Humanized antibody binding to Eva1 protein or functional fragment thereof, antibody drug conjugate and chimeric antigen receptor

Abstract

A humanized antibody or a functional fragment thereof that binds to a human-derived Eva1 protein, an antibody drug conjugate comprising the humanized antibody or the functional fragment thereof, or a chimeric antigen receptor comprising an antibody or a functional fragment thereof that binds to a human-derived Eva1 protein.

Description

Technical Field

[0001] The present invention relates to a humanized antibody or a functional fragment thereof that binds to human-derived Eva1 protein (human Eva1). The present invention also relates to an antibody-drug conjugate comprising the humanized antibody or a functional fragment thereof. Furthermore, the present invention relates to a chimeric antigen receptor comprising an antibody or a functional fragment thereof that binds to human Eva1. Background Art

[0002] Cancer is the main cause of death in developed countries together with coronary artery disease, and its ratio has a trend of increasing year by year. Therefore, it is hoped that a radical treatment for cancer will be developed rapidly. In the development of radical treatment for cancer, the existence of cancer stem cells has been taken seriously in recent years. Although cancer stem cells are only present in a very small part of the tissue of cancer, it is believed that they become the origin of creating most cancer cells by repeated self-replication and further differentiation. In addition, it is shown that while having such a strong tumor formation ability, cancer stem cells have a strong tolerance to chemotherapy and radiotherapy. Therefore, even if most of the cancer cells in the cancerous tissue can die by chemotherapy and radiotherapy, cancer stem cells remain, thereby causing recurrence, metastasis, etc. of cancer. However, if cancer stem cells can be used as a target to make them die, it can be expected to achieve the development of a useful treatment method for preventing the metastasis, recurrence, etc. of cancer.

[0003] In view of this situation, the present inventors have successfully identified the Eva1 protein as a protein highly expressed in gliomas and their cancer stem cells. Furthermore, it has been found that the expression of the Eva1 protein is correlated with the malignancy of gliomas, and there is a strong correlation between the survival rate of glioma patients and the expression of the Eva1 gene in the gliomas derived from the patients. In addition, it has been found that the inhibition of the function of the Eva1 gene is effective in inhibiting the proliferation ability, tumor formation ability and tissue infiltration ability of glioma cells, and the tumor mass formation ability of glioma stem cells (Patent Document 1).

[0004] In addition, in cancer, the treatment of glioma has remained basically unchanged for decades, with surgery as the basis and radiotherapy and chemotherapy as auxiliary therapy, especially for the most malignant glioblastoma multiforme (GBM, glioblastoma multiforme) in the malignant glioma in the central nervous system tissue, no effective treatment has been found. Furthermore, temozolomide is used as a standard treatment for malignant glioma, but the inventors have confirmed that glioma stem cells are non-sensitive even to temozolomide at several times the effective blood concentration. In addition, cancer cells exposed to such chemotherapeutic agents are not limited to gliomas, and most of them produce resistance to chemotherapeutic agents, and cross-resistance is also produced to other various chemotherapeutic agents. Furthermore, chemotherapeutic agents mostly also cause cell damage to normal cells. In order to reduce such side effects, the dosage or usage of chemotherapeutic agents is restricted in most cases.

[0005] Based on this situation, the use of antibodies as anticancer agents has attracted attention in recent years, and their importance has gradually been recognized. For example, if the antibody targets cancer-specific antigens, it is presumed that the administered antibody accumulates in cancer tissues, and thus it can be expected to attack cancer cells via the immune system using antibody-dependent cellular cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) activity.

[0006] In addition, by pre-binding cytotoxic substances, radionuclides and other drugs to antibodies (using the so-called antibody-drug conjugate (ADC) method), the bound drugs can be efficiently delivered to the tumor site. This reduces the amount of drugs delivered to other tissues, and it is expected that side effects will be reduced.

[0007] In addition, when the cancer-specific antigen has the activity of inducing cell death, an antibody with antagonistic activity is administered. In addition, when the cancer-specific antigen is involved in the proliferation and survival of cells, an antibody with neutralizing activity is administered. It is thereby possible to utilize the aggregation of tumor-specific antibodies and the activity of the antibodies, and to expect the proliferation of cancer to be stopped or regressed.

[0008] Furthermore, by administering T cells (CAR-T) expressing a chimeric antigen receptor (CAR) integrated with an antibody variable region, it is also expected that these cells can activate target antigen-positive cancer cells in an HLA-independent manner and induce cell death.

[0009] Considering that antibodies are suitable for use as anticancer agents based on such properties, the present inventors prepared high-affinity anti-human Eva1 mouse antibodies with the aim of creating antibody drugs targeting Eva1, and demonstrated their effectiveness (Patent Document 2).

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: International Publication No. 2012 / 043747

[0013] Patent Document 2: International Publication No. 2017 / 022668 Summary of the invention

[0014] Problems to be solved by the invention

[0015] The present invention aims to provide an antibody or a functional fragment thereof useful for cancer treatment or the like, or a conjugate comprising the antibody or a functional fragment thereof.

[0016] Means for solving problems

[0017] The present inventors attempted to humanize an anti-human Eva1 mouse antibody (B2E5-48 mouse antibody described in Patent Document 2) and combined four humanized heavy chain variable regions (H1 to H4) and four light chain variable regions (L1 to L4) to produce 16 humanized antibodies. As a result, they succeeded in obtaining humanized antibodies (combinations such as H2-L2, H2-L4, and H3-L4) having high binding affinity to the Eva1 protein and high production capacity (antibody titer).

[0018] In addition, a chimeric antigen receptor (CAR) incorporating the variable region of an anti-human Eva1 antibody was produced and its effectiveness was evaluated. As a result, T cells (CAR-T) expressing this CAR showed activation (cytokine production) and cell proliferation in response to human Eva1. Furthermore, it was found that it showed cytotoxic activity against tumor cells expressing the Eva1 protein.

[0019] In addition, in the CAR incorporating the variable region of the B2E5-48 mouse antibody, the length of the spacer between the antigen-binding region and the transmembrane region of the variable region was changed and evaluated, and it was found that when T cells expressing the CAR were cultured in the absence of IL-2, the CAR-T with a spacer length of 232 amino acids was the most excellent in cell proliferation and cytokine production. On the other hand, when cultured in the presence of IL-2, the cell proliferation of the CAR-T with a spacer consisting of 15 amino acids was the best.

[0020] Furthermore, it was found that in the CAR incorporating the variable region of the above-mentioned humanized antibody, the combination of L2-H2 is most useful from the perspective of responding to the activation of human Eva1 and cell proliferation. Furthermore, various intracellular domains were tried for CAR-T with an antigen recognition site of L2-H2, and the differences in reactivity caused by their differences were evaluated. As a result, it was found that CARs with intracellular domains of 4-1BB and CD79A / CD40 are excellent in cell proliferation. In addition, the CAR-T (L2-H2 hEva1-CAR-T) was administered to a cancer-bearing model, and its anti-tumor activity and safety were confirmed.

[0021] In addition, an antibody drug conjugate (ADC) containing the above humanized antibody was prepared and its effectiveness was evaluated. The results confirmed the anti-tumor activity of the ADC against various tumors such as glioma, pancreatic cancer, and gastric cancer. On the other hand, no adverse effects (reduction in body weight, shortened survival time, etc.) on the administered organisms were found.

[0022] The present invention is based on the above-mentioned research results, and more specifically provides the following solutions.

[0023] <1> A humanized antibody or a functional fragment thereof that binds to a human-derived Eva1 protein, comprising a light chain variable region and a heavy chain variable region,

[0024] The light chain variable region comprises: complementary determining regions 1 to 3 respectively comprising the amino acid sequence recorded in sequence number 1, the amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence recorded in sequence number 3,

[0025] The heavy chain variable region comprises complementarity determining regions 1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

[0026] <2> <1> The humanized antibody or its functional fragment,

[0027] The light chain variable region comprises: an amino acid sequence selected from the group consisting of an amino acid sequence set forth in SEQ ID NOs: 7 to 10, an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NOs: 7 to 10, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 10,

[0028] The heavy chain variable region comprises: an amino acid sequence selected from the amino acid sequences recorded in sequence numbers 13 to 16, an amino acid sequence having more than 80% homology with the amino acid sequences recorded in sequence numbers 13 to 16, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or several amino acids in at least any one of the amino acid sequences recorded in sequence numbers 13 to 16.

[0029] <3> The humanized antibody or a functional fragment thereof according to <1>,

[0030] The light chain variable region comprises: an amino acid sequence selected from the group consisting of an amino acid sequence recorded in SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence recorded in SEQ ID NO: 7, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or more amino acids in at least any one of the amino acid sequences recorded in SEQ ID NO: 7,

[0031] The heavy chain variable region comprises: an amino acid sequence selected from the amino acid sequence recorded in sequence number 13, an amino acid sequence having more than 80% homology with the amino acid sequence recorded in sequence number 13, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or several amino acids in at least any one of the amino acid sequences recorded in sequence number 13.

[0032] <4> An antibody-drug conjugate comprising the antibody or a functional fragment thereof according to any one of <1> to <3> and a drug bound thereto.

[0033] <5> A pharmaceutical composition comprising the antibody-drug conjugate described in <4>.

[0034] <6> A chimeric antigen receptor comprising a region that binds to a human-derived Eva1 protein, a transmembrane region, and an intracellular signaling region.

[0035] <7> The chimeric antigen receptor according to <6>, wherein the region that binds to the human-derived Eva1 protein comprises a region of the antibody or a functional fragment thereof according to any one of <1> to <3>.

[0036] <8> The chimeric antigen receptor according to <6>, wherein the region that binds to the human-derived Eva1 protein comprises a single-chain variable region fragment formed by combining a light chain variable region, a linker, and a heavy chain variable region in order from the amino terminal side, wherein the light chain variable region

[0037] comprising: complementary determining regions 1 to 3 respectively comprising the amino acid sequence recorded in SEQ ID NO: 1, the amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence recorded in SEQ ID NO: 3, and

[0038] An amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 7, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 7,

[0039] The heavy chain variable region

[0040] comprising: complementary determining regions 1 to 3 of the amino acid sequences of SEQ ID NOs: 4 to 6, respectively, and

[0041] An amino acid sequence comprising an amino acid sequence selected from the group consisting of an amino acid sequence recorded in sequence number 13, an amino acid sequence having more than 80% homology to the amino acid sequence recorded in sequence number 13, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or several amino acids in at least any one of the amino acid sequences recorded in sequence number 13.

[0042] <9> The chimeric antigen receptor according to any one of <6> to <8>, wherein the region binding to the human-derived Eva1 protein and the transmembrane region are bound via a spacer consisting of 10 to 300 amino acids.

[0043] <10> The chimeric antigen receptor according to any one of <6> to <9>, wherein the intracellular signaling region comprises a CD79A intracellular region and a CD40 intracellular region.

[0044] <11> A nucleotide encoding the chimeric antigen receptor according to any one of <6> to <10>.

[0045] <12> A vector comprising the nucleotide described in <11>.

[0046] <13> A cell expressing the chimeric antigen receptor according to any one of <6> to <10>.

[0047] <14> The cell according to <13>, which is an immune cell.

[0048] <15> A pharmaceutical composition comprising the cell described in <13> or <14>.

[0049] In addition, the sequence recorded in each serial number is as follows.

[0050] Table 1

[0051] Serial Number The name of the sequence 1 CDR1 of light chain (humanized B2E5-48) 2 CDR2 of light chain (humanized B2E5-48) 3 CDR3 of light chain (humanized B2E5-48) 4 CDR1 of heavy chain (humanized B2E5-48) 5 CDR2 of the heavy chain (humanized B2E5-48) 6 CDR3 of the heavy chain (humanized B2E5-48) 7 Variable region of light chain 2 (humanized B2E5-48) 8 Variable region of light chain 4 (humanized B2E5-48) 9 Variable region of light chain 1 (humanized B2E5-48) 10 Variable region of light chain 3 (humanized B2E5-48) 11 Variable region of light chain 2 (humanized B2E5-48) cDNA 12 Variable region of light chain 4 (humanized B2E5-48) cDNA 13 Variable region of heavy chain 2 (humanized B2E5-48) 14 Variable region of heavy chain 4 (humanized B2E5-48) 15 Variable region of heavy chain 1 (humanized B2E5-48) 16 Variable region of heavy chain 3 (humanized B2E5-48) 17 Heavy chain 2 variable region (humanized B2E5-48) cDNA 18 Heavy chain 3 variable region (humanized B2E5-48) cDNA

[0052] Table 2

[0053] Serial Number The name of the sequence 19 Signal peptide and light chain variable region (B2E5-48) cDNA 20 Signal peptide and variable region of light chain (B2E5-48) 21 Light chain variable region (B2E5-48) 22 CDR1 of light chain (B2E5-48) 23 CDR2 of light chain (B2E5-48) 24 CDR3 of light chain (B2E5-48) 25 Signal peptide and heavy chain variable region (B2E5-48) cDNA 26 Signal peptide and variable region of heavy chain (B2E5-48) 27 Heavy chain variable region (B2E5-48) 28 CDR1 of heavy chain (B2E5-48) 29 CDR2 of heavy chain (B2E5-48) 30 CDR3 of heavy chain (B2E5-48) 31 Signal peptide and light chain variable region (C3) cDNA 32 Signal peptide and variable region of light chain (C3) 33 Variable region of light chain (C3) 34 CDR1 (C3) of the light chain 35 CDR2 (C3) of the light chain 36 CDR3 of the light chain (C3) 37 Signal peptide and heavy chain variable region (C3) cDNA 38 Signal peptide and variable region of heavy chain (C3) 39 Heavy chain variable region (C3) 40 CDR1 (C3) of the heavy chain 41 CDR2 (C3) of the heavy chain 42 CDR3 of the heavy chain (C3) 43 Light chain variable region (A5D11-10) cDNA 44 Variable region of light chain (A5D11-10) 45 CDR1 of light chain (A5D11-10) 46 CDR2 of light chain (A5D11-10) 47 CDR3 of light chain (A5D11-10) 48 Heavy chain variable region (A5D11-10) cDNA 49 Heavy chain variable region (A5D11-10) 50 CDR1 of heavy chain (A5D11-10) 51 CDR2 of heavy chain (A5D11-10) 52 CDR3 of heavy chain (A5D11-10) 53 hEva1 cDNA 54 hEva1 55 mEva1 cDNA 56 mEva1

[0054] Table 3

[0055] Serial Number The name of the sequence 57 B2E5-48 CAR cDNA 58 B2E5-48CAR 59 Hinge region: 15aa 60 Hinge area: 30aa 61 Hinge area: 60aa 62 Hinge area: 125aa 63 Hinge area: 232aa 64 4-1BB IC 65 CD79A IC 66 CD40 IC

[0056] In addition, as shown in Table 1, sequence number 2 refers to the amino acid sequence of CDR2 (humanized B2E5-48) of the light chain (an amino acid sequence consisting of valine-threonine-serine in order from the N-terminus) (see also Table 7). However, this sequence is a sequence of less than 4 (3) amino acids, and is therefore treated as a "sequence that was actively skipped" in the sequence list.

