expression of CAR and hCTC28 in enhanced human B7-H3 CAR-T cells (CD4-positive and CD8-positive CAR-T cells). This graph shows the results of evaluating IL-12 production by B7-H3 CAR-T cells (CD4-positive and CD8-positive CAR-T cells) (Unt, untransduced T cells).Figure 32 is a graph showing the IFN-Y production rate of potentiated human B7-H3 CAR-T cells (CD4-positive and CD8-positive CAR-T cells). Figure 33 shows the results of SDS-PAGE analysis of humanized variants of B7-H3 targeting scFv antibodies. Figure 34 shows the results of a comparative test of the binding affinity of humanized and non-humanized anti-B7H3 antibody scFvs to human B7H3 on the cell surface in an MDA-MB-231 cell line expressing human B7H3 antigen. Figure 35 is a schematic diagram showing the structure of human CAR genes, i.e., huB7H3-BBz and hB7H3-BBz genes, constructed using humanized and non-humanized anti-B7H3 antibody scFvs (L, leader sequence; h, human; hu, humanized; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain). Figure 36 is a graph showing the CAR expression rate in human B7H3 CAR-T cells using humanized anti-B7H3 antibody scFv> (the numbers in the graph represent the percentage (%) of CAR-positive cells). CAR expression was detected with anti-chicken immunoglobulin Y (anti-chicken IgY) secondary antibody (ChlgY-FITC) for three non-humanized B7H3 CAR-T cells, and with anti-human F(ab')2 secondary antibody (F(ab')2-FTTC) for three humanized B7H3 CAR-T cells. UT (untransduced) is a negative control group with non-transduced T cells. For convenience, #12, #2-25, and #2-54 were designated as #12 BBz, #25 BBz, and #54 BBz, respectively. #12, #25, #54 Humanized B7H3 scFvs of B7H3 scFv are z32, z25, zl6, respectively. Figure 37 is a graph showing the cytotoxicity of humanized B7H3 CAR-T cells (E, Effector) against target cells (T, Target; MDA-MB-231).Figure 38 is a graph showing the IFN- x production of humanized B7H3 CAR- T cells. Figure 39 shows the results of analyzing the expression of CAR and KTC28 in three types of enhanced humanized B7H3 CAR- T cells (CD4-positive and CD8-positive CAR- T cells) (F(ab')2-FTTC, FTTC-labeled anti-human IgG F(ab')2; CTLA4-PE, PE-labeled anti-human CTLA4 antibody; CD4, APC / Cyanine7-labeled anti-human CD4 antibody; CD8, PerCP / Cy5.5-labeled anti-human CD8 antibody). UT (untransduced) is a negative control group with non-transduced T cells. For convenience, it is denoted as CTC28-IL12# C12. Figure 40 is a graph showing the results of evaluating the tumor killing capacity of humanized B7H3 CAR-T cells with enhanced potency (CD4-positive and CD8-positive CAR-T cells). Figure 41 is a graph showing the results of evaluating the IL-12 secretion capacity of humanized B7H3 CAR-T cells with enhanced potency (CD4-positive and CD8-positive CAR-T cells). Figure 42 is a graph showing the results of evaluating the IFN-x secretion capacity of humanized B7H3 CAR-T cells with enhanced potency (CD4-positive and CD8-positive CAR-T cells). Figure 43 is a result of comparing the in vitro potency of humanized #12 B7H3 CAR-T cells using different humanized variant scFv>s, using a CAR detection reagent with a B7H3 recombinant protein at a similar CAR expression rate.(B7H3—His represents the CAR expression rate when cell surface CAR expression was detected using 6xHis peptide-tagged B7H3 protein and APC-labeled secondary antibody (APC-labeled anti—6xHis antibody)#, and CTLA4 represents the CTC28 expression rate when cell surface CTC28 expression was detected using PE-labeled anti-human CTLA4 antibody.) UT (untransduced) is a negative control group and is a non-transduced T cell. Figure 44 shows the results of comparing the in vitro efficacy of #25 B7H3 CAR-T cells humanized using different humanized variant scFv>s, using B7H3 recombinant protein as a detection reagent, in a state where CAR expression rates were similarly adjusted. FIGS. 45 and 46 show the results of an in vitro efficacy comparison using different humanized variant scFv>s, using B7H3 recombinant protein as a detection reagent, in a state where the CAR expression rate was similarly adjusted for humanized #54 B7H3 CAR-T cells. Embodiment for implementing the invention Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. Example 1. Development of a novel B7-H3 target antibody In order to discover antibodies that are cross-reactive with mouse B7-H3 and human B7-H3, chickens were immunized several times with mouse and human B7-H3 proteins together, and then an scFv phage display library was created from RNA of immune organs such as spleen and bone marrow isolated from these chickens.Afterwards, phages that bind to human and mouse B7-H3 proteins were selected through several biopanning processes, and then antibody clones that bind to B7-H3 protein were selected through ELISA using crude scFv proteins and purified scFv proteins.

[0027] [Table 1] Number of antibody clones selected for each test As a result, we discovered about 30 antibody clones that bind with similar affinity to human B7-H3 and mouse B7-H3 proteins, among which the Kd value in ELISA was 10'. 9 - 10' 10 More than eight clones exhibiting high affinity in the range were discovered. Figures 1 and 2 show the results of evaluating the cross-reactivity of B7-H3 antibody clones to human B7-H3 and mouse B7-H3 proteins. Figure 1 is a graph showing the results of ELISA screening analyzing the affinity of the discovered scFv antibody clones to human B7-H3 (h4Ig, h2Ig) and mouse B7-H3 (m2Ig) (h4Ig, h2Ig: two isoforms of human B7-H3 protein). Figure 2 shows the results of ELISA affinity analysis of some purified B7-H3 target scFv proteins. As shown in Figure 2, the eight selected B7-H3 target antibodies all bound to human and mouse B7-H3 with similar affinity. Example 2. Tumor cell binding ability of the screened novel B7-H3 antibodies

[0028] To determine whether the eight scFv antibody clones exhibited the same binding affinity to native antigens expressed on the cell surface as to recombinant B7-H3 protein, flow cytometry analysis was performed using various cancer cell lines known to express B7-H3. In order to select antibodies with cross-reactivity to human B7-H3 and mouse B7-H3, the binding level was evaluated for a total of 10 cell lines, namely, 4 human cancer cell lines and 6 mouse cancer cell lines (Table 2). In the case of mouse cancer cell lines, a cell line that overexpressed B7-H3 (4T1-B7-H3) was constructed and included.

[0029] [Table 2] Ten cancer cell lines (4 human, 6 mouse) used for flow cytometry analysis Each cancer cell line was treated with B7-H3 scFv antibody clones at 1, 10 nM or 10, 100 nM, etc., and detected using a secondary antibody attached to a fluorescent material that binds to human Ck tagged to the scFv antibody. The mean fluorescence intensity (MFI) value was analyzed, and the antigen-specific binding affinity was evaluated as the increase in MFI compared to the anti-His scFv antibody used as a negative control. As a result, all eight types of B7-H3 scFv antibody clones showed an MFI more than twice that of the negative control in both human and mouse cell lines, and it was confirmed that they showed different reactive activities for cell surface antigens as well as cross-linking to human and mouse B7-H3, as in the ELISA using the recombinant antigen protein. Figure 3 is a graph showing the results of evaluating the B7-H3 scFv antibody clone reactivity for four types of human cell lines. According to Fig. 3, in the case of human cell lines, five antibody clones (1-21, 1-24, 2-3, 2-25, 2-54) showed significantly high MFI in all cell lines, and two antibody clones (12 and 2-53) and one antibody clone (2-28) showed intermediate and low MFI, respectively, so that they can be divided into three groups in total according to their binding affinity to cell surface antigens. Fig. 4 is a graph showing the results of evaluating B7-H3 scFv antibody clone reactivity for six mouse cell lines. According to Figure 4, in the case of mouse cell lines, the MFI value of antibody clone 2-28, which corresponds to a weak binder, was less than twice that of the negative control in some B7-H3 low-expression cell lines such as B16-F10 and MC38, but in E0771, Hepal-6, and 4T1-B7-H3, which have relatively high B7-H3 expression levels, the MFI was more than twice that of the negative control in all of them.In addition, since the binding affinity pattern of each antibody clone shown in the human cell line was confirmed to be the same in the mouse cell line, a total of 8 B7-H3 scFv antibody clones were finally secured, including 5 strong binders, 2 medium binders, and 1 weak binder that exhibit similar antigen binding affinity to human and mouse B7-H3 antigens (cross-reactivity). Example 3. Production of mouse B7-H3 CAR-T cells One clone (#12 clone) among the B7-H3 antibodies screened in Example 1 was selected to produce a retroviral vector expressing the mouse B7-H3 CAR. Figure 5 is a schematic diagram showing the structure of the mouse B7-H3 CAR (m, mouse; h, human; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain).