[0057] Effects of the Invention

[0058] According to the present invention, cancer and the like can be treated or prevented by targeting human Eva1. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a histogram showing the results of expression analysis of human Eva1 in normal human blood cells.

[0060] Figure 2A It is a histogram showing the results of expression analysis of human Eva1 in various tumor cells.

[0061] Figure 2B It is a histogram showing the results of expression analysis of human Eva1 in intrahepatic cholangiocarcinoma cases (samples 1 to 3).

[0062] Figure 3A This is a diagram showing the outline of a chimeric antigen receptor (Eva1-CAR) using an anti-Eva1 antibody sequence.

[0063] Figure 3B This is a diagram showing an overview of the production schedule of Eva1-CAR-expressing T cells (Eva1-CAR-T).

[0064] Figure 3C It is a dot graph showing the analysis results of the Eva1-CAR gene introduction efficiency before screening (tEGFR selection) using tEGFR expression as an indicator.

[0065] Figure 3D It is a dot plot showing the analysis results of the introduction efficiency of the Eva1-CAR gene before tEGFR selection. In the figure, the dots represent the source of the cells into which the Eva1-CAR gene was introduced (5 healthy people). The bold line represents the mean value, and the whiskers represent the standard error.

[0066] Figure 3E This is a graph showing the analysis results of Eva1-CAR-T on the production of cytokines (IFN-γ) in various tumor cells. In the graph, the horizontal axis shows the results of each introduced Eva1-CAR on CFPAC-1, NCI-H1975, HuCCT-1, MCF7, HepG2, Huh7, OVTOKO, T98G, and PA1 from the left.

[0067] Figure 3F : is a diagram showing the outline of 6 types of spacers with different lengths in Eva1-CAR. Figure 3F The numbers "H00" to "H232" shown in Figure 3G~3M in which they correspond.

[0068] Figure 3G It is a dot plot showing the analysis results of the introduction efficiency of the Eva1-CAR gene before tEGFR selection. In the figure, the dots represent the source of the cells into which the Eva1-CAR gene was introduced (5 healthy people). The bold line represents the mean value, and the whiskers represent the standard error.

[0069] Figure 3H This is a dot plot showing the analysis results of the introduction efficiency of the Eva1-CAR gene after tEGFR selection. In the figure, the dots represent the source of the cells into which the Eva1-CAR gene was introduced (5 healthy people). The bold line represents the mean value, and the whiskers represent the standard error.

[0070] Fig. 3I This is a graph showing the analysis results of cytokine production by Eva1-CAR-T on various tumor cells. In the figure, the left graph shows the analysis results of IFN-γ production, and the right graph shows the analysis results of IL-2 production. The results of each introduced Eva1-CAR on HuCCT-1, CFPAC-1, HepG2, and OVTOKO are shown in order from the left.

[0071] Figure 3J This is a graph showing the expansion efficiency of Eva1-CAR-T after antigen stimulation in the absence of IL-2. In the figure, the horizontal axis shows the number of days after the start of antigen stimulation (co-culture with Eva1-positive HuCCT-1). The vertical axis shows the number of Eva1-CAR-T cells.

[0072] Figure 3K This is a graph showing the expansion efficiency of Eva1-CAR-T after antigen stimulation in the absence of IL-2. In the graph, the horizontal axis shows the type of CAR introduced. The vertical axis shows the number of Eva1-CAR-T cells.

[0073] Figure 3L This is a graph showing the expansion efficiency of Eva1-CAR-T after antigen stimulation in the presence of IL-2. In the figure, the horizontal axis shows the number of days after the start of antigen stimulation (co-culture with Eva1-positive HuCCT-1). The vertical axis shows the number of Eva1-CAR-T cells.

[0074] Figure 3MThis is a graph showing the expansion efficiency of Eva1-CAR-T after antigen stimulation in the presence of IL-2. In the graph, the horizontal axis shows the type of introduced CAR. The vertical axis shows the number of Eva1-CAR-T cells.

[0075] Figure 4A It is a dot plot showing the analysis results of the introduction efficiency of the Eva1-CAR (hEva1-CAR) gene using the anti-Eva1 humanized antibody sequence before tEGFR selection. In the figure, the dots represent the source of the cells into which the hEva1-CAR gene was introduced (4 healthy people). The whiskers represent the standard error, and the line in the middle of the whiskers represents the average value.

[0076] Figure 4B This is a dot plot showing the analysis results of the amplification efficiency of hEva1-CAR-T after tEGFR selection. In the figure, the dots represent the source of cells into which the hEva1-CAR gene was introduced (4 healthy subjects). The whiskers represent the standard error, and the line in the middle of the whiskers represents the mean value.

[0077] Figure 4C This is a graph showing the results of analyzing IFN-γ production by hEva1-CAR-T on various tumor cells. The results for HuCCT-1, CFPAC-1, HepG2, and OVTOKO are shown in order from the left for each introduced Eva1-CAR.

[0078] Figure 4D This is a graph showing the results of analyzing IL-2 production by hEva1-CAR-T on various tumor cells. The results for HuCCT-1, CFPAC-1, HepG2, and OVTOKO are shown in order from the left for each introduced Eva1-CAR.

[0079] Figure 4E It is a graph showing the amplification efficiency of hEva1-CAR-T after antigen stimulation. In the figure, the left side shows the results in the absence of IL-2, and the right side shows the results in the presence of IL-2. In each figure, the horizontal axis represents the number of days after the start of antigen stimulation (co-culture with Eva1-positive HuCCT-1). The vertical axis represents the number of hEva1-CAR-T cells.

[0080] Figure 4F This is a diagram showing the outlines of six types of hEva1-CAR-T (antigen recognition site: L2H2) having different intracellular domains and spacer lengths.

[0081] Figure 4GIt is a dot graph showing the analysis results of the efficiency of hEva1-CAR gene introduction before tEGFR selection. In the figure, the dots represent the sources of cells into which the hEva1-CAR gene has been introduced (5 healthy subjects). The middle line represents the average value. "28", "41" and "79" in the horizontal axis indicate that the intracellular domains of each CAR are "CD28IC", "4-1BBIC" and "CD79A / CD40IC", respectively. In addition, "-S" and "-L" respectively indicate that the spacer is Figure 4F "H15" and "H232" shown.

[0082] Figure 4H This is a graph showing the analysis results of hEva1-CAR-T on IFN-γ production of various tumor cells. The results of each imported Eva1-CAR on OVTOKO, CFPAC-1, HepG2, HuCCT, and MCF7 are shown in order from left to right. "28", "41", and "79" in the horizontal axis indicate that the intracellular domains of each CAR are "CD28IC", "4-1BBIC", and "CD79A / CD40IC", respectively. In addition, "-S" and "-L" respectively indicate that the spacer is Figure 4F "H15" and "H232" shown.

[0083] Fig. 4I This is a graph showing the analysis results of hEva1-CAR-T on IL-2 production of various tumor cells. The results of each imported Eva1-CAR on OVTOKO, CFPAC-1, HepG2, HuCCT, and MCF7 are shown in order from left to right. "28", "41", and "79" in the horizontal axis indicate that the intracellular domains of each CAR are "CD28IC", "4-1BBIC", and "CD79A / CD40IC", respectively. In addition, "-S" and "-L" respectively indicate that the spacer is Figure 4F "H15" and "H232" shown.

[0084] Figure 4J It is a graph showing the expansion efficiency of hEva1-CAR-T after antigen stimulation. In the figure, the graph on the left shows the results in the absence of IL-2, and the graph on the right shows the results in the presence of IL-2. In each figure, the horizontal axis shows the number of days after the start of antigen stimulation (co-culture with Eva1-positive HuCCT-1). The vertical axis shows the number of hEva1-CAR-T cells. In addition, "28", "41" and "79" in the explanation indicate that the intracellular domains of each CAR are "CD28IC", "4-1BBIC" and "CD79A / CD40IC", respectively. In addition, "-S" and "-L" respectively indicate that the spacer is Figure 4F"H15" and "H232" shown.

[0085] Figure 4K It is a graph showing the results of the co-culture experiment of hEva1-CAR-T and Eva1-positive myeloma cells (RPMI-8226). In the figure, the figure on the left shows the results of hEva1-CAR-T made from the peripheral blood of donor 2, and the figure on the right shows the results of hEva1-CAR-T made from the peripheral blood of donor 3. The vertical axis of each figure shows the proportion of tumor cells in all cells. The horizontal axis shows the time after the start of co-culture. In addition, "28", "41" and "79" in the explanation indicate that the intracellular domains of each CAR are "CD28IC", "4-1BBIC" and "CD79A / CD40IC", respectively. In addition, "s" and "L" respectively indicate that the spacer is Figure 4F "H15" and "H232" shown.

[0086] Figure 4L It is shown that hEva1-CAR-T and 51 The results of co-culture of various tumor cell lines labeled with Cr and analysis of cytotoxic activity are shown in the figure. 51 The ratio of damaged cells to the amount of Cr released. The horizontal axis combines various tumor cell lines with whether the expression of Eva1 is negative or positive. In addition, the "41" and "79" in the legend indicate that the intracellular domains of each CAR are "4-1BBIC" and "CD79A / CD40IC", respectively. In addition, "S" and "L" indicate that the spacer is Figure 4F "H15" and "H232" shown.

[0087] Figure 4M Is displayed based on Figure 4L The results shown in the figure are the results of analyzing the correlation between the expression level of human Eva1 in various tumor cell lines and the cytotoxic activity of hEva1-CAR-T against various tumor cell lines. In the figure, the vertical axis shows the expression level of human Eva1 in the culture supernatant. 51 The ratio of damaged cells to the amount of Cr released. The horizontal axis shows the mean fluorescence intensity (MFI) obtained by flow cytometry analysis using anti-human Eva1 antibodies in various tumor cell lines, which is related to the expression level of Eva1. In addition, "41" and "79" in the legend indicate that the intracellular domains of each CAR are "4-1BBIC" and "CD79A / CD40IC", respectively. In addition, "S" and "L" respectively indicate that the spacer is Figure 4F "H15" and "H232" shown.

[0088] Figure 4NThis is a diagram showing the results of analyzing the anti-tumor activity of L2H2hEva1-CAR-T made from peripheral blood from two different donors (donor A and donor B) by bioluminescent imaging after introducing the L2H2hEva1-CAR-T into mice carrying a human lung adenocarcinoma cell line (H1975) that has been introduced with the firefly luciferase gene. As L2H2hEva1-CAR, a short spacer (indicated by "short hinge" in the figure) is used to separate the antigen recognition site (L2H2) and the transmembrane / intracellular domain (containing the domain of 4-1BB or CD79A / CD40). Figure 4F As a control, an administration example of tEGFR-introduced cells (simulated CAR-T cells into which only the tEGFR gene was introduced) was also prepared and analyzed. In addition, the "Day" in the figure indicates the number of days after the administration of CAR-T. In addition, there were two fewer cells in the "Day 14" of the administration example of L2H2 hEva1-CAR-T containing 4-1BB in the transmembrane / intracellular domain, because they died due to problems with the experimental technology. In addition, the emission spectrum is Figure 4P and 4R It is also applied in.

[0089] Fig.4O This is a graph showing the results of introducing L2H2hEva1-CAR-T made from peripheral blood from two different donors (donor A and donor B) into mice carrying a human pancreatic cancer cell line (CFPAC1) that has been introduced with the firefly luciferase gene, and analyzing the anti-tumor activity of the CAR-T by bioluminescence imaging. As L2H2hEva1-CAR, a cell in which the antigen recognition site (L2H2) and the transmembrane / intracellular domain (containing the domain of 4-1BB or CD79A / CD40) are connected by a short hinge is used. As a control, an example of tEGFR-introduced cell administration was also prepared and analyzed. In addition, "Day" in the figure indicates the number of days after the administration of CAR-T. The emission spectrum is Figure 4P and 4R It is also applied in.

[0090] Figure 4PThis is a graph showing the results of analyzing the anti-tumor activity of L2H2hEva1-CAR-T made from peripheral blood from two different donors (donor A and donor B) by bioluminescent imaging after introducing it into mice carrying a human pancreatic cancer cell line (CFPAC1) or a human lung adenocarcinoma cell line (H1975) that has been introduced with the firefly luciferase gene. As L2H2 hEva1-CAR, a cell in which the antigen recognition site (L2H2) and the transmembrane / intracellular domain (containing the domain of 4-1BB or CD79A / CD40) are connected by a short hinge is used. As a control, an example of administration of tEGFR-introduced cells was also prepared and analyzed. In addition, "Day" in the figure indicates the number of days after the administration of CAR-T. The emission spectrum is Figure 4P and 4R It is also applied in.

[0091] Figure 4Q It is a graph showing that L2H2hEva1-CAR-T made from peripheral blood from 2 different donors (donor A, donor B) is introduced into a mouse carrying a human pancreatic cancer cell line (CFPAC1) or a human lung adenocarcinoma cell line (H1975) that has been introduced with a firefly luciferase gene, and the anti-tumor activity of the CAR-T is analyzed over time by bioluminescence imaging and the results (luminescence intensity) are obtained. As L2H2 hEva1-CAR, an antigen recognition site (L2H2) and a transmembrane / intracellular domain (containing a domain of 4-1BB or CD79A / CD40) are connected with a short hinge. As a control, an example of tEGFR introduction into cells was also made and analyzed.

[0092] Figure 4R It is a figure showing the results of analyzing the anti-tumor activity of the CAR-T by bioluminescent imaging. As L2H2 hEva1-CAR, the antigen recognition site (L2H2) and the transmembrane / intracellular domain (containing the domain of 4-1BB or CD79A / CD40) were connected with a short hinge. As a control, an example of tEGFR introduction into cells was also made and analyzed. In addition, "Day" in the figure indicates the number of days after CAR-T is administered.

[0093] Figure 4SIt is a figure showing the results of the analysis of L2H2 hEva1-CAR-T made from peripheral blood derived from donor C and introduced into a human cholangiocarcinoma cell line (HuCCT cell) carrying a firefly luciferase gene. As L2H2 hEva1-CAR, a short hinge was used to connect the antigen recognition site (L2H2) and the transmembrane / intracellular domain (containing the domain of 4-1BB or CD79A / CD40). As a control, an example of tEGFR introduction into cells was also made and analyzed. In addition, in the figure, the figure on the left shows the results (luminescence intensity) obtained by analyzing the anti-tumor activity of the CAR-T over time by bioluminescence imaging. The figure on the right shows the results of analyzing the tumor volume in the cancer-bearing mice to which the CAR-T was administered over time.

[0094] Figure 5 This is a diagram showing an overview of the production process of an antibody-drug conjugate (B2E5-ADC) comprising an anti-human Eva1 humanized antibody.

[0095] Fig. 6A The graph shows the results of the cytotoxicity experiment of B2E5-ADC against glioma (hGIC E6, hGIC E16).

[0096] In the figures, the vertical axis of each graph shows the survival rate of tumor cells, and the horizontal axis shows the concentration of ADC or antibody added to the culture medium.

[0097] Figure 6B This is a graph showing the results of a cytotoxicity experiment of B2E5-ADC on pancreatic cancer (BxPC3). In the graph, the vertical axis of each graph shows the absorbance reflecting the degree of damage in tumor cells. The horizontal axis shows the concentration of ADC or drug (DM1) added to the culture medium.

[0098] Figure 6C The graphs show the results of the cytotoxicity experiment of B2E5-ADC on gastric cancer (MKN74). In the graphs, the vertical axis of each graph shows the survival rate of tumor cells, and the horizontal axis shows the concentration of ADC or antibody added to the culture medium.

[0099] Figure 7 This is a graph showing the results of a toxicity test of B2E5-ADC on mice. More specifically, it is a graph showing the changes in body weight over time in wild-type (C57BL / 6) or nude mice (SKN / slc) after a single administration of B2E5-ADC (100, 33, 10 μg) via tail vein (intravenous injection) or lumbar puncture (intrathecal injection). In the figure, circles, triangles, and squares represent data for one individual mouse. DETAILED DESCRIPTION

[0100] <Anti-Eva1 Antibody>

[0101] The present invention relates to an antibody or a functional fragment thereof that binds to the Eva1 protein of human origin (human Eva1).

[0102] In the present invention, "Eva (Epithelial V-like antigen) 1" is a single-transmembrane cell membrane protein and a molecule involved in the cytoskeleton system called MPZL2 (Myelin protein Zero-like 2). If it is Eva1 of human origin, it is typically a protein composed of the amino acid sequence recorded in sequence number 54 (a protein encoded by the nucleotide sequence recorded in sequence number 53). However, the DNA sequence of the gene mutates in nature (i.e., not artificially) through mutation, etc., and the amino acid sequence of the protein encoded by it is also changed accordingly. Therefore, the "Eva1 protein" involved in the present invention is not limited to the protein composed of the typical amino acid sequence, but also includes such natural mutants.

[0103] The "antibody" in the present invention includes all classes and subclasses of immunoglobulins. "Antibodies" include polyclonal antibodies and monoclonal antibodies. "Polyclonal antibodies" are antibody preparations containing different antibodies against different epitopes. In addition, the so-called "monoclonal antibody" refers to an antibody obtained from a substantially uniform group of antibodies. In contrast to polyclonal antibodies, monoclonal antibodies are antibodies that recognize a single determinant cluster on an antigen. The antibodies of the present invention are preferably monoclonal antibodies. The antibodies of the present invention are antibodies separated and / or recovered (i.e., separated) from components of the natural environment.

[0104] The reactivity of the antibody of the present invention to human Eva1 is preferably 10 as a KD (dissociation constant) value. -6 Below, more preferably 6×10 -7 As follows. The so-called "reactivity" refers to the binding affinity and / or specificity to human Eva1 as an antigen. The reactivity can be evaluated by an immunological method as long as a person skilled in the art is skilled in the art. For example, as shown in the examples described below, it can be obtained as a KD value by analysis using the surface plasmon resonance method. In addition, the antibody of the present invention may be an antibody that binds to Eva1 proteins of other animal origin (for example, mouse Eva1 protein (typically a protein composed of the amino acid sequence recorded in SEQ ID NO: 56, a protein encoded by the nucleotide sequence recorded in SEQ ID NO: 55)) in addition to antibodies that bind to human Eva1 protein.

[0105] The antibody of the present invention may have at least one cytotoxic activity selected from ADCC activity and CDC activity, or may have both ADCC activity and CDC activity.

[0106] In the present invention, the so-called "ADCC activity (antibody-dependent cellular cytotoxicity)" refers to the activity of the effector cells (immune cells such as NK cells and monocytes) further binding to the Fc region of the antibody when the antibody binds to the cell surface antigen of the target cell, thereby activating the cell and releasing factors to kill the target cell. On the other hand, the so-called "CDC activity (complement-dependent cytotoxicity)" refers to the activity of the complement system activating when the antibody binds to the target cell, which results in the lysis of the target cell.

[0107] In addition, the antibody of the present invention is preferably an antibody that has ADCC activity against target cells expressing human Eva1 at 0.01 μg / mL. Whether the antibody of the present invention has such ADCC activity can be measured, for example, by the method described in the Examples of Patent Document 2. More specifically, when Daudi cells expressing human Eva1 are used as target cells and the ADCC activity is evaluated by the cell lysis rate, the ADCC activity when the antibody of the present invention is used at 0.01 μg / mL is preferably a cell lysis rate of 30% or more, more preferably 50% or more, and even more preferably 70% or more.

[0108] In addition, the antibody of the present invention is preferably an antibody that has CDC activity on target cells expressing human Eva1 at 0.30 μg / mL, more preferably 0.01 μg / mL. Whether the antibody of the present invention has CDC activity can be measured, for example, by the method described in the Examples of Patent Document 2. More specifically, when Daudi cells expressing human Eva1 are used as target cells and the CDC activity is evaluated by the cell lysis rate, the CDC activity when using 0.30 μg / mL of the anti-Eva1 antibody of the present invention preferably has a cell lysis rate of 20% or more, more preferably 40% or more, further preferably 60% or more, and more preferably 80% or more.

[0109] The antibody of the present invention can also have anti-tumor activity. In the present invention, the so-called "anti-tumor activity" refers to at least any one of the activity of inhibiting the proliferation of cancer cells, the activity of inducing the death of cancer cells, and the activity of inhibiting the metastasis of cancer cells. The anti-tumor activity can be evaluated by, for example, using the analysis of a cancer-bearing model (mice inoculated with B16 melanoma cells, etc.) as described in the examples of patent document 2. More specifically, after B16 melanoma cells are intravenously administered to mice, etc., anti-Eva1 antibodies, etc. are intravenously administered every day or every few days starting from the next day or a few weeks later. Then, by measuring the number of colonies of B16 melanoma cells formed in the lungs, the anti-cancer activity in vivo can be evaluated. As a negative control, a control antibody with the same isotype can be administered, and PBS, etc. can also be administered. Then, when the number of colonies formed in the anti-Eva1 antibody-administered group is less than the number of colonies formed in the negative control-administered group (when the number of colonies formed in the negative control-administered group is taken as 100%, it is preferably 60% or less, more preferably 40% or less, further preferably 20% or less, and more preferably 10% or less), the anti-Eva1 antibody can be judged to have anti-tumor activity.

[0110] The antibodies of the present invention are not particularly limited in origin, type, shape, etc. as long as they are reactive with human Eva1, and specifically include antibodies derived from non-human animals (e.g., mouse antibodies, rabbit antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies.

[0111] Examples of the "non-human animal-derived antibody" in the present invention include antibodies against human Eva1 disclosed in Patent Document 2 that retain the following variable regions.

[0112] (a) An antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21.

[0113] (b) an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0114] (c) an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:49 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:44.

[0115] Furthermore, the present invention relates to, for example, an antibody against human Eva1 that retains the following CDRs.

[0116] (a) An antibody that retains heavy chain CDR1 to 3 defined by a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:27 and light chain CDR1 to 3 defined by a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:21.

[0117] (b) An antibody that retains heavy chain CDR1 to 3 defined by a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:39 and light chain CDR1 to 3 defined by a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:33.

[0118] (c) An antibody that retains heavy chain CDR1 to 3 defined by a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:49 and light chain CDR1 to 3 defined by a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:44.

[0119] In addition, regarding the antibody, the method for determining CDR based on the amino acid sequence of the variable region is not particularly limited, and examples thereof include well-known numbering schemes such as Chothia, IMGT, Kabat, and Aho. More specifically, as an example determined by the Chothia numbering method, an antibody against human Eva1 that retains the following CDRs can be cited.

[0120] (a-1) An antibody that retains the amino acid sequences described in SEQ ID NOs: 28 to 30 as heavy chain CDR1 to 3, respectively, and retains the amino acid sequences described in SEQ ID NOs: 22 to 24 as light chain CDR1 to 3, respectively.

[0121] (b) An antibody that retains the amino acid sequences described in SEQ ID NOs: 40 to 42 as heavy chain CDR1 to 3, respectively, and retains the amino acid sequences described in SEQ ID NOs: 34 to 36 as light chain CDR1 to 3, respectively.

[0122] (c) An antibody that retains the amino acid sequences described in SEQ ID NOs: 50 to 52 as heavy chain CDR1 to 3, respectively, and retains the amino acid sequences described in SEQ ID NOs: 45 to 47 as light chain CDR1 to 3, respectively.

[0123] In addition, examples of antibodies identified by the IMGT numbering system include antibodies against human Eva1 that retain the following CDRs.

[0124] (a-2) An antibody that retains the amino acid sequences described in SEQ ID NOs: 4 to 6 as heavy chain CDR1 to 3, respectively, and retains the amino acid sequence described in SEQ ID NO: 1, the amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence described in SEQ ID NO: 3 as light chain CDR1 to 3, respectively.

[0125] In addition, in the present invention, not only antibodies that maintain the variable region and / or CDR composed of a specific amino acid sequence are included, but also antibodies whose amino acid sequences are modified without reducing the desired activity (reactivity to antigens, etc.). Such amino acid sequence mutants, for example, can be made by introducing mutations of DNA encoding the above-mentioned variable regions, etc., or by peptide synthesis. Such modifications include, for example, replacement, deletion, addition and / or insertion of residues in the amino acid sequence of the antibody. Regarding the site where the amino acid sequence of the antibody is changed, as long as it has the same activity as the antibody before the change, it can be the constant region of the heavy chain or light chain of the antibody, in addition, it can also be the variable region (framework region (FR) and CDR). It is believed that changes in amino acids other than CDR have relatively little effect on reactivity with antigens, but now, methods for screening antibodies with improved affinity for antigens by changing the amino acids of CDR are also known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), International Publication No. 2002 / 051870, J. Biol. Chem., 280: 24880-24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008), MAbs. Mar-Apr; 6 (2): 437-45 (2014)). In addition, it is now possible to model antibodies with improved affinity for antigens by using integrated computational chemistry systems (e.g., Molecular Operating Environment, manufactured by CCG, Canada) (e.g., see http: / / www.rsi.co.jp / kagaku / cs / ccg / products / application / protein.html). Furthermore, as described in Protein Eng Des Sel. 2010 Aug; 23(8): 643-51, there are known examples where CDR1 of the heavy chain variable region and CDR3 of the light chain variable region are not involved in affinity for antigens. Similarly, Molecular Immunology 44: 1075-1084 (2007) reported that CDR2 of the light chain variable region in most antibodies is not involved in affinity for antigens. In this way, even if all of CDR1 to 3 of the heavy chain variable region and the light chain variable region are not required, the same activity can be exerted in the affinity of the antibody for the antigen.In fact, Biochem Biophys Res Commun. 2003 Jul 18; 307(1): 198-205, J Mol Biol. 2004 Jul 9; 340(3): 525-42, and J Mol Biol. 2003 Aug 29; 331(5): 1109-20 report examples of maintaining affinity for antigens by retaining at least one CDR of the original antibody.