[0030] B7-H3 CAR retrovirus was transduced into T cells of C57BL / 6 mice (hereinafter referred to as B6 mice) to produce B7-H3 target CAR-T cells (hereinafter referred to as B7-H3 CAR-T cells). As a result, effective CAR expression in mouse B7-H3 CAR-T cells was confirmed through flow cytometry, and the results are shown in Fig. 6. Fig. 6 is a graph showing the CAR expression rate in mouse B7-H3 CAR-T cells (the numbers in the graph represent the proportion (%) of CAR-positive cells). Example 4. In vitro activity and tumor killing capacity of mouse B7-H3 CAR-T cells In order to determine the activity of the CAR-T cells produced in Example 3, they were co-cultured with E07, a breast cancer cell line derived from B6 mice expressing B7-H3, for 24 hours, and then the amount of mouse IFN- x in the supernatant was measured by ELISA, and the results are shown in Fig. 7. Figure 7 is a graph showing the results of evaluating IFN- x production of mouse B7-H3 CAR-T cells. The test results confirmed that IFN- x production was significantly increased in mouse B7-H3 CAR-T cells compared to untransduced T cells, which were the negative control group. Next, in order to confirm whether CAR-T cells could directly kill target tumor cells, they were co-cultured with luciferase-transduced E0771 cells (E0771-Luc) for 18 hours, and the tumor killing ability of CAR-T cells was confirmed by checking the luminescence of living target cells, and the results are shown in Figure 8. Figure 8 is a graph showing the cytotoxicity of mouse B7-H3 CAR-T cells against target cells (E0771). The test results confirmed that mouse B7-H3 CAR-T cells effectively killed target cells in a dose-dependent manner depending on the number of CAR-T cells. Example 5.In vivo efficacy testing of mouse B7-H3 CAR-T cells To determine the basic in vivo efficacy of mouse B7-H3 CAR-T cells (clone #12) with proven in vitro efficacy, a commonly used lymphodepletion-post-CAR-T cell administration model was used.

[0031] E07 cells, a B7-H3 positive mouse breast cancer cell line, were injected into C57BL / 6 (B6) mice at 5xl0 5 Dogs were subcutaneously inoculated. Six days later, total body irradiation (3 Gy) was administered to eliminate lymphocytes. Then, on the following day, day 7, 5xl0 B7-H3 CAR-T cells were injected. 6It was administered intravenously. Afterwards, in order to confirm the anti-tumor effect and toxicity in the mouse body, the tumor size and the mouse body weight were measured twice a week, and the results are shown in Fig. 9. Fig. 9 is a graph showing the tumor size (a) and the change in body weight (b) after administration of mouse B7-H3 CAR-T cells. From Fig. 9(a), it can be seen that when mouse B7-H3 CAR-T cells were administered, tumor growth was significantly suppressed. On the other hand, as shown in Fig. 9(b), in the case of body weight loss of the mouse representing the toxicity of CAR-T cells, a temporary weight loss of less than 10% (7% on the 3rd day) was observed in the early stage after CAR-T cell administration, but it was soon observed to recover. This is a temporary phenomenon that appears according to CAR-T cell activity, and is well known in CD19 CAR-T cell therapy targeting hematological tumors, and can rather be used as an indicator to predict good efficacy in the future. Since no additional weight loss or death was observed after the initial small weight loss, it was confirmed that the toxicity of B7-H3 CAR-T cells was not a major problem. Example 6. Production of B7-H3 CAR-T cells with enhanced efficacy Although the basic in vivo efficacy and safety of mouse B7-H3 CAR-T cells using the #12 clone from Example 5 were confirmed, mouse B7-H3 CAR-T cells with enhanced efficacy were additionally produced to further enhance the efficacy.

[0032] To enhance the efficacy of B7-H3 CAR-T cells, we chose a strategy of expressing the CTLA4-CD28 chimera (CTC28), a T cell function-enhancing receptor designed by the inventors, and the previously reported inducible IL-12 gene together with the CAR gene in T cells.

[0033] CTC28 is a fusion protein that links the extracellular domain and transmembrane domain of CTLA4, a T-cell inhibitory receptor, to the intracellular signal transduction domain of CD28, a T-cell activating receptor. When overexpressed in T cells, it is an artificial T-cell activating receptor that competitively inhibits CTLA4 while simultaneously transmitting CD28 activation signals into the cells, thereby inducing T-cell activation (Blood (2012) 119(24):5678-87; Korean Patent No. 10—1471647). IL-12 is a representative effector function-enhancing cytokine of T cells, and CAR-T cell technology designed to secrete IL-12 through IL-12 overexpression has proven its efficacy in various preclinical CAR-T cell models (Clin Cancer Res, 2012, 8(6) 672—83; Oncolmmunology, 2015, 4:3, e994446; J Immunol, 2019, 203 (1) 198— 207). In particular, inducible IL-12-secreting CAR-T cells, which secrete IL-12 only when the CAR protein binds to the target and is activated, are conceptually designed to control the systemic side effects caused by IL-12 by secreting IL-12 only in the tumor microenvironment when CAR-T cells accumulate in the tumor site. To generate CAR-T cells loaded with these potentiating receptors and cytokines, three new potentiating B7-H3 CAR retroviral vectors were generated: a retrovirus co-expressing CTC284 B7-H3 CAR (B7H3-CTC28 CAR), a retrovirus co-expressing inducible IL-12 (IL-12) and B7-H3 CAR (B7H3-iIL12 CAR), and a retrovirus co-expressing these three genes (B7H3-CTC28-iIL12 CAR).Figure 10 is a schematic diagram showing the structure of a retroviral vector expressing a CTLA4-CD28 chimeric (CTC28) gene and / or an inducible IL-12 gene together with a B7-H3 CAR gene (PGK, PGK promoter; 6xNFAT (six tandem NFAT binding element promoter: six tandem NFAT binding elements); P2A, P2A peptide). The three types of retroviruses were introduced into mouse T cells to confirm the expression of B7-H3 CAR and CTC28, and the results are shown in Figure 11. Figure 11 is a graph showing the CAR and CTC28 expression rates of potentiated B7-H3 CAR-T cells. In the potentiated B7-H3 CAR-T cells, a similar level of CAR expression was confirmed as that of the non-potentiated B7-H3 CAR-T cells of the #12 clone (denoted as B7H3), and in addition, high expression of CTC28 was confirmed in B7H3-CTC28 CAR-T cells expressing CTC28, B7H3- CTC28-iIL12 CAR-T cells. Furthermore, in order to confirm the activity and inducible IL-12 expression of the produced potentiated B7-H3 CAR-T cells, CAR-T cells were co-cultured with tumor cells (E0771) for 24 hours, and the amounts of IL-12 and IFN-x in the supernatant were measured by ELISA, and the results are shown in Fig. 12. Fig. 12 is a graph showing the IL-12 production rate of the potentiated B7-H3 CAR-T cells. In the case of IL-12, since it is regulated by an inducible promoter that is expressed only when CAR-T cells are activated, in the case of CAR-T cells that were not co-cultured with tumor cells (Effector:Target ratio (E:T) was l:0), IL-12 was hardly detected (Fig. 12(a)).However, in the case of E:T=1:1 where target cells exist, IL-12 was induced, and significantly higher IL-12 expression was confirmed in the B7H3-iIL12 CAR-T and B7H3-CTC28-iIL12 CAR-T groups compared to the control group of non-transduced T, B7H3 CAR-T, and B7H3-CTC28 CAR-T groups (Fig. 12(b)). This result shows that not only is the expression of IL-12 well regulated by the inducible promoter, but also the IL-12 expression rate is excellent when expression is induced.