[0126] In addition, with respect to the antibodies of the present invention, the number of amino acids changed in the variable region is preferably within 10 amino acids, more preferably within 5 amino acids, and further preferably within 3 amino acids (e.g., within 2 amino acids, 1 amino acid). Furthermore, from the viewpoint of having little effect on the reactivity with the antigen, the change is preferably performed entirely outside the CDR, i.e., the FR. In addition, the number of amino acids changed in the CDR is preferably within 5 amino acids, more preferably within 3 amino acids (e.g., within 2 amino acids, 1 amino acid).

[0127] The change of amino acid is preferably a conservative replacement. The so-called "conservative replacement" in the present invention refers to replacement with other amino acid residues having chemically identical side chains. The group of amino acid residues having chemically identical side chains is well known in the technical field to which the present invention belongs. For example, it can be classified into acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine / arginine / histidine), amino acids having hydrocarbon chains in neutral amino acids (glycine / alanine / valine / leucine / isoleucine / proline), amino acids having hydroxyl groups (serine / threonine), sulfur-containing amino acids (cysteine / methionine), amino acids having amide groups (asparagine / glutamine), amino acids having imino groups (proline), and amino acids having aromatic groups (phenylalanine / tyrosine / tryptophan).

[0128] In addition, as long as the antibody whose amino acid sequence is modified and whose variable region contains an amino acid sequence having 80% or more homology to the variable region composed of the above-mentioned specific amino acid sequence at the amino acid sequence level also has the same activity as the antibody before modification, it is included in the antibody of the present invention. The homology may be at least 80%, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more). In addition, the sequence homology can be determined using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This program is based on the algorithm BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing amino acid sequences by BLASTP, the parameters are, for example, score=50 and wordlength=3. In addition, when using the Gapped BLAST program to analyze amino acid sequences, it can be performed in the manner described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific methods of these analysis methods are well known.

[0129] In addition, "having equivalent activity" means, for example, that the reactivity to the antigen is equivalent to that of the target antibody (e.g., the B2E5-48 mouse antibody described in Patent Document 2) (e.g., 70% or more, preferably 80% or more, more preferably 90% or more).

[0130] In addition, the change of the antibody of the present invention is, for example, a change in the post-translational processing of the antibody such as changing the number or position of the glycosylation site. Thus, for example, the ADCC activity of the antibody can be improved. The so-called glycosylation of the antibody is typically N-bound or O-bound. The glycosylation of the antibody depends largely on the host cell used to express the antibody. The change of the glycosylation profile can be carried out by known methods such as the introduction or deletion of specific enzymes related to sugar production (Japanese Patent Publication No. 2008-113663, U.S. Patent No. 5047335, U.S. Patent No. 5510261, U.S. Patent No. 5278299, International Publication No. 99 / 54342). Furthermore, in the present invention, for the purpose of increasing the stability of the antibody, deamidation can also be suppressed by replacing the deamidated amino acid or the amino acid adjacent to the deamidated amino acid with other amino acids. In addition, glutamic acid can also be replaced with other amino acids to increase the stability of the antibody. The present invention provides such stabilized antibodies.

[0131] The so-called "chimeric antibody" in the present invention is an antibody formed by connecting the variable region of a certain antibody to the constant region of an antibody of a different species. Chimeric antibodies can be obtained, for example, by immunizing mice with antigens, cutting out the variable part (variable region) of the antibody that binds to the antigen from the gene of the mouse monoclonal antibody, combining it with the gene of the antibody constant part (constant region) derived from human bone marrow, integrating it into an expression vector and introducing it into a host to produce it, thereby obtaining (for example, Japanese Patent Publication No. 8-280387, U.S. Patent No. 4816397, U.S. Patent No. 4816567, U.S. Patent No. 5807715).

[0132] The constant region of a chimeric antibody generally uses a constant region of an antibody of human origin. For example, the heavy chain can use Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε as a constant region. In addition, the light chain can use Cκ and Cλ as constant regions. The amino acid sequences of these constant regions and the base sequences encoding them are well known. In addition, in order to improve the stability of the antibody itself or the production stability of the antibody, one or more amino acids in the constant region of an antibody of human origin can be replaced, deleted, added and / or inserted.

[0133] In the present invention, "humanized antibodies" are antibodies obtained by transplanting the gene sequence of the CDR of an antibody of non-human animal origin (CDR transplantation) to an antibody gene of human origin, and the method for making the same is known as overlap extension PCR (e.g., European Patent Application Publication No. 239400, European Patent Application Publication No. 125023, International Publication No. 90 / 07861, International Publication No. 96 / 02576). The variable region of an antibody is usually composed of 3 CDRs sandwiched by 4 FRs. CDR is a region that substantially determines the binding specificity of an antibody. The amino acid sequence of CDR is rich in diversity, but on the other hand, the amino acid sequences constituting FRs show high homology between antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of a certain antibody can be transplanted to other antibodies by transplanting CDRs. In addition, from the perspective of maintaining the function of CDRs, in the transplantation of non-human CDRs to human FRs, human FRs with high homology to the FRs of the non-human animal origin are selected. That is, the amino acids in the CDR not only recognize the antigen but also coordinate with the amino acids of the FR located near the CDR and participate in the maintenance of the loop structure of the CDR. Therefore, it is preferable to use human FRs composed of an amino acid sequence with high homology to the amino acid sequence of the FR adjacent to the CDR to be transplanted.

[0134] The search for known human FRs with high homology to FRs of non-human animal origin can be performed, for example, using a search system specialized for antibodies that can be used on the Internet (http: / / www.bioinf.org.uk / abysis / ). Mutations can be introduced into sequences other than the CDR of antibodies of non-human origin in a manner consistent with the sequence of the human FRs obtained in this way. Alternatively, in the case where a gene (cDNA) encoding the amino acid sequence of the human FR obtained by the search can be obtained, non-human CDRs can be introduced into its sequence. The introduction of mutations can be performed using techniques known in the art such as nucleic acid synthesis and site-directed mutagenesis.

[0135] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody thus prepared, it is possible to appropriately select the FR of the human-derived antibody such that the CDR forms a good antigen-binding site when connected via the CDR. In addition, as required, by replacing the amino acid residues of the FR in such a way that the CDR of the humanized antibody forms an appropriate antigen-binding site according to the method described in Sato, K. et al., Cancer Res, 1993, 53, 851-856, etc., further measuring and evaluating the antigen-binding activity of the mutant antibody in which the amino acids are replaced, a mutant FR sequence having the desired property can be selected.

[0136] In the present invention, examples of “humanized antibodies” include humanized antibodies having a light chain variable region comprising CDRs of the amino acid sequence described in SEQ ID NO: 1, an amino acid sequence consisting of Val-Thr-Ser, and an amino acid sequence described in SEQ ID NO: 3, and a heavy chain variable region comprising CDRs1 to 3 of the amino acid sequences described in SEQ ID NOs: 4 to 6, respectively, and which bind to the human-derived Eva1 protein.

[0137] More specifically, as shown in the examples described below,

[0138] The light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 7 to 10, amino acid sequences having 80% or more homology to the amino acid sequences set forth in SEQ ID NOs: 7 to 10, and amino acid sequences in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 10,

[0139] The heavy chain variable region comprises a humanized antibody having an amino acid sequence selected from the group consisting of an amino acid sequence recorded in SEQ ID NOs: 13 to 16, an amino acid sequence having 80% or more homology to the amino acid sequence recorded in SEQ ID NOs: 13 to 16, and an amino acid sequence in which one or several amino acids are replaced, deleted, added and / or inserted in at least any one of the amino acid sequences recorded in SEQ ID NOs: 13 to 16.

[0140] In addition, regarding changes in the variable region and homology, as described in "Antibodies from non-human animals", it is preferred that in the humanized antibody of the present invention, the sequence of the CDR is not changed, and the sequences of the light chain variable region CDR1 to 3 and the heavy chain variable region CDR1 to 3 are specifically the amino acid sequence recorded in sequence number 1, the amino acid sequence consisting of Val-Thr-Ser, the amino acid sequence recorded in sequence number 3, and the amino acid sequence recorded in sequence numbers 4 to 6, respectively.

[0141] In addition, among the humanized antibodies, as shown in the Examples described below, from the viewpoint of higher reactivity to antigens, preferably

[0142] The (H2-L2) light chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 7, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 7,

[0143] A humanized antibody wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 13, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 13,

[0144] (H1-L4) the light chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 8, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 8,

[0145] A humanized antibody wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 15, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 15, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 15,

[0146] The (H2-L3) light chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 10, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 10, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 10,

[0147] A humanized antibody wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 13, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 13,

[0148] (H2-L4) the light chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 8, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 8,

[0149] A humanized antibody having a heavy chain variable region comprising an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 13, and an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 13

[0150] As a suitable example.

[0151] In addition, considering the high production capacity, we can list

[0152] The (H2-L2) light chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 7, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 7,

[0153] A humanized antibody wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 13, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 13,

[0154] (H2-L4) the light chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 8, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 8,

[0155] A humanized antibody wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 13, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 13,

[0156] The (H3-L4) light chain variable region comprises an amino acid sequence selected from the group consisting of an amino acid sequence as set forth in SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence as set forth in SEQ ID NO: 8, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences as set forth in SEQ ID NO: 8,

[0157] A humanized antibody having a heavy chain variable region comprising an amino acid sequence selected from the group consisting of an amino acid sequence set forth in SEQ ID NO: 16, an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 16, and an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in at least any one of the amino acid sequences set forth in SEQ ID NO: 16

[0158] As a suitable example.

[0159] Furthermore, the antibody of the present invention may be an antibody that competes with an antibody that retains a variable region or CDR comprising the above-mentioned specific amino acid sequence or a modified product thereof in binding to human Eva1. In other words, an antibody that binds to an epitope reactive with an antibody that retains a variable region or CDR comprising the above-mentioned specific amino acid sequence or a modified product thereof may be an antibody.

[0160] The so-called "epitope" in the present invention refers to the antigenic determinant present in the antigen, that is, the site on the antigen to which the antigen-binding domain in the antibody binds. Therefore, the epitope in the present invention can be a polypeptide composed of a plurality of continuous amino acids in the primary sequence of amino acids (linear epitope), or a polypeptide formed by non-adjacent amino acids in the primary sequence of amino acids coming close to each other through three-dimensional structures such as the folding of peptides or proteins (discontinuous epitope, conformational epitope). In addition, as the epitope, it is typically composed of at least 3 (e.g., 4), and most commonly at least 5 (e.g., 6 to 20, 7 to 15, 8 to 10) amino acids.

[0161] In addition, when an antibody is obtained, a person skilled in the art can identify the peptide region (epitope) on the antigen to which the antibody is reactive and prepare various antibodies that bind to the peptide region. Furthermore, whether two antibodies bind to the same or sterically polymerized epitope can be determined by a competitive assay.

[0162] In the present invention, the "functional fragment" of an antibody refers to a fragment that is a part of an antibody (partial fragment) and is reactive with human Eva1. Specifically, Fab, Fab', F(ab')2, variable region fragment (Fv), disulfide bond Fv, single-chain variable region fragment (single-chain Fv, scFv), sc(Fv)2, bispecific antibodies, multispecific antibodies, and polymers thereof can be cited.

[0163] Here, "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin composed of one light chain and a portion of a heavy chain. It can be obtained by papain digestion of the antibody or by recombinant methods. "Fab'" contains one or more cysteines in the hinge region of the antibody and differs from Fab by the addition of only a few residues at the carboxyl terminus of the heavy chain CH1 domain. "F(ab')2" refers to a divalent antigen-binding fragment of an immunoglobulin composed of portions of both light chains and both heavy chains.

[0164] "Variable region fragment (Fv)" is the smallest antibody fragment with complete antigen recognition and binding sites. Fv is a dimer formed by strongly connecting the heavy chain variable region and the light chain variable region through non-covalent bonds. "Single-chain variable region fragment (single-chain Fv, scFv)" contains the heavy chain variable region and light chain variable region of the antibody, and these regions exist in a single polypeptide chain. "sc(Fv)2" is a fragment made of a single chain by combining two heavy chain variable regions and two light chain variable regions with a connector or the like. The so-called "bispecific antibody" is a small antibody fragment with two antigen binding sites, which contains a heavy chain variable region combined with a light chain variable region in the same polypeptide chain, and each region forms a pair with the complementary region of another chain. "Multispecific antibody" is a monoclonal antibody with binding specificity to at least two different antigens. For example, it can be prepared by the simultaneous expression of two immunoglobulin heavy chain / light chain pairs with different specificities of the two heavy chains.

[0165] The antibodies of the present invention can be produced by the hybridoma method, and can also be produced by the recombinant DNA method. As a representative hybridoma method, the method of Kohler and Milstein (Kohler & Milstein, Nature, 256: 495 (1975)) can be cited. The antibody-producing cells used in the cell fusion step in this method are spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats) immunized with antigens (human Eva1, partial peptides thereof, proteins fused to them with Fc proteins, etc., or cells expressing them, etc.). Antibody-producing cells obtained by allowing the antigen to act on the above-mentioned cells or lymphocytes that have been pre-isolated from animals that have not been immunized in culture medium can be used. Various well-known cell strains can be used as myeloma cells. As long as the antibody-producing cells and myeloma cells can be fused, they can also be derived from different animal species, but are preferably derived from the same animal species. Hybridomas are produced, for example, by cell fusion between spleen cells obtained from mice immunized with an antigen and mouse myeloma cells, and then hybridomas producing monoclonal antibodies specific to human Eva 1 can be obtained by screening. Monoclonal antibodies against human Eva 1 can be obtained by culturing hybridomas or from the ascites of a mammal to which the hybridomas have been administered.

[0166] The recombinant DNA method is a method of cloning a DNA encoding the antibody of the present invention described above from a hybridoma, B cell, etc., integrating it into an appropriate vector, and introducing it into a host cell (e.g., a mammalian cell line such as HEK cell, Escherichia coli, yeast cell, insect cell, plant cell, etc.), so as to produce the antibody of the present invention as a recombinant antibody (e.g., PJ Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem. 192: 767-775 (1990)). In the expression of the DNA encoding the antibody of the present invention, the DNA encoding the heavy chain or the light chain may be separately integrated into an expression vector to transform the host cell, or the DNA encoding the heavy chain and the light chain may be integrated into a single expression vector to transform the host cell (see International Publication No. 94 / 11523). The antibody of the present invention can be cultured in the above-mentioned host cells, separated / purified from the host cells or culture fluid, and obtained in a substantially pure and uniform form. The separation / purification of the antibody can use the method used in the conventional polypeptide purification. If a transgenic animal (cow, goat, sheep, pig, etc.) with an antibody gene integrated is produced using transgenic animal production technology, monoclonal antibodies derived from the antibody gene can also be obtained in large quantities from the milk of the transgenic animal.

[0167] The present invention may also provide a DNA encoding the antibody of the present invention, a vector comprising the DNA, a host cell that retains the DNA, and a method for producing the antibody comprising culturing the host cell and recovering the antibody.

[0168] <Antibody Drug Conjugate>

[0169] As shown in the examples described below, human Eva1, which is the target of the antibody of the present invention, is expressed in various cancers. In addition, the antibody of the present invention (particularly humanized antibody) is also useful as ADC. Thus, the present invention can take the form of an antibody-drug conjugate (ADC) in which the antibody of the present invention (particularly humanized antibody) or its functional fragment is combined with a drug.

[0170] In the ADC of the present invention, the "drug" that can be bound to the antibody is not particularly limited, and examples thereof include cytotoxic substances known in the art. As "cytotoxic substances", anticancer agents such as irinotecan (CPT-11), irinotecan metabolite SN-38 (10-hydroxy-7-ethylcamptothecin), doxorubicin, paclitaxel, 5-fluorouracil, nimustine, ranimustine, temozolomide and other alkylating agents, gemcitabine, hydroxyurea and other metabolic antagonists, etoposide, vincristine and other plant alkaloids, mitomycin, bleomycin and other anticancer antibiotics, cisplatin and other platinum preparations, sorafenib, erlotinib and other molecular target agents, methotrexate, cytarabine, 6-thioguanine, 6-mercaptopurine, cyclophosphamide, ifosfamide, busulfan, MMAE (monomethyl auristatin E), DM1 (maytansine), calicheamicin can be listed. As cytotoxic substances, radioactive isotopes may also be mentioned, for example, 32 P. 14 C. 125 I. 3 H. 131 I. 186 Re, 188 Re, 10 B. 111 In, 90 Y. As a cytotoxic substance, it can be a photosensitive substance that enables cytotoxic activity to function, more specifically, a substance that is activated by light irradiation so that it itself changes into a form that shows cytotoxicity (cytotoxicity), and it can also be a substance that produces a cytotoxic substance (cytotoxic substance). For example, chlorins, chlorin e6, porfimer sodium, talaporfin sodium, verteporfin, and their precursors and derivatives can be listed. In addition, as a cytotoxic substance, toxic peptides can be listed, for example, ribosome inactivating proteins (RIPs) such as saponin, ricin, and Shiga toxin.

[0171] In addition, the binding of the antibody to the cytotoxic substance can also be carried out by methods known in the art, and can be either direct binding or indirect binding. For example, as direct binding, a covalent bond can be used. As indirect binding, binding via a linker can be used.

[0172] In the present invention, it is preferred that the antibody and the cytotoxic substance are bound via a linker. By binding two molecules via a linker, the antigenicity of the cytotoxic substance can be reduced, which is preferred in the administration to the subject. General techniques for linkers are described in, for example, Hermanson, G.T. Bioconjugate Techniques, Academic Press, 1996; Harris, J.M. and Zalipsky, S., Poly (ethylene glycol), Chemistry and Biological Applications, ACS Symposium Series, 1997; Veronese, F. and Harris, J.M., Peptide and protein PEGylation. Advanced Drug Delivery Review 54 (4), 2002.

[0173] In addition, the linker includes not only a straight-chain linker (a bivalent linker) but also a branched linker (a linker with a valency of more than 3). A straight-chain linker has a cytotoxic substance at one end and an antibody at the other end. A branched linker usually has a cytotoxic substance at each branch (each chain) and an antibody at the other end. Examples of the linker include N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (6-amidocaproate) (LC-SMCC), N-succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), 3-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxy The peptide linkers include succinimidyl ester (MBS), N-(α-maleimidoacetoxy)-succinimidyl ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl) isocyanate (PMPI), 6-maleimidocaproyl (MC), maleimidopropionyl (MP), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), valine-citrulline (Val-Cit) and alanine-phenylalanine (ala-phe).

[0174] The technology of combining antibodies with cytotoxic substances via a connector is well known in the art. For example, the combination of the antibody and the connector is a covalent bond or a non-covalent bond (ionic bond, hydrophobic bond, etc.), preferably a covalent bond. When the conjugate of the present invention is applied to a subject, the combination is preferably a combination in which the cytotoxic substance is not easily free in the blood. As such a combination, a bond between a maleimide group and a sulfhydryl group, a bond obtained by reacting a halogenated ester with a sulfhydryl group, an amide bond between a carboxyl group and an amino group, a disulfide bond between a sulfhydryl group and a sulfhydryl group, a Schiff base produced by an amino group and an aldehyde group, a thioester bond between a sulfhydryl group and a carboxylic acid, an ester bond between a hydroxyl group and a carboxyl group, a bond produced by an amino group and a square acid derivative (e.g., dimethyl square acid), a bond between a diene aldehyde group and an amino group, etc. can be cited. As specific examples of such binding, there can be cited the binding of a maleimide group present at one end of the linker to a thiol group contained in a cysteine ​​residue on an antibody, the dehydration substitution binding of a succinimide group present at one end of the linker to an amino group contained in a lysine residue on an antibody (e.g., International Publication No. 2008 / 096760), the dehydration condensation binding of an amino group present at one end of the linker to a carboxylic acid contained in aspartic acid or glutamic acid on an antibody (using, for example, WSCDI), etc. For a specific method for binding an antibody to a linker, for example, please refer to International Publication No. 2010 / 055950.

[0175] On the other hand, the binding of the antibody or linker to the cytotoxic substance is a covalent bond or a non-covalent bond (ionic bond, hydrophobic bond, etc.), preferably a covalent bond. In particular, when the conjugate of the present invention is administered to a subject, the binding is preferably a binding in which the cytotoxic substance is not easily free in the blood. From such a viewpoint, the binding of the antibody or linker to the cytotoxic substance is not limited, but preferably an ester bond, a carbamate bond, a carbonate bond, a thiocarbamate bond, and more preferably an ester bond.

[0176] In the ADC of the present invention, the number of cytotoxic substances bound to each antibody molecule (drug binding number) is considered to be effective and safe, and the reaction conditions such as the amount of raw materials / reagents used in the reaction are specified as a certain number. However, unlike the chemical reaction of low molecular weight compounds, it is common to obtain a mixture of drugs with different numbers of binding. The number of drugs bound to one antibody molecule is specified and indicated as an average value, that is, the average drug binding number. Therefore, in the specification of this application, unless otherwise specified, the number of drugs bound refers to the average value.

[0177] The number of drug bindings per antibody molecule can be controlled by adjusting the type and concentration of the reducing agent used, the concentration of the linker and the drug relative to the antibody (molar excess, etc.), and the temperature or time of the binding reaction. As a "reducing agent", there is no particular limitation as long as it can reductively cleave the disulfide bonds in the antibody to generate a binding via the above-mentioned sulfhydryl group, and examples thereof include dithiothreitol (DTT, DL-DTT), 2-mercaptoethylamine, mercaptoethanol, and TCEP.

[0178] In the present invention, the average number of drug binding per antibody is not particularly limited, for example, 1 to 10, preferably 3 to 8, and more preferably 3 to 5. In addition, the number of drug binding per antibody molecule can be determined by measuring the absorbance in the absorption peak of the cytotoxic substance (e.g., DM1) and the absorbance at 280nm of the absorption peak of the antibody, weighting the influence of each absorption peak on each other and establishing simultaneous equations for calculation, finding the concentration of the antibody and the cytotoxic substance, determining the ratio of the number of molecules, and thereby finding it. In addition, the reagent for sulfhydryl colorimetric quantification, such as DTNB, can also be used to find it.

[0179] In addition, the ADC of the present invention may absorb moisture, adhere to adsorbed water, or form a hydrate by being left in the air or recrystallized. Such compounds and salts containing water are also included in the present invention.

[0180] One or more of the atoms constituting the ADC of the present invention may also contain an unnatural ratio of atomic isotopes. Examples of atomic isotopes include, for example, 2 H. 3 H. 125 I. 14 C. In addition, the ADC of the present invention can be detected in bioimaging technology, etc. Therefore, it can also be combined with a labeling substance. The labeling substance is appropriately selected according to the type of detection method used, and examples thereof include radioactive labeling substances, fluorescent labeling substances, paramagnetic labeling substances, superparamagnetic labeling substances, and enzyme labeling substances. In addition, the binding of such a labeling substance to ADC can be direct or indirect. As an indirect binding, the binding via a secondary antibody bound to a labeling substance, a polymer (protein A, protein B, etc.) bound to a labeling substance can be cited.

[0181] More specifically, as a radioisotope for labeling ADC, a positron-releasing nuclide can be used. 64 Cu, 18 F. 55 Co. 66 Ga, 68 Ga, 76 Br, 89 Zr and 124I. ADCs labeled with positron-releasing nuclei can be labeled using known methods (Nucl Med Biol. 1999; 26 (8): 943-50., J Nucl Med. 2013; 54 (11): 1869-75., etc.). In addition, after the ADCs labeled with positron-releasing nuclei are administered to a subject, the radiation emitted from the radioactive nuclei is measured in vitro by PET (positive ion emission tomography) and converted into images by computer tomography. In this way, the delivery and accumulation of the ADCs administered to the subject to the target site can be tracked.

[0182] <Chimeric Antigen Receptor>

[0183] As shown in the examples described below, human Eva1, which is the target of the antibody of the present invention, is expressed in various cancers. In addition, the variable region of the antibody of the present invention is useful in chimeric antigen receptors (CAR) and T cells expressing them (so-called CAR-T). Therefore, the present invention can also take the form of a CAR comprising a region that binds to the human-derived Eva1 protein, a transmembrane region, and an intracellular signaling region.

[0184] In the present invention, "Chimeric Antigen Receptor (CAR)" refers to a chimeric protein in which an extracellular region that binds to a human-derived Eva1 protein, a transmembrane region, and an intracellular signaling region are arranged in order from the N-terminal side and are directly or indirectly linked.

[0185] In the present invention, the “region that binds to the human-derived Eva1 protein (antigen-binding region)” can be, for example, the above-mentioned antibody or a functional fragment thereof, but scFv is generally used as CAR.

[0186] As described above, "scFv" is a structure formed by connecting the light chain variable region and the heavy chain variable region of a monoclonal antibody (immunoglobulin) via a connector, and maintains the ability to bind to an antigen. The order on the N-terminal side in scFv can be a light chain variable region, a connector, and a heavy chain variable region, or a heavy chain variable region, a connector, and a light chain variable region, but the former is preferred.

[0187] As a "connector" in scFv, for example, a peptide connector can be used. The length of the connector is not particularly limited. For example, a connector with 5 to 25 amino acids can be used. The length of the connector is preferably 8 to 25 amino acids, and more preferably 15 to 20 amino acids. Suitable examples of peptide connectors include those composed of glycine or glycine and serine (GGS connector, GS connector, GGG connector, etc.). In addition, a connector composed of 18 amino acids at positions 134 to 151 recorded in sequence number 58 can also be listed as a suitable example. Glycine and serine, which are amino acids constituting these connectors, have the advantages that their own size is small and it is not easy to form a higher-order structure in the connector.

[0188] The monoclonal antibody serving as the basis of scFv is not particularly limited, but examples thereof include the above-mentioned antibodies derived from non-human animals, human antibodies, and humanized antibodies. In addition, suitable examples are also as described above, but as shown in the Examples described below, from the viewpoint of adapting to activation of human Eva1 and cell proliferation, the above-mentioned combination of (H2-L2) is preferred, and an scFv in which L2, a linker, and H2 are linked in order from the N-terminal side is further preferred.

[0189] In the present invention, the "transmembrane region" is not particularly limited as long as it has the function of penetrating the cell membrane and can anchor CAR to the cell membrane. Proteins that are the source of polypeptides that bear transmembrane regions include, for example, CD28, CD3ζ, CD3ε, 4-1BB, CD45, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, and the α and β chains of T cell receptors.

[0190] In the present invention, "intracellular signal region" is a region configured in the cell when CAR is configured on the cell membrane, and is a signal required for the effector function of the immune cell to be transduced, that is, when the antigen binding region is combined with the antigen (human Eva1), the region (so-called primary signal) required for the activation of the immune cell can be transduced. As such an intracellular signal region, there are generally: only an activation signal transduction domain such as a T cell receptor (TCR) and a CD3 complex (first generation CAR), a costimulatory signal transduction domain (e.g., an intracellular domain of CD28 or 4-IBB) derived from a costimulatory molecule in addition to the activation signal transduction domain (second generation CAR), and a plurality of costimulatory signal transduction domains (e.g., an intracellular domain of CD28 and 4-IBB) in addition to the activation signal transduction domain (third generation CAR). In addition, the intracellular signaling region including the CD79A intracellular region and the CD40 intracellular region previously developed by the present inventors can be cited (see Mol Ther 2021 Sep 1; 29(9): 2677-2690).