[0034] Since IL-12 is known to be a cytokine that acts on T cells to increase the expression of IFN- x, it was expected that the expression of IFN- x would increase in the B7H3-iIL12 CAR-T and B7H3-CTC28-iIL12 CAR-T groups expressing inducible IL-12 compared to the control group. As shown in Fig. 13, the IFN- x ELISA results confirmed that under the conditions of E: T = 1:1, the expression of IFN- x in the B7H3-iIL12 CAR-T and B7H3— CTC28— iIL12 CAR— T groups was significantly increased compared to the control B7H3 CAR-T group. Fig. 13 is a graph showing the IFN- x production rate of potentiated B7-H3 CAR-T cells. Therefore, it was confirmed that IL-12 secreted from CAR-T cells can significantly increase the activity of CAR-T cells themselves through an autocrine mechanism. Next, to confirm the tumor-killing ability of the enhanced B7-H3 CAR-T cells, they were co-cultured with luciferase-transduced E0771-Luc cells for 18 hours, and the luminescence of live E0771-Luc cells was confirmed, and the results are shown in Fig. 14. Fig. 14 is a graph showing the cytotoxicity of the enhanced B7-H3 CAR-T cells against target cells (breast cancer cells). The test results confirmed that all three types of enhanced B7-H3 CAR-T cells effectively killed target cells in a dose-dependent manner of the CAR-T cell number to a similar degree as the conventional B7-H3 CAR-T cells. Example 7. In vivo efficacy and safety evaluation of enhanced B7-H3 CAR-T cells To verify the in vivo efficacy and safety of enhanced B7-H3 CAR-T cells with confirmed in vitro efficacy, B6 mice and the E0771 tumor model, which were previously used in the in vivo experiment of B7-H3 CAR-T cells, were used.According to the results of previous studies on CAR-T cells expressing IL-12, it was known that IL-12-expressing CAR-T cells showed anti-tumor effects even without lymphodepletion, a process known to induce inflammatory responses and CAR-T cytotoxicity in the body. Therefore, CAR-T cells were administered without lymphodepletion in this in vivo experiment.

[0035] E07 cells, a B7-H3 positive mouse breast cancer cell line, were injected into C57BL / 6 (B6) mice at 5xl0 5 Dogs were subcutaneously inoculated. After 7 days, 5xl0 of B7-H3 CAR-T cells and three types of potentiated B7-H3 CAR-T (B7H3— CTC28, B7H3— iIL12, B7H3— CTC28— iIL12) were injected 6 It was administered intravenously. At this time, in order to enhance the T cell efficacy of CTC28, the effect is maximized when the T cell subtypes CD4 T cells and CD8 T cells are injected in equal amounts (Blood (2012) 119(24):5678-87), so each group of CAR-T cells was transduced with the corresponding retrovirus into CD4 T cells and CD8 T cells, respectively, and then CD4 CAR-T cells and CD8 CAR-T cells were injected in a 1:1 ratio (2.5xl0 each). 6Each (each) was mixed and administered. Afterwards, in order to confirm the antitumor effect and toxicity in the mouse body, the tumor size and the mouse body weight were measured twice a week, and the results are shown in Fig. 15. Fig. 15 is a graph showing the change in tumor (breast cancer) size and body weight after administration of B7-H3 CAR-T cells with enhanced efficacy to small tumors. As a result of the test, when the lymphocyte removal process was omitted, no tumor inhibition effect was observed in the B7-H3 CAR-T cell alone and B7H3-CTC28 CAR-T cell groups, but a significant tumor inhibition effect was observed in the B7H3-iIL12 and B7H3-CTC28-iIL12 CAR-T cell groups to which IL- 12 was introduced (Fig. 15 (a)). In terms of toxicity, in the B7H3-iIL12 CAR-T cell administration group and the B7H3-CTC28-iIL12 CAR-T cell administration group that showed antitumor effect, a temporary slight weight loss of less than 10% (4% on day 5) was observed in the early stage after CAR-T cell administration, but it was soon observed to recover (Fig. 15 (b)). Since this is a manageable early side effect commonly observed in the currently commercially available CD19 CAR-T cells, the safety of the potency-enhanced CAR-T cells was confirmed. Therefore, it can be seen that B7H3-iIL12 CAR-T cells and B7H3— CTC28— iIL12 CAR-T cells exhibit excellent efficacy and safety. The above results are based on the results obtained when the tumor size at the time of CAR-T cell administration was 100 mm. 3 Small size below (50mm 3 ), additional in vivo experiments were conducted to confirm whether the enhanced efficacy B7-H3 CAR-T cells exhibited enhanced anti-tumor effects against more advanced tumors.

[0036] 5xl0 E07 cells in B6 mice 5 After subcutaneous vaccination, the tumor grew larger on the 14th day (350mm) 3)In the previous experiment, B7H3-iIL12 CAR-T cells and B7H3-CTC28-iIL12 CAR-T cells were confirmed to be effective at 5xl0 6It was administered by intravenous injection. Afterwards, in order to confirm the anti-tumor effect and toxicity in the mouse body, the tumor size and the body weight of the mouse were measured twice a week, and the results are shown in Fig. 16. Fig. 16 is a graph showing the change in tumor (breast cancer) size and body weight after administration of B7-H3 CAR-T cells with enhanced efficacy to advanced tumors (n / 5: n = number of mice in which the tumor was completely eliminated out of a total of 5 mice). As a result of the test, a more enhanced tumor inhibition effect was observed in the group injected with B7H3-CTC28- iIL12 CAR-T cells than in B7H3-iIL12 CAR-T cells (Fig. 16 (a)). As a result of additional observation, on the 44th day after tumor inoculation (30 days after CAR-T cell administration), the tumor was completely removed in one of the five mice in the group injected with B7H3-iIL12 CAR-T cells, but the tumor recurred and died in the remaining four mice. However, in the group injected with B7H3-CTC28-iIL12 CAR-T cells, tumors were completely removed in four out of five mice, and the tumor recurred in only one mouse, showing a complete response rate of 80% (Fig. 16(a)). In terms of toxicity, the B7H3-iIL12 CAR-T cell administration group showed a 7% body weight loss on the fourth day after CAR-T cell administration, but it soon recovered (Fig. 16(b)). In the B7H3-CTC28-iIL12 CAR-T cell administration group, which showed better efficacy, showed an 11% body weight loss on the fourth and seventh days after CAR-T cell administration, but the body weight was quickly recovered (Fig. 16(b)). In other words, it was confirmed that there were no major safety issues because it did not exceed the range of manageable early side effects seen in existing CAR-T cell therapies. Example 8. In vivo efficacy evaluation of mouse B7-H3 CAR-T cells using two anti-B7-H3 CAR-T cells other than clone #12.

[0037] In addition to the #12 clone, two types of scFv (#2-25, #2-54) were used to produce the mouse B7-H3 CAR gene, and mouse #25-CTC28-iIL12 CAR-T cells (using the #2-25 clone) and #54—CTC28—iIL12 CAR-T cells (using the #2-54 clone) were produced together with the potency-enhancing platform CTC28-iIL12. The efficacy and safety of these CAR-T cells were evaluated using the same experimental method and schedule as in Example 7, and the results are shown in Figs. 17 and 18. Fig. 17 is a graph showing the change in tumor (breast cancer) size and body weight after administration of #12-CTC28-iIL12 CAR-T cells {#25—CTC28—iIL12 CAR-T cells}. Figure 18 is a graph showing the change in tumor (breast cancer) size and body weight after administration of {#12-CTC28-iIL12 CAR-T cells} and {#54—CTC28—iIL12 CAR-T cells}. As a result of the test, both {#25-CTC28-iIL12 CAR-T cells} and {#54—CTC28—iIL12 CAR-T cells} showed a great anti-tumor effect and manageable temporary initial body weight loss similar to the #12 clone, thereby proving efficacy and safety. In particular, in the case of #25-CTC28-iIL12 CAR-T cells, even in a state where initial body weight loss was almost invisible, anti-tumor effect similar to #12-CTC28-iIL12 CAR-T cells was confirmed, suggesting the possibility of developing safer and more effective CAR-T cells (Figure 17). Next, the in vivo efficacy of B7H3-CTC28-iIL12 CAR-T cells using #2-25 clone was evaluated against hepatocellular carcinoma cells, a cancer type other than breast cancer. Hepal-6 cells, a B7-H3 positive hepatocellular carcinoma cell line, were injected into C57BL / 6 (B6) mice at 5xl0 6Dogs were inoculated subcutaneously. After 14 days, 5xl0 of #25 CAR-T cells and three types of potentiated B7-H3 CAR-T (#25— CTC28, #25- iIL12, #25- CTC28- iIL12) were injected 5It was administered intravenously. Figure 19 is a graph showing the in vivo efficacy of #25-CTC28-iIL12 CAR-T cells against liver cancer cells. As a result of the test, both #25-iIL12 CAR-T cells and #25-CTC28-iIL12 CAR-T cells showed a strong anti-tumor effect, and in particular, the efficacy of #25-CTC28-iIL12 CAR-T cells was confirmed to be very excellent. In addition, the initial weight loss was also minimal, and safety was also secured. Therefore, the efficacy and safety of B7H3-CTC28-iIL12 CAR-T cells using the anti-B7-H3 antibody discovered in the present invention were proven for severely progressed intractable tumors. This result shows the possibility of developing human B7-H3 CAR-T cells using the same anti-B7-H3 antibody scFv> in the future. Example 9. Cloning of human CAR gene using anti-B7-H3 antibody and production of human CAR-T cells In order to produce human CAR-T cells using a novel anti-B7-H3 antibody scFv, a human CAR gene (hB7H3 CAR; hB7H3-BBz) was produced using eight anti-B7-H3 antibody scFvs selected through an antibody discovery process. The produced CAR gene was cloned into a lentiviral vector to produce a CAR expression lentiviral plasmid. Figure 20 is a schematic diagram showing the structure of the human CAR gene, i.e., the hB7H3 CAR (hB7H3-BBz) gene produced using anti-B7-H3 antibody Apa (h, human). After transfecting 293T cells with the produced lentiviral plasmid together with packaging plasmids, the lentivirus secreted into the culture medium was harvested and concentrated by ultracentrifugation.After separating only T cells from lymphocytes collected from normal blood using a CD4 / CD8 MACS column, T cells were activated for 24 hours by adding T cell activation beads (TransAct, Miltenyi), and then transduced with concentrated lentivirus for 2 days. After culturing for approximately 5 days in a T cell culture medium containing IL-7 and IL-15, cell surface CAR expression was analyzed by flow cytometry (#1-24 and #2-3 of the 8 antibodies were excluded from further analysis due to low cell surface CAR expression rates (see Table 3 below). For CAR expression analysis, fluorescently labeled human B7-H3 recombinant protein was added to CAR-T cells and used for detection.

[0038] [Table 3] List of novel antibody candidates according to the human B7-H3 CAR-T cell production process. Figure 21 is a graph showing the CAR expression rate in human B7-H3 CAR-T cells. As a result of the test, cell surface expression of CAR molecules was successfully confirmed in six types of CAR-T cells, and similar to the antigen binding affinity of existing antibodies, high ligand binding affinity was confirmed for #1-21, 2-25, 2-54, moderate binding affinity for #12, #2-53, and weak binding affinity for #2-28. Example 10. Evaluation of in vitro efficacy of human B7-H3 CAR-T cells In order to confirm the in vitro activity and tumor killing ability of the produced human B7-H3 CAR-T cells, IFN-x secretion ability and cytotoxicity to target cells were evaluated.

[0039] B7-H3 CAR-T cells were co-cultured with a B7-H3 positive human breast cancer cell line (MDA-MB-231) transduced with luciferase for 24 hours, and the tumor killing ability was measured by measuring the luciferase expression of surviving cells, and the results are shown in Fig. 22. Fig. 22 is a graph showing the cytotoxicity of human B7-H3 CAR-T cells against target cells. As a result of the test, it was confirmed that all six types of CAR-T cells effectively killed target cells in a dose-dependent manner depending on the number of cells. In addition, in the case of #2-28 CAR-T cells, corresponding to the low binding affinity to B7-H3, the tumor killing ability was slightly lower than that of other clones, confirming that there is a correlation between the binding affinity to B7-H3 of CAR-T cells and the tumor killing ability. Next, the amount of IFN-x secreted into the supernatant after co-culture with tumor cells