[0191] As described above, the "intracellular signaling region" can be used as long as it can transduce the primary signal, and the activation signal transduction domain of the protein involved in the signal can be used. It is known that the immunoreceptor tyrosine-based activation motif (ITAM) is involved in primary signal transduction. Proteins having ITAM include, for example, CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

[0192] In addition, as described above, and as shown in the examples described later, "an intracellular signaling region comprising a CD79A intracellular region and a CD40 intracellular region" is also suitable for use as the intracellular signaling region in the present invention. In this case, the configuration relationship between the CD79A intracellular domain and the CD40 intracellular domain is not particularly limited, and any of the modes in which the N-terminal side is the CD79A intracellular region and the N-terminal side is the CD40 intracellular region can be adopted. The configuration relationship is preferably the former. The CD79A intracellular region and the CD40 intracellular region are preferably directly connected or connected via a connector. The connector is not particularly limited as long as the NFκB signal activation effect is not significantly impaired and is arbitrary. As a connector, preferably a connector composed of glycine or glycine and serine (GGS connector, GS connector, GGG connector, etc.). The length of the connector is not particularly limited, for example, 1 to 20 amino acids, preferably 1 to 10 amino acids, and more preferably 1 to 5 amino acids.

[0193] The number of intracellular signaling regions containing CAR in the present invention is not limited to 1, and multiple intracellular signaling regions may be included (for example, 1 to 5, 1 to 3, or 1 or 2 signal transduction factors). In this case, the multiple intracellular signaling regions included may be the same or different.

[0194] The CAR of the present invention may include other domains in addition to the above-mentioned antigen binding region, transmembrane region and intracellular signaling region. Examples of other domains include costimulatory transduction regions, spacer sequences, signal peptides, etc., but are not limited to these.

[0195] It is known that the costimulatory molecules expressed on T cells, by binding to the ligands specific to each costimulatory molecule expressed on antigen presenting cells, transduce costimulatory signals into the cell to assist the activation of T cells (secondary signal transduction). The so-called "costimulatory transduction region" in the present invention refers to the intracellular domain involved in the costimulatory signal transduction of such costimulatory molecules. As examples of costimulatory molecules, CD28, 4-1BB (CD137), OX40 (CD134), ICOS, CD2, CD4, CD5, CD8α, CD8β, CD154, etc. can be listed. The CAR of the present invention can include the costimulatory transduction regions of these costimulatory molecules.

[0196] The number of costimulatory transduction regions that the CAR of the present invention may include is not limited to 1, but may be multiple. The CAR of the present invention may include, for example, 1 to 5, 1 to 3, or 1 or 2 costimulatory transduction regions. In the case where the CAR of the present invention includes multiple costimulatory signal transduction domains, the domains may be the same or different.

[0197] The "spacer" in the present invention refers to a sequence connecting the various regions, etc., and is not particularly limited as long as it does not inhibit the functions of the various regions. In addition, the number of amino acids is not particularly limited, and is usually 1 to 500 amino acids, preferably 5 to 300 amino acids, and more preferably 10 to 100 amino acids.

[0198] In particular, in the CAR of the present invention, the antigen binding region and the transmembrane region are preferably directly connected or connected via a relatively short spacer. The number of constituent amino acids of the spacer between the antigen binding region and the transmembrane region is, for example, 1 to 400, preferably 10 to 300. In addition, as shown in the embodiments described below, when the cells expressing the CAR of the present invention are cultured in the absence of IL-2, from the perspective of cell proliferation and cytokine production, the constituent amino acids are more preferably 150 to 300, and further preferably 200 to 250. On the other hand, when the cells expressing the CAR of the present invention are cultured in the presence of IL-2, the constituent amino acids are more preferably 10 to 30.

[0199] The sequence of the spacer is not particularly limited. For example, a hinge portion or a portion thereof of IgG, preferably human IgG (e.g., subtype IgG1, IgG4), a hinge portion and a portion of CH2, or a portion of a factor used in a transmembrane domain (e.g., CD28) can be used as a spacer. In addition, by utilizing a hinge portion or a portion thereof, it is expected that a flexible spacer can be formed. As the spacer, a spacer consisting of an amino acid sequence described in sequence number 59, 60, 61, 62, or 62 (positions 268 to 499 described in sequence number 58) can be specifically cited.

[0200] In the present invention, "signal peptide" refers to a peptide for promoting the secretion of CAR or for indicating the localization to the cell membrane, also referred to as a leader sequence. The signal peptide can usually bind directly or indirectly to the N-terminus of the antigen binding region. In addition, the signal peptide can also be arbitrarily cut from the antigen binding region in cell processing and the localization of CAR to the cell membrane. As an example of the signal peptide, signal peptides such as human GM-CSF receptor, α chain and β chain of T cell receptor, CD8α, CD8β, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR can be listed.

[0201] Above, examples of each region in the CAR of the present invention are described, but as for the specific amino acid sequences of these regions, as long as a person skilled in the art can search the known literature, NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ) and other databases and obtain them appropriately. Furthermore, as shown in Table 3 above, specific sequences are also disclosed in the sequence table of the present application. In addition, it is not limited to such typical amino acid sequences (for example, NCBI reference sequences), and the regions involved in the present invention, as long as the functions undertaken by the regions are maintained, may also include modified bodies and homologs relative to these typical amino acid sequences. In addition, the modified bodies and homologs have generally high homology relative to typical amino acid sequences. High homology is generally 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more (for example, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0202] In addition, the CAR of the present invention can be a molecule composed of one polypeptide, or a molecule composed of a complex of two or more polypeptides. In addition, the CAR of the present invention can be a molecule composed of a polypeptide or a complex thereof, or a molecule formed by connecting other substances (e.g., fluorescent substances, radioactive substances, inorganic particles, etc.) to a polypeptide or a complex thereof.

[0203] The CAR of the present invention may be chemically modified. The C-terminus of the polypeptide constituting the CAR of the present invention may be any of a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH2) or an ester (-COOR). Here, as R in the ester, for example, C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl can be used; for example, C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; for example, C6-12 aryl groups such as phenyl and α-naphthyl; for example, phenyl-C1-2 alkyl groups such as benzyl and phenethyl; C7-14 aralkyl groups such as α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl; pivaloyloxymethyl, etc. The polypeptide constituting the CAR of the present invention may be amidated or esterified at the carboxyl group (or carboxylate) other than the C-terminus. As the ester at this time, for example, the ester at the C-terminus mentioned above can be used. Furthermore, the polypeptide constituting the CAR of the present invention may also include a polypeptide in which the amino group of the amino acid residue at the N-terminal is protected by a protecting group (e.g., C1-6 acyl groups such as formyl and acetyl groups, etc.), a polypeptide in which the N-terminal glutamine residue generated by cleavage in vivo is pyroglutamated, and a polypeptide in which the substituents on the side chains of the amino acids in the molecule (e.g., -OH, -SH, amino, imidazole, indolyl, guanidino, etc.) are protected by appropriate protecting groups (e.g., C1-6 acyl groups such as formyl and acetyl groups, etc.).

[0204] In addition, the CAR of the present invention may have other functional proteins. As other functional proteins, there is no particular limitation and they can be appropriately selected according to the function that the CAR of the present invention is intended to be given. For example, as functional proteins used for the purpose of facilitating the purification and detection of CAR, Myc tags, His tags, HA tags, FLAG tags (registered trademark, Sigma-Aldrich), fluorescent protein tags (GFP, etc.), etc. can be listed.

[0205] The chimeric antigen receptor of the present invention may also be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and any acidic salt or basic salt may be used. For example, examples of acidic salts include inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts such as acetates, propionates, tartrates, fumarates, maleates, malates, citrates, methanesulfonates, and p-toluenesulfonates; amino acid salts such as aspartates and glutamates; and the like. In addition, examples of basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and the like.

[0206] The chimeric antigen receptor of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid, and the like.

[0207] In addition, there are several reports on experiments and clinical studies using CAR (e.g., Rossig C, et al. Mol Ther 10:5-18, 2004; Dotti G, et al. Hum Gene Ther 20:1229-1239, 2009; Ngo MC, et al. Hum Mol Genet 20(R1):R93-99, 2011; Ahmed N, et al. Mol Ther 17:1779-1787, 2009; Pule MA, et al. Nat Med 14:1264-1270, 2008; Louis CU, et al. Blood 118:6050-6056, 2011; Kochenderfer JN, et al. Blood 116:4099-4102, 2010; Kochenderfer JN, et al. Blood 119:2709-2720, 2012; Porter DL, et al. N Engl J Med 365:725-733, 2011; Kalos M, et al. Sci Transl Med 3:95ra73, 2011; Brentjens RJ, et al. Blood 118:4817-4828, 2011; Brentjens RJ, et al. Sci Transl Med 5:177ra38, 2013), and the CAR of the present invention can be constructed with reference to these reports. In addition, specific sequences and the like are also shown in the examples described below, and thus the CAR of the present invention can be constructed with reference to these reports.

[0208] <Nucleotide encoding chimeric antigen receptor>

[0209] The present invention provides a polynucleotide encoding the CAR of the present invention. Regarding the information of the nucleotide sequence encoding the antigen binding region such as scFV, a monoclonal antibody that recognizes the target antigen can also be prepared, and the amino acid sequence of the monoclonal antibody is determined by a known method such as the Edman method, and obtained based on the amino acid sequence. In addition, regarding the above-mentioned humanized antibodies, etc., specific sequences are also disclosed in the sequence table of this application. In addition, it can also be obtained by analyzing the nucleotide sequence of the hybridoma that produces the monoclonal antibody.

[0210] In addition, the specific nucleotide sequences encoding each region in the CAR of the present invention including the antigen binding region, as long as the technicians in this field can also retrieve the known literature, NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ) and other databases and obtain them appropriately. Furthermore, the nucleotide sequences encoding the above-mentioned regions, etc. are not limited to the known ones, as long as the nucleotide sequences encoding the above-mentioned regions, etc. can be used. According to the degeneracy of gene coding, there are multiple codons corresponding to one amino acid. Therefore, there are many nucleotide sequences encoding the same amino acid sequence. As long as the nucleotide sequence of the polynucleotide of the present invention encodes the CAR of the present invention, it can be any of the multiple nucleotide sequences generated by the degeneracy of gene coding. Further, in order to optimize the expression in the cell expressing CAR, in the nucleotide sequence encoding each region in the CAR of the present invention, the codon selected for encoding amino acids can be modified.

[0211] Furthermore, as long as one is skilled in the art, based on such nucleotide sequence information, a polynucleotide encoding each region, etc. can be prepared using a chemical synthesis method (phosphoramidite method, etc.), a method using PCR amplification, and other known techniques. Furthermore, the polynucleotide encoding each region, etc. obtained in this way can be directly connected or connected via a spacer to obtain a polynucleotide encoding the CAR of the present invention. In addition, the connection of the polynucleotide encoding each region, etc. is shown in the embodiments described later, for example, a known method such as overlap extension PCR can be used.

[0212] <Vector containing a polynucleotide encoding a chimeric antigen receptor>

[0213] The present invention provides a vector comprising the above-mentioned polynucleotide. The vector of the present invention may be linear or circular, and examples thereof include viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors.

[0214] Examples of viral vectors include retrovirus vectors such as lentivirus, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, silbis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

[0215] Examples of the plasmid vector include plasmid vectors for animal cell expression such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0216] Episomal vectors are vectors that can replicate autonomously outside the chromosome. The specific means of using episomal vectors are well known (refer to Yu et al., Science, 2009, 324, 797-801 pages). As episomal vectors, for example, vectors containing sequences required for autonomous replication derived from EBV, SV40, etc. as vector elements can be cited. As vector elements required for autonomous replication, specifically genes for replication origins and proteins that encode proteins that bind to the replication origins and control replication, for example, for EBV, replication origins oriP and EBNA-1 genes can be cited, and for SV40, replication origins ori and SV40LT genes can be cited.

[0217] Examples of the artificial chromosome vector include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors.

[0218] Among these vectors, retroviral vectors are preferred from the viewpoints that genes can be easily and efficiently introduced even into slowly growing cells and that stable expression strains can be easily established.

[0219] In addition to the polynucleotide encoding the above-mentioned CAR, the vector of the present invention may also contain expression control sequences such as promoters, enhancers, poly A addition signals, terminators, replication origins, nucleotide sequences encoding proteins that bind to the replication origin and control replication, nucleotides encoding other proteins, etc.

[0220] The polynucleotide encoding the above-mentioned CAR and the nucleotide encoding other proteins described later are configured in a manner that can work downstream of the promoter, so that each polynucleotide can be efficiently transcribed. As the "promoter", for example, CMV (cytomegalovirus) promoter, SRα promoter, SV40 early promoter, LTR of retrovirus, RSV (Rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1α promoter, metallothionein promoter, heat shock promoter, etc. can be cited.

[0221] As "nucleotides encoding other proteins", for example, genes encoding marker genes such as fluorescent protein genes, reporter genes, drug resistance genes, genes of molecules involved in T cell activation such as cytokines, etc. can be listed. In addition, for example, by using DNA encoding IRES, 2A peptide sequences (e.g., 2A peptide (T2A) derived from Thosea asigna), the other proteins can be expressed in the polycistronic with the above-mentioned CAR.

[0222] Furthermore, in order to appropriately induce apoptosis of the administered CAR-expressing cells in the body of a subject (cancer patient, etc.) to which CAR-expressing cells are administered, the vector of the present invention may contain a suicide gene as a "nucleotide encoding other proteins". Examples of suicide genes include herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 (iCasp9). In addition, as drugs that activate the function of the gene, ganciclovir can be mentioned for HSV-TK, and AP1903, a dimerization-inducing compound (chemical induction of dimerization: CID), can be mentioned for iCasp9 (see Cooper LJ. et al., Cytotherapy. 2006; 8(2): 105-17, Jensen MC et al., Biol Blood Marrow Transplant. 2010 Sep; 16(9): 1245-56, Jones BS., Front Pharmacol. 2014 Nov 27; 5: 254., Minagawa K., Pharmaceuticals (Basel). 2015 May 8; 8(2): 230-49, Bole-Richard E., Front Pharmacol. 2015 Aug 25; 6: 174).

[0223] <Cells expressing chimeric antigen receptor>

[0224] The present invention provides a cell expressing the CAR of the present invention. The cell of the present invention can be obtained by introducing the polynucleotide or vector encoding the CAR of the present invention into the cell.

[0225] The "cell" into which the polynucleotide or vector of the present invention is introduced is preferably a cell of mammalian origin, and for example, cells of human origin, or cells of non-human mammalian origin such as rodents (mice, rats, etc.), cattle, sheep, horses, dogs, pigs, monkeys, etc. can be used. The type of cell is not particularly limited, and cells taken from body fluids such as blood, bone marrow fluid, tissues such as spleen, thymus, lymph nodes, tumors, cancerous ascites, etc. can be used. As a preferred example, immune cells can be listed as cells for CAR expression.

[0226] As "immune cells", CD4 positive CD8 negative T cells, CD4 negative CD8 positive T cells, T cells prepared from iPS cells, αβ-T cells, γδ-T cells, NK cells, NKT cells, etc. can be listed. As long as lymphocytes or precursor cells as described above are included, various cell groups can be used. PBMC (peripheral blood mononuclear cells) taken from peripheral blood is one of the preferred immune cells. That is, in a preferred embodiment, PBMC is subjected to a gene introduction operation. PBMC can be prepared according to or in accordance with conventional methods.

[0227] It is preferred to pre-activate CAR expression cells before the introduction of the polynucleotide of the present invention. For example, anti-CD3 antibodies and anti-CD28 antibodies can be used to stimulate and activate CAR expression cells. More specifically, the culture vessel (e.g., a culture dish) coated with anti-CD3 antibodies and anti-CD28 antibodies can be used for culture, thereby applying stimulation using anti-CD3 antibodies and anti-CD28 antibodies. The stimulation can also be performed using magnetic beads coated with anti-CD3 antibodies and anti-CD28 antibodies (e.g., Dynabeads T-Activator CD3 / CD28 provided by VERITAS).

[0228] In order to improve the survival rate / proliferation rate of cells, it is preferred to use a culture solution to which a T cell proliferation factor is added during the activation process. IL-2, IL-15, IL-7, etc. are used as T cell proliferation factors. T cell proliferation factors such as IL-2, IL-15, IL-7 can be prepared according to conventional methods. In addition, commercial products can also be used. The use of T cell proliferation factors of animal species other than humans is not excluded, but T cell proliferation factors usually use human-derived (or recombinant) ones.

[0229] Typically, for its application (administration to an object), the polynucleotide of the present invention is introduced into the cell (polynucleotide of the present invention is introduced into lymphocytes) and then proliferated. For example, the polynucleotide of the present invention is cultured into lymphocytes (subcultured as needed) using a culture solution supplemented with a T cell proliferation factor. In addition to this culture, the same treatment (reactivation) as in the case of activation of CAR expression cells can also be performed.

[0230] In addition, a medium supplemented with serum (human serum, fetal bovine serum, etc.) can be used in the culture of the cells of the present invention, but by using a serum-free medium, cells having the advantages of high safety during clinical application and less prone to differences in culture efficiency caused by differences between serum batches can be prepared. When serum is used, it is preferred to use autologous serum, i.e., serum collected from a subject to whom the cells of the present invention are administered.

[0231] In addition, the cell of the present invention may also be a cell in which the function of a protein (immune checkpoint substance, etc.) that suppresses excessive immune response, etc. is suppressed. As the protein, for example, Programmed Death 1 (PD1), PD-L1, PD-L2, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5, etc.), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9 can be cited.

[0232] Furthermore, in order to facilitate allogeneic transplantation, the cells of the present invention may be cells in which the functions of endogenous proteins such as TCR and HLA are suppressed.

[0233] As a method for inhibiting the function of a protein, a knockout method using a gene encoding the protein as a target, a method using a dsRNA (double-stranded RNA, such as siRNA) complementary to the transcription product of the gene, a method using an antisense RNA complementary to the transcription product, and a method using an RNA having a ribozyme activity that specifically cleaves the transcription product can be cited. In addition, a genome editing method that is a method for transforming a target gene using a site-specific nuclease (e.g., DNA double-stranded cleavage enzymes such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-Cas9, etc.) is also suitable for use in order to inhibit the function of the substance.

[0234] As a method for introducing the polynucleotide or vector of the present invention into cells, liposome transfection, microinjection, calcium phosphate method, DEAE-dextran method, electroporation, gene gun method, etc. can be cited. In addition, when the vector of the present invention is a retroviral vector, appropriate packaging cells can be selected based on the LTR sequence and packaging signal sequence possessed by the vector, and retroviral particles can be prepared using the packaging cells. As packaging cells, for example, PG13, PA317, GP+E-86, GP+envAm-12, Psi-Crip can be cited. In addition, 293 cells and 293T cells with high transfection efficiency can also be used as packaging cells.

[0235] In addition, after the polynucleotide or vector of the present invention is introduced into cells, the expression of CAR in the cells can be confirmed by known methods such as flow cytometry, RT-PCR, Northern blotting, Western blotting, ELISA, fluorescent immunostaining, etc.

[0236] <Drug Composition>

[0237] The present invention also provides a method for treating or preventing a disease associated with the Eva1 protein (e.g., cancer), comprising administering to a subject a pharmaceutical composition (e.g., an anticancer agent) having an antibody of the present invention, a cell expressing ADC or CAR (hereinafter also simply referred to as “the antibody of the present invention, etc.”) as an active ingredient, and a therapeutically or preventively effective amount of the antibody of the present invention, etc.

[0238] The disease associated with the Eva1 protein that is the target of the antibody of the present invention may be any disease in which the expression of the Eva1 protein is involved in the onset, development of symptoms, aggravation, etc., and examples thereof include cancer.

[0239] In addition, the "cancer" that is the target of the antibody of the present invention only needs to express at least human Eva1, and as long as the protein is expressed, it may be a solid tumor, or it may be a leukemia, lymphoma, etc. More specifically, gliomas (tumors arising from neural stem cells, neural progenitor cells, and glial cells, for example, glioblastoma multiforme (GBM), astrocytoma (astrocytoma), medulloblastoma, ependymoma, oligodendroglioma, choroid plexus papilloma, especially anaplastic astrocytoma, anaplastic oligoastrocytoma, anaplastic oligodendroglioma), pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, bile duct cancer, hepatocellular carcinoma, breast cancer, lung cancer, kidney cancer, urothelial carcinoma, testicular cancer, prostate cancer, head and neck cancer, thyroid cancer, skin cancer, cervical cancer, uterine end cancer, ovarian cancer, hemangioma, myeloma, plasmacytoma, melanoma (melanoma). In addition, such cancer may be primary or metastatic, and the cancer involved in the present invention also includes cancer stem cells.

[0240] The "pharmaceutical composition" of the present invention may include other pharmacologically acceptable ingredients in addition to the antibody of the present invention, etc. As such other ingredients, for example, carriers, emulsifiers, wetting agents, pH buffering agents, culture media, excipients, disintegrants, buffers, isotonic agents, suspending agents, solubilizers, pain-relieving agents, stabilizers, preservatives, and preservatives may be cited. More specifically, as other pharmacologically acceptable ingredients, in the case of liquid preparations such as injections, aqueous solutions (physiological saline, water for injection, phosphate buffer, aqueous glucose solution, aqueous glycerol solution, etc.), aluminum hydroxide, etc. may be cited as examples. In addition, in the case of freeze-dried preparations, sugars (mannitol, lactose, sucrose, etc.), albumin, etc. may be cited as examples, but are not limited to these. Furthermore, the pharmaceutical composition of the present invention may be in the form of a kit that can be mixed before the above-mentioned ingredients are administered. In addition, when used as an injection, it may be in the form of a syringe.

[0241] The pharmaceutical composition of the present invention may contain only the antibody of the present invention as an active ingredient, or may contain the antibody and at least one other anticancer agent. In addition, the antibody of the present invention may be administered together with other anticancer agents, thereby enhancing the antitumor effect. Other anticancer agents used for such purposes may be administered to the subject simultaneously, separately, or continuously with the antibody of the present invention, or may be administered at different administration intervals. As such "anticancer agents", for example, immune checkpoint inhibitors (anti-PD-1 antibodies, etc.), gemcitabine, S-1, erlotinib, 5-FU, temozolomide, leucovorin, irinotecan (CPT-11), oxaliplatin, albumin paclitaxel, carboplatin, paclitaxel, pemetrexed, sorafenib, vinblastine, LH-RH analogs (leuprolide, goserelin, etc.), estramustine / phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), and the drugs described in International Publication No. 2003 / 038043 are listed, and the drugs are not limited as long as they are drugs with antitumor activity. Furthermore, in the case of a pharmaceutical composition comprising the cells of the present invention, as other components, dimethyl sulfoxide (DMSO), serum albumin, etc. may be included for the protection of cells, and antibiotics, etc. may be included to prevent the mixing of bacteria. Furthermore, for the purpose of cell activation, proliferation or differentiation induction, cytokines, growth factors, T cell activating factors such as steroids, vitamins, immunostimulators, and immune checkpoint inhibitors may be included, and the cells of the present invention may be used in combination with these other components.

[0242] The method of administering the pharmaceutical composition varies depending on the type of substance administered, the form of the composition, the age, weight, sex, health status, etc. of the subject, and can be administered by any administration route of non-oral administration (e.g., intravenous administration, arterial administration, local administration) or oral administration. The preferred method of administration is non-oral administration, more preferably intravenous administration. In addition, local administration may be performed instead of systemic administration. As a local administration, direct injection into the target tissue, organ, or organ can be exemplified.

[0243] The dosage of the pharmaceutical composition can be varied according to the age, weight, sex, health status, degree of symptom development of the subject and the components of the pharmaceutical composition administered. In addition, the administration schedule can also be appropriately adjusted according to these conditions. In addition to a single administration, it can also be continuously or regularly administered multiple times. Generally, when the antibody is administered intravenously, an adult is administered 0.1 to 1000 mg per kg of body weight per day, preferably 1 to 100 mg. In addition, when ADC is administered, the amount of the drug contained is converted to 0.001 to 1000 mg per kg of body weight per day for an adult, preferably 0.01 to 100 mg. As the number of administrations of these antibodies or ADCs, for example, it can be administered multiple times at intervals of once or once a day to once a year (for example, every week, every 10 to 30 days, every 1 month, every 3 to 6 months, every half a year, every 1 year).

[0244] In addition, the dosage of cells to be administered may be 1×10 3 ~1×10 10 (preferably 1×10 4 ~1×10 9 , more preferably 1×10 5 ~1×10 8 The administration interval may be, for example, every week, every 10 to 30 days, every month, every 3 to 6 months, every half year, every year, etc. In addition, the cells of the present invention can proliferate autonomously in the body of the subject to be administered, so the administration may be only once. In addition, the number of cells of the present invention in the body may be detected after administration, and the administration time may be determined based on the result.

[0245] In addition, when the treatment target is the brain, the presence of the blood-brain barrier (BBB) ​​often becomes a problem. However, in people with glioblastoma multiforme (GBM) and the like, the BBB in the normal brain is not formed in the blood vessels in the brain tumor where angiogenesis occurs, so the antibody can be delivered to the GBM by intravenous injection or the like.

[0246] In addition, even when the BBB is functioning, the antibody of the present invention can be administered by bypassing the BBB. For example, a method of inserting a cannula by positioning a brain surgery method, and directly administering to a glioma through the cannula can be cited. In addition, a method of embedding a drug delivery system containing the antibody of the present invention into the brain can be cited (Gill et al., Nature Med. 9: 589-595 (2003) etc.). Further included is the use of a vector containing a gene encoding the antibody of the present invention to transfect neurons across the BBB (U.S. Patent Application Publication No. 2003 / 0083299 etc.).

[0247] In addition to the above-mentioned method of avoiding the BBB, a method of administering the antibody of the present invention by containing or binding to a brain barrier permeable substance can also be used. As a brain barrier permeable substance, for example, liposomes bound to an antibody binding fragment that binds to a receptor on the vascular endothelium of the BBB (U.S. Patent Application Publication No. 20020025313, etc.), low-density lipoprotein particles (U.S. Patent Application Publication No. 20040204354, etc.), apolipoprotein E (U.S. Patent Application Publication No. 20040131692, etc.), transferrin (U.S. Patent Application Publication No. 2003 / 0129186, etc.), and a glycoprotein composed of 29 amino acids derived from rabies virus (see Kumar et al., Nature, July 5, 2007, Vol. 448, pp. 39-43) can be cited.

[0248] Furthermore, by controlling the activity of receptors or channels to administer the antibody of the present invention, the antibody can also be delivered to gliomas through the BBB. As this method, for example, the use of glucocorticoid blocking drugs to increase the permeability of the blood-brain barrier (U.S. Patent Application Publication No. 2002 / 0065259, U.S. Patent Application Publication No. 2003 / 0162695, U.S. Patent Application Publication No. 2005 / 0124533, etc.), activating potassium channels (U.S. Patent Application Publication No. 2005 / 0089473, etc.), inhibiting ABC drug transporters (U.S. Patent Application Publication No. 2003 / 0073713, etc.), and cationizing the antibody of the present invention (U.S. Patent No. 5,004,697, etc.) can be cited.