[0040] The results were measured using ELISA and are shown in Fig. 23. Fig. 23 is a graph showing the IFN- x production rate of human B7-H3 CAR-T cells (E: T, ratio of the number of effector (CAR- T cells) : number of targets (tumor cells)). As a result of the test, only the #2-28 CAR-T cells, which showed weak binding affinity similar to the tumor killing ability, showed a relatively low IFN-Y production amount compared to other clones, but very high IFN-Y production ability was confirmed in all 6 types of CARs including the #2-28 CAR-T cells. Therefore, it was confirmed that the in vitro efficacy of all 6 types of selected CAR-T cells was excellent. Example 11. Production of CAR-T cells loaded with human B7-H3 CAR gene and human potentiation gene In order to enhance the potency of the human B7-H3 CAR-T cells, three antibody clones (#12, #2-25, #2-54) whose efficacy and safety were proven in a mouse CAR-T cell model were used. CAR-T cells co-expressing human CTLA4-CD28 chimera (hCTC28) and human IL-12 (hIL-12) (hB7H3-CTC28-iIL12; designated as #12BBz-C12, #25BBz-C12, and #54BBz-C12, respectively) were produced. Here, a lentiviral vector co-containing the hB7H3 CAR, inducible IL-12, and CTLA4-CD28 chimera (CTC28) genes was constructed, and a schematic diagram of its structure is shown in Figure 24 (h, human; PGK, PGK promoter; 6xNFAT promoter, six tandem NFAT binding elements; P2A, P2A peptide). The constructed lentiviral plasmid was transfected into 293T cells together with packaging plasmids, and the lentivirus secreted into the culture medium was harvested and concentrated by ultracentrifugation.After separating only T cells from lymphocytes collected from normal blood using a CD4 / CD8 MACS column, T cell activation beads (TransAct, Miltenyi) were added to activate the T cells for 24 hours, and then concentrated lentivirus was added to transduce them for 2 days. After culturing them for about 5 days in a T cell culture medium containing IL-7 and IL-15, cell surface CAR expression was analyzed by flow cytometry, and the results are shown in Fig. 25. Fig. 25 is a graph showing the CAR and CTC28 expression rates of enhanced-efficacy human B7-H3 CAR-T cells (UT, untransduced; ChlgY, FITC-labeled anti-chicken IgY Fab; CTLA4-PE, PE-labeled anti-human CTLA4 antibody). As a result of the test, CAR expression similar to that of the preceding hB7H3— BBz CAR-T cells (#12BBz— C12, #25BBz— C12, #54BBz— C12) was confirmed in three types of potentiated CAR-T cells (#12BBz— C12, #25BBz— C12), and it was confirmed that cell surface CTC28 was expressed only in the potentiated CAR-T cells. Example 12. In vitro efficacy evaluation of potentiated human B7- H3 CAR- T cells To evaluate the in vitro efficacy of the produced potentiated human CAR-T cells, cell killing activity and cytokine secretion activity were confirmed. The results of the tumor killing activity evaluation of the potentiated human B7-H3 CAR-T cells are shown in Fig. 26. When the MDA-MB-231-Luc cell line used in the previous in vitro efficacy evaluation experiment was used as the target cell, it was confirmed that the potentiated CAR-T cells showed an effective tumor killing effect similar to that of the preceding hB7H3-BBz CAR-T cells.Since the potentiated human CAR-T cells have an inducible IL-12 platform that allows IL-12 to be expressed only in activated CAR-T cells, the potentiated CAR-T cells and the target cells, MDA-MB-231 cells, were co-cultured for 24 hours at an E:T ratio of 1:5, and the expression of IL-12 and IFN-x was confirmed using ELISA, and the results are shown in Figs. 27 and 28. Fig. 27 is a graph showing the results of evaluating the IL-12 secretion ability of the potentiated human B7-H3 CAR-T cells. From Fig. 27, it can be seen that significant IL-12 expression was confirmed only when co-cultured with tumor cells in three types of potentiated CAR-T cells, confirming the inducible secretion ability of IL-12. Fig. 28 is a graph showing the results of evaluating the IFN-x secretion ability of the potentiated human B7-H3 CAR-T cells. In the case of IFN- x, similar to the results of mouse CAR- T cells, a significant increase in the secretion amount was observed in the potentiated CAR-T cells compared to the preceding hB7-H3 CAR-T cells, suggesting that the activation of CAR-T cells is greatly enhanced by the additional loading of potentiating receptors and IL-12. Example 13. In vitro efficacy evaluation of potentiated human B7-H3 CD4 and CD8 CAR- T cells According to previous reports, when CD4:CD8 CAR-T cells are administered at a ratio of 1:1, an enhanced anti-tumor effect is observed in wVc (Sommermeyer et al. 2016, Turtle et al. 2016), and accordingly, some commercially available CAR-T cells are administered after separately manufacturing CD4 and CD8 CAR-T cells (Lisocabtagene maraleucel).In addition, as mentioned when producing mouse CAR-T cells, in order to enhance the T cell efficacy of CTC28, the effect is maximized when CD4 T cells and CD8 T cells, which are T cell subtypes, are injected in equal amounts (Blood (2012) 119(24):5678-87). Therefore, for the enhanced human hB7-H3 CAR-T cells, after transducing CD4 T cells and CD8 T cells with lentivirus, respectively, CD4 CAR-T cells and CD8 CAR-T cells were analyzed, and the results are shown in Fig. 29. Fig. 29 shows the results of analyzing the expression of CAR and hCTC28 in three types of human B7-H3 CAR-T cells (ChlgY, FITC-labeled anti-chicken IgY Fab; CTLA4-PE, PE-labeled anti-human CTLA4 antibody). As a result of the test, effective CAR protein and hCTC28 protein were confirmed on the cell surface in both CD4 and CD8 T cells. In addition, an in vitro efficacy evaluation experiment was conducted to confirm the cell killing ability and cytokine secretion ability of the three types of potentiated hB7-H3 CAR-T cells produced (#12, #2-25, #2-54). The MDA-MB-231-Luc cell line used in the existing in vitro efficacy evaluation experiment was used as the target cell, and the potentiated CAR-T cells were produced by isolating CD4 T cells and CD8 T cells using the MACS technique and used as effector cells. After co-culturing the target cells and effector cells for 16 hours, the tumor killing ability was measured, and the results are shown in Figure 30. Figure 30 is a graph showing the results of evaluating the tumor killing ability of the potentiated human B7-H3 CAR-T cells. The test results confirmed that all three types effectively killed target cells in a dose-dependent manner in CD4 CAR-T and CD8 CAR-T cells.Since the potentiated CAR-T cells were applied with an inducible IL-12 technique that allows IL-12 to be expressed only in activated CAR-T cells, the potentiated CAR-T cells and the target cells, MDA-MB-231 cells, were co-cultured for 24 hours at an E:T ratio of 1:5, and the expression of IL-12 and IFN-x was confirmed using ELISA, and the results are shown in Figs. 31 and 32. Fig. 31 is a graph showing the results of evaluating the IL-12 production of potentiated human B7-H3 CAR-T cells (Unt, non-transduced T cells). As a result of the test, IL-12 secretion was confirmed only when co-cultured with tumor cells in all three CAR-T cells, confirming the inducible IL-12 secretion ability of these cells. Fig. 32 is a graph showing the IFN-x production rate of potentiated human B7-H3 CAR-T cells. In the case of IFN- x, the appropriate activation of CAR-T cells was confirmed by the confirmation of significant secretion amount by the activation of CAR-T cells when co-cultured with tumor cells in CD4 and CD8 CAR-T cells. Example 14. Humanization of B7-H3 targeting scFv antibody To reduce the immunogenicity potential of chicken-derived anti-B7-H3 scFv antibody clones, humanization was performed by applying CDR grafting and backmutation to three clones (#12, #2-25, #2-54) that have proven efficacy and safety in mouse and human CAR-T cell models. Kabat numbering for chicken-derived antibodies was automatically assigned using ANARCI software (Dunbar and Deane, Bioinformatics 2016 32 298), and CDRs were defined according to the criteria proposed by Andrew CR Martin's group published on the website www.bioinf.org.uk.The sequences of each chicken-derived antibody were aligned to the human germline sequence repertoire, IMGT, and the human germline sequence with the smallest amino acid differences at the corresponding framework positions was selected as the humanization template. For the light chain humanization template, IGKV1-16*01 was selected for clone #12, IGKV1-9*03 for clone #2-25, and IGKV1-9*03 for clone #2-54. For the heavy chain humanization template, IGHV3-23*02 was selected for clone #12, IGHV3-23*01 for clone #2-25, and IGHV3-53*01 for clone #2-54. Each chicken CDR sequence was in silico grafted onto the corresponding humanization template, and then several human-chicken backmutation sites that may be important for maintaining the CDR structure were selected to introduce the corresponding mutations. In addition, we scanned the risk sites of post-translational modification (PTM) such as Asparagine deamidation (Asn-Gly and Asn-Ser) in CDR, Aspartate isomerization (Asp-Gly) in CDR, Unpaired Cysteine ​​in CDR and framework, N-linked glycosylation sites (Asn-Xxx-Ser / Thr, short Xxx-pro) in CDR and framework, etc., and designed several PTM-eliminating mutants by selecting alternative amino acids to evaluate the effect of these potential PTM sites on antibody affinity. The genes of the mutants were synthesized and inserted into the pCEP4-His expression vector to construct scFv expression vectors tagged with 6xhistidine at the C-terminus. Expi293F cells (ThermoFisher, A14527) were transfected using the manufacturer's protocol provided by the Epifectamine 293 transfection kit (ThermoFisher, A14524). Six days after transfection, 100 mL of Expi293F cell culture was harvested, and the culture supernatant expressing the antibody was separated by centrifugation and k.of f was applied to the ranking analysis. k of humanized variants O For ff ranking, the culture supernatant expressing antibodies was diluted 10-fold and analyzed for single kinetics# by surface plasmon resonance (SPR). Briefly, the CM5 sensor chip was activated using an EDC / NHS mixture, and anti-human Fc antibody (anti-human IgG, Fc specific) was injected into the CM5 sensor chip to induce covalent binding, followed by inactivation with 1 M ethanolamine-HC1. Human B7H3 protein (Ligand) fused to human IgG1 Fc region or mouse B7H3 protein (Ligand) fused to human IgG1 Fc region was injected into the CM5 sensor chip immobilized with anti-human Fc antibody and captured. Afterwards, the antibody (Analyte) was injected to perform the association phase and dissociation phase, and the analysis was performed using the software within Biacore 8K (Version 3.0.12.15655). As a result of koff ranking analysis, three variants with koff values ​​similar to the parent antibody were selected from clone #12, three from clone #2-25, and five from clone #2-54, and these were subjected to full kinetics SPR analysis. Table 4 presents the amino acid sequences of the humanized antibody variants selected from single kinetics analysis, with the amino acids in bold corresponding to backmutation sites.

[0041] [Table 4] VH / VL sequences of humanized variants

[0042] Antibody purification was performed for full kinetics SPR analysis and purity analysis. The culture supernatant of the selected mutants was first purified using a Ni-chelating affinity column (GE lifesciences, HisTrap excel, 29048586), and the buffer was exchanged into PBS by dialysis through cellulose tubing. Subsequently, the dialyzed product was purified again using a size exclusion chromatography (SEC) column (GE lifesciences, Superdex 200 increase 10 / 300). The final purified product eluted with PBS was sterile filtered through a 0.22 lim filter, aliquoted into 0.2 mL portions, and stored at -20°C. The concentration of the purified antibody protein was determined by the OD280 value measured with a Nanodrop, and the purity was determined by SDS-PAGE and HPLC-SEC analysis values. Figure 33 and Table 5 show the SDS-PAGE and SEC-HPLC results, respectively. According to Figure 33, the humanized variants exhibited a molecular weight of approximately 25-30 kDa^ under both SDS-PAGE reducing and non-reducing conditions, and according to Table 5, the purity of the antibody was over 90%, and the peak eluted at approximately 10.4 minutes, indicating that the molecular weight of the antibody was appropriate.

[0043] [Table 5] SEC-HPLC measurements of humanized variants of B7-H3 target scFv antibodies

[0044] For the full kinetics SPR analysis, six concentrations of protein prepared by serially diluting purified antibody proteins were applied as analytes. As shown in Table 6, all selected humanized variants showed a difference in KD (binding affinity) of less than 3-fold for human B7H3 or mouse B7H3 protein compared to before humanization. In addition, the binding affinity of humanized and non-humanized anti-B7H3 antibody scFv to human B7H3 on the cell surface was tested in the MDA-MB-231 cell line expressing human B7H3 antigen, and is shown in Figure 34. The test results confirmed that binding was at a similar level to before humanization, indicating that the humanization of the antibody was successfully completed.

[0045] [Table 6] Full kinetics SPR measurements of humanized variants of B7-H3 target scFv antibodies

[0046] Among the humanized variants, one variant each of #12, #2-25, and #2-54 was selected as the final humanized antibody by comprehensively evaluating purity, expression level, and antigen binding affinity. Table 7 shows the amino acid sequences of the final selected humanized antibodies (scFv) hu#12 (#12-z32), hu#25 (#2-25-z25), hu#54 (#2-R) and the framework region. CDR and framework sequences of the selected humanized antibodies Example 15. Comparison of the efficacy of humanized B7H3 CAR and non-human B7H3 CAR When CAR-T therapy is performed on patients, the efficacy of CAR-T therapy may be reduced due to the immune response to existing chicken-derived antibodies. If this is made into a humanized CAR-T, the immune response can be minimized, thereby reducing side effects and making it a safer and more effective treatment. Therefore, CARs using humanized B7H3 scFvs z32, z25, and zl6 were produced against CARs of three non-humanized B7H3 scFvs (#12, #2-25, #2-54), and the aim was to determine whether the in vitro efficacy of the humanized B7H3 CAR was similar to that of the non-humanized B7H3 CAR.

[0047] (1) Humanized B7H3 CAR gene cloning and virus production CAR genes for each of the three humanized and non-humanized B7H3s were constructed. The constructed CAR genes were cloned into a lentiviral vector to construct a CAR expression lentiviral plasmid. Figure 35 is a schematic diagram showing the structure of the CAR gene constructed using humanized B7H3 antibody scFv and non-humanized B7H3 antibody scFv (L, leader sequence; h, human; hu, humanized; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain). The newly humanized CAR was named B7H3-z32 (or z25, zl6)-BBz. The non-humanized CAR was named B7H3 #12 (or #25, #54)-BBz. For convenience, #12, #2-25, and #2-54 were designated as #12, #25, and #54, respectively. The constructed lentiviral plasmids were transfected into 293T cells together with envelope and packaging plasmids, and the lentivirus secreted into the culture medium was harvested and concentrated by ultracentrifugation.

[0048] (2) Production of humanized B7H3 CAR-T cells T cells were separated from lymphocytes collected from normal blood using a CD4 / CD8 MACS (Magnetic-activated cell sorting) column, and then T cell activation beads (TransAct, Miltenyi Biotec) were added to activate the T cells for 24 hours. After that, the concentrated lentivirus was added and transduced for 2 days. After culturing for about 5 days in a T cell culture medium containing IL-7 and IL-15, cell surface CAR expression was analyzed by flow cytometry. For CAR expression analysis, fluorescently labeled anti-human IgG F(ab')2 antibody was used for three types of humanized B7H3 CAR-T cells, and fluorescently labeled anti-chicken IgY Fab (ChlgY) antibody was used for three types of non-humanized B7H3 CAR-T cells. CAR expression rates in humanized and non-humanized B7H3 CAR-T cells are shown in Figure 36 (numbers in the graph represent the percentage of CAR-positive cells). Similar CAR expression of approximately 40–45% was confirmed in these CAR-T cells.

[0049] (3) In vitro tumor cell killing ability and activity of humanized B7H3 CAR-T cells To compare the in vitro tumor killing ability and activity of the produced humanized B7H3 CAR-T cells with that of non-humanized B7H3 CAR-T cells, cytotoxicity and IFN-Y secretion ability against target cells were evaluated.

[0050] B7H3 CAR- T cells were co-cultured with luciferase-transduced B7H3-positive human breast cancer cell line (MDA-MB-231) for 16 hours, and the tumor killing ability was analyzed by measuring the luciferase expression of the surviving cells, and the results are shown in Fig. 37. Fig. 37 is a graph showing the cytotoxicity of B7H3 CAR- T cells against target cells. The test results confirmed that the cytotoxicity was increased in both human B7H3 CAR- T cells compared to the untransduced T cells, which were the negative control group. It was confirmed that both humanized B7H3 and non-humanized B7H3 CAR- T cells effectively killed target cells in a similar dose-dependent manner depending on the number of cells. Next, the manufactured CAR-T cells (Effector) were co-cultured with the human breast cancer cell line MDA-MB-231 (Target) at an E:T ratio of 1:5 for 24 hours, and the amount of IFN-x secreted into the supernatant was measured by ELISA (Enzyme-Linked Immunosorbent Assay) and the results are shown in Fig. 38. Fig. 38 is a graph showing the IFN-Y production amount of each of the three types of B7H3 CAR-T cells before and after humanization. According to Fig. 38, when the activation of CAR-T cells before and after becoming B7H3 scFv was compared, it was confirmed that the amount of IFN-x secretion was maintained at a fairly similar level even after humanization. Therefore, it was confirmed that the CAR expression and CAR-T cell activity of the three types of humanized B7H3 CAR-T cells were stably maintained compared to before humanization. Example 16.In vitro efficacy evaluation of humanized B7H3 CAR-T cells with enhanced potency Next, the CTLA4-CD28 chimera (CTC28), a T cell function enhancing receptor developed by the present inventors, and the previously reported inducible IL-12 gene were expressed in T cells together with the humanized B7H3 CAR gene to determine whether the potency of the humanized B7H3 CAR-T cells was enhanced in vitro, similar to before humanization.

[0051] (1) Cloning of humanized B7H3 CAR gene with enhanced efficacy and virus production Lentiviral plasmids carrying human CAR genes using three types of humanized scFv, z32, z25, and zl6, along with human CTC28 and human inducible IL12, were constructed. The constructed lentiviral plasmids were transfected into 293T cells together with envelope and packaging plasmids, and the lentivirus secreted into the culture medium was harvested and concentrated by ultracentrifugation.

[0052] (2) Production of humanized B7H3 CAR-T cells with enhanced efficacy

[0053] According to reports that when CD4:CD8 CAR- T cells are administered at a 1:1 ratio, an enhanced anti-tumor effect is observed in wVo (Sommermeyer et al. 2016, Turtle et al. 2016), some commercially available CAR-T supporters (Lisocabtagene maraleucel) are administered after separately manufacturing CD4 and CD8 CAR- T cells. In addition, according to a previous study by the present inventors (Blood (2012) 119(24):5678-87), the effect of CTC28 in T cell therapy is maximized when CD4 T cells and CD8 T cells are each transduced with CTC28 and these CD4 and CD8 T cells are mixed and administered at a 1:1 ratio. Therefore, in the present invention, CD4 CAR- T cells and CD8 CAR- T cells were separately manufactured and their efficacy was analyzed. CD4 T cells and CD8 T cells were separated from lymphocytes collected from normal blood using CD4 and CD8 MACS columns, and T cell activation beads (TransAct, Miltenyi Biotec) were added to activate the T cells for 24 hours. Afterwards, concentrated lentivirus was added to CD4 T cells and CD8 T cells, respectively, and transduced for 2 days. After culturing for approximately 5 days in CD4 and CD8 T cell culture medium containing IL-7 and IL-15, cell surface CAR expression on CD4 CAR-T cells and CD8 CAR-T cells, respectively, was analyzed by flow cytometry. The expression of CAR and CTC28 in three types of humanized B7H3 CAR- T cells was analyzed using fluorescently labeled anti-human IgG, F(ab')2 antibody and fluorescently labeled anti-human CTLA4 antibody, respectively, and the results are shown in Figure 39 (F(ab')2-FITC, FITC-labeled anti-human IgG F(ab')2; CTLA4-PE, PE—labeled anti-human CTLA4 antibody; CD4, APC / Cyanine7—labeled anti-human CD4 antibody; CD8, PerCP / Cy5.5—labeled anti-human CD8 antibody). Test results showed that three types of potentiated humanized CAR-T cells (z32— BBz— CTC28— IL12, z25— BBz— CTC28— IL12, zl6— BBz— CTC28—) were potent in both CD4 and CD8 T cells.

[0054] IL12) showed a similar level of CAR expression when compared to the preceding huB7H3-BBz CAR-T cells (z32-BBz, z25-BBz, zl6-BBz), and it was confirmed that cell surface CTC28 was expressed only in the potentiated CAR-T cells.

[0055] (3) Evaluation of in vitro tumor cell killing capacity and activity of humanized B7H3 CAR-T cells with enhanced efficacy

[0056] To evaluate the in vitro efficacy of three types of potentiated humanized B7H3 CAR-T cells produced from CD4 and CD8 T cells, cytotoxicity against target cells and IL12 and IFN-x secretion capacity were compared with those of pre-existing B7H3-BBz CAR-T cells.

[0057] B7H3 CD4 and CD8 CAR- T cells were co-cultured with a B7H3-positive human breast cancer cell line (MDA-MB-231) expressing the luciferase gene for 16 hours, and the tumor killing ability was analyzed by measuring the luciferase expression of surviving cells, and the results are shown in Fig. 40. Fig. 40 is a graph showing the cytotoxicity of B7H3 CD4 and CD8 CAR- T cells (E, effector) against target cells (T, target). The test results confirmed that cytotoxicity was increased in all humanized B7H3 CAR-T cells compared to untransduced (UT, untransduced) T cells, which was the negative control group. It was confirmed that the potency of the three types of humanized B7H3 and the preceding CD4 and CD8 CAR-T cells effectively killed target cells in a similar dose-dependent manner in a cell number-dependent manner. Since the potentiated CAR-T cells are applied with an inducible IL-12 platform that allows IL-12 to be expressed only in activated CD4 and CD8 CAR-T cells, the potentiated humanized CAR-T cells (E) and target cells, MDA-MB-231 cells (T), were co-cultured for 24 hours at an E:T ratio of 1:5, and the amounts of IL-12 and IFN-x secreted into the supernatant were confirmed using ELISA.

[0058] Only in the three types of potency-enhanced B7H3 CD4 and CD8 CAR- T cells, significant IL-12 expression was confirmed when co-cultured with tumor cells, and the results of the IL-12 induction secretion ability are shown in Figure 41.

[0059] The results of the IFN- x analysis are shown in Fig. 42. According to Fig. 42, a significant increase in the secretion of IFN- X was observed in both potentiated B7H3 CD4 and CD8 CAR- T cells compared to the pre-human B7H3 CAR- T cells, suggesting that the activation of CAR- T cells was significantly enhanced by the additional loading of potentiated CTC28 and IL-12. Therefore, it was confirmed that the potency enhancement effect by co-loading of CTC28 and IL-12 was maintained in humanized CAR- T cells as before humanization.

[0060] (4) Efficacy evaluation of humanized B7H3 CAR-T cells using different humanized variant scFvs In addition to the z32, z25, and zl6 clones, there are additional humanized clones for the #12, #2-25, and #2-52 scFv antibody parental clones used in each of the three B7H3 CAR-T cells (Table 6). Therefore, we aimed to verify whether humanized CAR-T cells using these additional humanized clones exhibit similar efficacy to humanized CAR-T cells using the representative z32, z25, and zl6 clones using CAR-T cells of the enhanced efficacy CTC28-IL12 CAR type (abbreviated as C12 type). In this example, in order to make the CAR expression rates in the pre-humanization mother clone used as a control and the clones after humanization similar, human B7H3 recombinant protein, a CAR ligand commonly used in both CAR-D cells before and after B7H3 scFv humanization, was added as a detection reagent and analyzed under cell conditions showing similar CAR expression rates. Accordingly, CAR expression in humanized C12 B7H3 CAR-T cells with enhanced efficacy and non-humanized C12 B7H3 CAR-T cells was confirmed by flow cytometry using B7H3 recombinant protein (6xHis-tagged B7H3 protein (h4Ig form)) and a secondary antibody (APC-conjugated anti-6xHis Tag antibody) that binds to it, and conditions showing similar CAR expression rates were established. In addition, the expression of CTC28 was also confirmed by flow cytometry using fluorescently labeled anti-human CTLA4 antibody.These C12 B7H3 CAR- T cells were co-cultured with luciferase-transduced B7H3-positive human breast cancer cell line (MDA-MB-231) for 16 hours, and the tumor killing ability was analyzed by measuring the luciferase expression of surviving cells. In addition, the amount of IFN- X secreted into the supernatant was measured by ELISA after co-culture with CAR- T cells and MDA-MB-231 cells at a ratio of 1:5 for 24 hours. In addition, the secretion amount of IL12 was also confirmed by ELISA.

[0061] For the #12 parental clone, the expression of CAR and CTC28 in humanized #12 C12 B7H3 CAR- T cells and non-humanized #12 C12 B7H3 CAR- T cells using different humanized variant scFv> was confirmed by flow cytometry using B7H3 recombinant protein and CTLA4 antibody, respectively, and the results of comparing the in vitro tumor cell killing capacity and cytokine secretion capacity of these CAR- T cells are shown in Fig. 43. According to Figure 43, z32, z35, z36 B7H3 C12 CAR-T cells and #12 B7H3 C12 CAR-T cells, which showed similar antigen binding affinity of about 50%, showed similar cell killing ability, and the secretion amount of IFN-αIL12 of z32, z35, z36 B7H3 C12 CAR-T cells was slightly increased compared to non-humanized #12 B7H3 C12 CAR-T cells.

[0062] For the #2-25 parent clone (abbreviated as #25), the results of comparative analysis of humanized #25 B7H3 C12 CAR- T cells with non-humanized #25 B7H3 C12 CAR- T cells as mentioned above are shown in Fig. 44. According to Fig. 44, zl8, z22, z25 B7H3 C12 CAR- T and #25 B7H3 C12 CAR- T, which showed similar antigen binding affinity of about 50%, showed similar cell killing ability, and the secretion of IFN-γ and IL12 of z22, z25 B7H3 C12 CAR- T cells was slightly increased compared to non-humanized #25 C12 B7H3 CAR- T cells.

[0063] For the #2-54 parent clone (abbreviated as #54), the results of comparative analysis of humanized #54 B7H3 C12 CAR- T cells and non-humanized #54 B7H3 C12 CAR- T cells as mentioned above are shown in Fig. 45. According to Fig. 45, zl6 B7H3 C12 CAR- T and non-humanized #54 C12 B7H3 CAR- T, which showed similar antigen binding affinity of about 50%, showed similar cell killing ability, and the amount of IFN-γ and IL12 secreted by zl6 B7H3 C12 CAR- T cells was similar to that of non-humanized #54 B7H3 C12 CAR- T cells. Subsequently, zl6 B7H3 C12 CAR— T cells and z3, z4, zl4, zl5 B7H3 C12 B7H3 CAR— T cells showed similar cell killing ability, and the secretion amounts of IFN- x and IL12 were also similar to each other. Therefore, the in vitro efficacy of CAR— T cells of humanized B7H3 scFvs z32, z25, zl6, as well as their humanized mutant CAR- T cells, was similar to or not inferior to that of CAR- T cells of non-humanized B7H3 scFvs (#12, #2-25, #2-54), and was confirmed to be superior to them all. As described above, in the present invention, novel anti-B7-H3 antibodies having a similar affinity for human B7-H3 to mouse B7-H3 were developed, and it was confirmed that they had excellent in vivo anti-tumor effects in mouse CAR-T cells while having low cytotoxicity. In addition, human CAR-T cells having the same antibody as the anti-B7-H3 antibody used in mouse CAR-T cells were produced, and it was confirmed that they exhibited effective in vitro anti-tumor effects. In addition, humanized anti-B7-H3 antibodies were developed, and human CAR-T cells were produced using the same, and it was confirmed that they exhibited effective in vitro anti-tumor effects.Furthermore, the inventors of the present invention confirmed that B7-H3 target CAR-T cells, which are loaded with a functional receptor called CTLA4-CD28 chimera and IL-12, developed previously, can exhibit a strong anti-tumor effect with relatively low toxicity.

[0064] [Table 8] Sequence of chicken-derived anti-B7-H3 antibodies

[0065] * In the above table, the underline indicates CDR.

[0066] [Table 9] Sequence of human B7H3 CAR

[0067] * h, human; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain

[0068] * In the CAR sequences in the table above, the hCD8 EC sequence is indicated in bold, and the h41BB cyt sequence is indicated in italics.

[0069] [Table 10] Sequence of human CTLA4-CD28 chimera

[0070] * h, human; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain

[0071] [Table 11] Sequence of human single-chain IL-12 (IL-12f)

[0072] [Table 12] Sequence of humanized anti-B7-H3 antibody

[0073] [Table 13] Sequence of humanized B7H3 CAR domain

[0074] * In the CAR sequences in the table above, the hCD8 EC sequence is indicated in bold, and the h41BB cyt sequence is indicated in italics.

Claims

Scope of the claim

1. A heavy chain variable region (VH) comprising a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 13, 22, 31, 40, and 49, a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 14, 23, 32, 41, 50, and 135, and a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 24, 33, 42, 51, and 136; and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 16, 34, 43, and 52, a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 17, 26, 35, 44, and 53, and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 18, 27, An antibody or antigen-binding fragment thereof that specifically binds to B7-H3, comprising a light chain variable region (VL) comprising a light chain CDR3 comprising an amino acid sequence selected from the group consisting of 36, 45 and 54. [ The claim 1, wherein (i) the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 33; A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51; or A heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 134, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 135, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 136; (ii) a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 36; An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 45; or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

54. [

3. The antibody of claim 1, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 9; or a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 18; A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 24; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 33; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 36; A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 45; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 54;Or an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 136; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 138, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 139.; [

4. In claim 1, ( i ) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 11, 20, 29, 38, 47, 105, 114, 123, 132, 141, 150, 159, 168, 177, 186 and 195, or an amino acid sequence having at least 90% identity thereto; and / or ( ii ) Sequence numbers 3, 12, 21, 30, 39, 48, 106, 115, 124, 133, 142, 151, 160, 169, An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of 178, 187 and 196, or an amino acid sequence having at least 90% identity thereto. [

5. The antibody according to claim 4, comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 3; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 12; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 20; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 21; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 30; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 39; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 47; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 48; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 105; and SEQ ID NO: A light chain variable region comprising an amino acid sequence of SEQ ID NO: 106; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 114; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 115; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 123; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 124; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 132; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 133; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141; and A light chain variable region comprising an amino acid sequence of SEQ ID NO: 142; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 150; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 151; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 159; and A light chain variable region comprising an amino acid sequence of SEQ ID NO: 160; or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 168; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 169; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 177; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 178; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 186; and An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 187; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 195; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

196. ［ Claim 1: An antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 10, 19, 28, 37, 46, 104, 113, 122, 131, 140, 149, 158, 167, 176, 185 and 194, or an amino acid sequence having at least 90% identity thereto. ［

7. An antibody or antigen-binding fragment thereof, according to claim 1, which is a full-length antibody, a single-chain variable fragment; scFv, (scFv)2, Fv, Fab, F(ab'), F(ab')2, a nanobody, or a combination thereof. ［ Claim 8] A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid of claim 8.

10. A host cell comprising the expression vector of claim 9. ［ Claim 11] (a) an anti-B7-H3 antigen binding domain comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 7; (b) extracellular domain, (c) a transmembrane domain, and (d) an intracellular domain, a chimeric antigen receptor (CAR) specific for B7-H3. ［

12. The chimeric antigen receptor according to claim 11, wherein the extracellular domain is an extracellular domain derived from IgGl, IgG2, IgG4, IgD, CD8 or CD28. ［

13. The chimeric antigen receptor according to claim 12, wherein the extracellular domain is an extracellular domain derived from CD8 or comprises the amino acid sequence of SEQ ID NO:

58. ［

14. The chimeric antigen receptor according to claim 11, wherein the transmembrane domain is a T cell receptor (TCR) alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta (ζ), CD3 epsilon (ε), CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS, and a transmembrane domain derived from a protein selected from the group consisting of CD154, or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

15. The chimeric antigen receptor according to claim 14, wherein the transmembrane domain is a transmembrane domain derived from CD8 or comprises the amino acid sequence of SEQ ID NO:

58.

16. The chimeric antigen receptor according to claim 11, wherein the intracellular domain comprises an intracellular signaling domain and / or a costimulatory domain.

17. The chimeric antigen receptor according to claim 16, wherein the intracellular domain is an intracellular signaling domain selected from the group consisting of CD3 zeta (ζ), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b and CD66d; and / or A chimeric antigen receptor comprising a co-stimulatory domain selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4—1BB (CD137), OX40 (CD134), ICOS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C and B7—H3.

18. A chimeric antigen receptor according to claim 17, wherein the intracellular signaling domain is a CD3 zeta intracellular signaling domain or a CD3 zeta (&) intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:

61.

19. A chimeric antigen receptor according to claim 17, wherein the costimulatory domain is a 4-1BB costimulatory domain or a 4-1BB costimulatory domain comprising the amino acid sequence of SEQ ID NO:

60.

20. A chimeric antigen receptor according to claim 11, further comprising a signal peptide at the N-terminus of the antigen binding domain.

21. A chimeric antigen receptor according to claim 20, wherein the signal peptide is a CD8a signal peptide or a CD8a signal peptide comprising an amino acid sequence of SEQ ID NO:

55.

22. In claim 11, the chimeric antigen receptor comprises an anti-B7-H3 antigen binding domain comprising an anti-B7-H3 scFv, a CD8-derived extracellular domain, a CD8-derived transmembrane domain, A chimeric antigen receptor comprising a 4-1BB costimulatory domain and a CD3 & intracellular signaling domain.

23. A chimeric antigen receptor according to claim 22, wherein the chimeric antigen receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 63, 70, 77, 84, 91, 204, 211, and 218, or an amino acid sequence having at least 90% identity thereto.

24. A nucleic acid encoding the anti-B7-H3 chimeric antigen receptor of claim 11.

25. An expression vector comprising the nucleic acid of claim 24.

26. A host cell comprising the expression vector of claim 25.

27. ​​An immune cell expressing the chimeric antigen receptor of claim 11 on its surface. [

28. The immune cell of claim 27, wherein the immune cell is a T cell, NK cell, NKT cell or macrophage.

29. A pharmaceutical composition for treating cancer, comprising the immune cell of claim 28.

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