[0249] In the above, the method for administering the antibody of the present invention to the disease is not limited to this. In addition, for other diseases, similar to the above-mentioned brain diseases, a person skilled in the art can appropriately select a known method according to the disease to allow the antibody of the present invention to act on the affected area.

[0250] In the present invention, as a "subject" to which the antibody of the present invention is administered, for example, a person suffering from the above-mentioned cancer, a person at risk of recurrence of the above-mentioned cancer, a person at risk of suffering from the above-mentioned cancer, etc., can be cited. In addition, in the present invention, the so-called "treatment" includes alleviating the characteristic symptoms or accompanying symptoms (mildening) of the target disease such as cancer, preventing or delaying the deterioration of symptoms, etc. The so-called "prevention" refers to preventing or delaying the onset, expression, recurrence of a disease (disorder) or its symptoms, or reducing the risk of onset, expression, or recurrence.

[0251] Example

[0252] Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

[0253] <Example 1> Humanized Anti-Human Eva1 Antibody

[0254] An attempt was made to humanize an anti-human Eva1 mouse antibody (B2E5-48 mouse antibody described in Patent Document 2) using the materials and methods described below. The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of this antibody are shown in SEQ ID NOs: 21 and 27, respectively.

[0255] (Materials and methods)

[0256] 1.1 Analysis of variable regions and identification of CDRs

[0257] Using the IMGT Domain Gap Align tool (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi), the CDRs in the variable regions of the specific B2E5-48 mouse antibody were analyzed for the most consistent germline sequences.

[0258] 1.2 Molecular modeling

[0259] For VL and VH, molecular models were constructed using Absolute Antibody's in-house software based on homology to known antibody crystal structures. Images were created using PyMol.

[0260] 1.3 Analysis of risky sequences

[0261] Regarding the sequence of the antibody, specific risks were analyzed based on the motifs of the disclosed proteins. The analysis was performed using a system (Absolute Antibody Sequence Liability Tool) built using Microsoft Excel. In this software, risks were evaluated based on the motifs shown in the following table. In addition, in the following table, X represents any amino acid other than proline.

[0262] Table 4

[0263]

[0264] 1.4 Gene synthesis and cloning

[0265] As shown in Table 6 below, a total of 4 humanized heavy chains and 4 humanized light chains were designed. They were synthesized separately and cloned into human IgG1 heavy chain expression vector and human κ light chain expression vector. During transfection, the humanized sequences were combined as much as possible, and a total of 16 humanized antibodies were produced. In addition, as a control, a chimeric human IgG1 (a product in which the constant region of the B2E5-48 mouse antibody was replaced with a constant region of the human IgG1 type) was produced.

[0266] More specifically, the DNA sequences encoding the variable regions of the heavy and light chains were designed by adding appropriate restriction enzyme recognition sites at the 5' and 3' ends in a manner that allows cloning into AbsoluteAntibody's cloning vectors and expression vectors. Furthermore, these DNA sequences were optimized for codons in consideration of expression in human cells. Based on these sequences, after chemical synthesis, they were cloned into the Absolute Antibody's vectors of appropriate species and types. In addition, the correctness of the sequence was confirmed by sequencing by Sanger using DNASTAR Lasergene software to analyze the raw data. After this confirmation, plasmid DNA of appropriate size was prepared to prepare a sufficient amount of high-quality DNA required for transfection.

[0267] 1.5 Expression and purification

[0268] HEK293T cells were passaged to the stage most suitable for temporary transfection. Heavy and light chain expression vectors were introduced into the cells and cultured for 6 days. The culture was centrifuged at 4000rpm, the culture supernatants were recovered and filtered with a 0.22M filter. The first stage of purification was performed by protein A affinity chromatography, after elution with citric acid pH 3.0 buffer, it was neutralized with 0.5M Tris, pH 9.0. The eluted protein was exchanged into PBS using a desalting column buffer. The antibody concentration was determined by UV spectroscopy and concentrated as needed. In addition, the chimeric human IgG1 was prepared by expressing it not only in HEK293T cells but also in CHO cells.

[0269] 1.6 Antibody Analysis

[0270] The purity of the antibody was determined by SDS-PAGE and HPLC. SEC-HPLC was performed using an Agilent 1100 series device using an appropriate size exclusion column (SEC). The expression titer of the antibody was determined using Protein A HPLC.

[0271] 1.7 Surface plasmon resonance analysis

[0272] The dissociation constant and association constant of the humanized antibody and the like were analyzed by surface plasmon resonance (SPR, product name: BiaCore T200 (manufactured by GE Healthcare)).

[0273] Specifically, first, human IgG capture antibody was immobilized on a CM5 chip by amino coupling method. This immobilization was performed on both the reference unit and the active unit. Then, kinetic analysis of anti-Eva1 antibody and hEva1 was performed by single cycle method using the following measurement conditions.

[0274] Measurement conditions

[0275] / Sample compartment temperature:10℃

[0276] / Running buffer: HBS-EP+buffer, pH7.4

[0277] / Capturing molecule: Human IgG antibody, reference & active unit

[0278] / Ligand: Various antibodies 5nM, active units only

[0279] / Analyte: hEva1-mouse Fc1, 5, 25, 125, 250nM, reference & active unit

[0280] / Regeneration buffer: 3M MgCl2, reference & active units

[0281] / Blank (Analyte zero concentration) 3 cycles

[0282] In addition, the cycle conditions are shown in the following table.

[0283] Table 5

[0284]

[0285] The following shows the results obtained using the above materials and methods.

[0286] (result)

[0287] 2.1 Sequence analysis

[0288] The sequences of VH and VL of the B2E5-48 mouse antibody were analyzed by comparing them with the sequences of all known antibody germ cells using the IGMT Gap Align tool. The results are not shown in the figure, but these sequences are the closest to mouse sequences, especially VH is the closest to the IGHV1-53 family sequence, and VL is the closest to the IGKV4-68 sequence. In addition, CDRs were assigned using the IMGT definition.

[0289] 2.2 Selection of germ cell lines

[0290] The sequences of VH and VL were compared with the database of human germ cell lines of Absolute Antibody and analyzed. The results are shown in Table 6 below as germ cell lines selected as humanized frameworks. In this table, the homology (%) of the germ cell lines of the heavy chain and light chain selected as humanized frameworks and the original mouse VH and VL is shown.

[0291] Table 6

[0292]

[0293] 2.4 CDR Transplantation Aggregation

[0294] The sequences of VH and VL were analyzed using a CDR transplantation algorithm and transplanted into a human germ cell series in which the CDRs of the B2E5-48 mouse antibody were selected. CDRs are primarily defined as being involved in binding to antigens, but amino acids located outside of a region known as a framework region may also be directly involved in binding or play a role in properly configuring the CDR. Thus, in order to maintain binding, a structure induction method was used to determine which amino acid in the amino acids of the framework region was left as the mouse-derived (original) amino acid residue. An overview of the sequence generated by the results is shown in Table 7. In addition, in the table, the underlined sequences in the sequence are represented as CDRs in each variable region.

[0295] Table 7

[0296]

[0297] 2.5 Analysis of risky sequences

[0298] In order to confirm that there were no serious risks (problems) in the humanized sequence, the original mouse antibody sequence and the humanized sequence were substituted into the Absolute Antibody Sequence Liability Tool.

[0299] As a result, the glycosylation motifs and free cysteine, which are the sequences with the highest medium risk shown in Table 4, were not detected in the mouse antibody and the humanized antibody. On the other hand, the deamidated and isomerized motifs were confirmed to exist in these antibodies. However, although these motifs are considered to be high-risk as shown in Table 4, they are common in ordinary antibodies. In addition, although modifications based on these motifs may cause confusion during manufacturing, in most cases they can be eliminated by management methods, etc. Furthermore, if necessary, these motifs can also be removed by mutation introduction. In addition, sequences with medium and low risks were tested from the perspective of obtaining more complete antibodies, but basically no problems have been reported in the antibody manufacturing process. It was determined that there were no special problems with the sequences of the mouse antibody and the humanized antibody.

[0300] 3. Antibody Production and Analysis

[0301] As described above, 16 humanized antibodies were attempted to be produced. As a result, no antibodies were detected in the supernatant of 6 of them (H1-L1, H1-L2, H1-L3, H2-L1, H3-L1, H4-L1), and the antibody concentration (Titer (mg / L) below) in the supernatant of the other 3 (H2-L3, H3-L3, H4-L2) was also significantly low. It is considered that these normal antibodies were not formed and were not sufficiently secreted into the culture supernatant.

[0302] Table 8

[0303]

[0304] In addition, the binding affinity of the antibody was analyzed by SPR method. The results are shown in the above table. The average dissociation constant (K D ) are both 10- 7 In particular, among humanized antibodies, H2-L2, H1-L4, H2-L3, and H2-L4 were at the same level as chimeric antibodies (produced by HEK293T cells). In addition, considering the above antibody concentrations, it is suggested that H2-L2, H2-L4, and H3-L4 are useful in the antibody uses described below.

[0305] <Reference example>Expression of human Eva1 protein

[0306] (Analysis of Eva1 protein expression in normal human blood cells)

[0307] In order to analyze the usefulness of anti-Eva1 antibodies for medical use, first, the expression of Eva1 protein in various blood cells that are sites of CAR expression was evaluated in order to analyze the usefulness in chimeric antigen receptor (CAR) described below.

[0308] Specifically, healthy control peripheral blood was hemolyzed (BD FACS Lysing Solution), and then stained for CD4, CD8, CD19, neutrophils (by scatter plot gate), and monocytes (by scatter plot gate), and stained with mouse anti-human Eva1 antibody and secondary antibody, and analyzed by flow cytometry.

[0309] The results are as follows Figure 1 As shown, no Eva1 protein expression was found in any of CD4, CD8 T lymphocytes, CD19 B lymphocytes, neutrophils, and monocytes. This suggests that blood cells expressing CAR with Eva1 protein as an antigen are likely not to target these cells.

[0310] (Analysis of Eva1 protein expression in tumors)

[0311] Next, the expression of Eva1 protein in various tumors was evaluated. Specifically, first, various tumor cell lines were detached from the culture flask to prepare a cell suspension, and then Eva1 protein was stained in the same manner as described above (Analysis of Eva1 protein expression in normal human blood cells).

[0312] The results are as follows Figure 2A As shown, although the expression of Eva1 protein was not confirmed in OVTOKO, PA-1, and T98G, the expression of Eva1 protein was confirmed in various other tumor cell lines.

[0313] In addition, the expression of Eva1 protein in cells isolated from surgical specimens of intrahepatic cholangiocarcinoma was also analyzed. Specifically, a cell suspension was prepared from the tumor part collected from the surgical specimen of intrahepatic cholangiocarcinoma (MACS Tumor Dissociation Kit, human, Miltenyi Biotec). Tumor cells were further purified using the Tumor cell isolation kit and human, Miltenyi Biotec, and Eva1 protein was stained in the same way as above, and flow cytometric analysis was performed.

[0314] The results are as follows Figure 2B As shown, Eva1 was positive in 3 of 3 cases of intrahepatic cholangiocarcinoma.

[0315] <Example 2> Chimeric Antigen Receptor Using Mouse Anti-Eva1 Antibody Sequence

[0316] (Preparation of Eva1-CAR using anti-human Eva1 mouse antibody sequence)

[0317] Next, in order to analyze the usefulness of a chimeric antigen receptor (CAR) targeting the Eva1 protein, such a CAR was prepared. Figure 3A As shown, a single-chain antibody was constructed using VL and VH of an anti-human Eva1 mouse antibody (the above-mentioned B2E5-48 mouse antibody). Then, VL-connector-VH and VH-connector-VL were made, and the CD28 transmembrane region (CD28TM), CD28 intracellular domain (CD28IC), and CD3zeta were connected to construct CAR molecules via a 15aa short spacer domain (consisting of amino acids at positions 268 to 282 recorded in sequence number 58 or an amino acid sequence recorded in sequence number 59) or a 232aa long spacer domain (consisting of amino acids at positions 268 to 499 recorded in sequence number 58 or an amino acid sequence recorded in sequence number 63).

[0318] Furthermore, EGFR without an intracellular domain (truncated EGFR, tEGFR) is bound to the T2A sequence. This is transcribed as one mRNA, and then after translation, it is cleaved into CAR and tEGFR molecules at the T2A sequence, distributed in a 1:1 ratio. tEGFR can be used as a gene transfer marker and in the selection of cells into which the gene has been transferred.

[0319] Specifically, Eva1-CAR (the order of the variable regions from the N-terminal side is VL, VH, and has the long spacer domain) has the structure shown in the following Table 9.

[0320]

[0321] In Table 9, the numbers in the "cDNA" item indicate the positions (bp) in the DNA sequence described in SEQ ID NO: 57, and the numbers in the "polypeptide" item indicate the positions (amino acids) in the sequence described in SEQ ID NO: 58.

[0322] (Production of Eva1-CAR-T using anti-human Eva1 mouse antibody sequence)

[0323] Then, if Figure 3BAs shown, blood was collected from healthy donors on day 0 and purified into CD8 positive cells using CD8 microbeads (Miltenyi Biotec). Then, CD3 / CD28 beads (Invitrogen) were mixed at a ratio of T cells: beads = 1: 3 to activate T cells. On the 3rd day, gene introduction was performed using a retrovirus capable of gene introduction into the sequence encoding the CAR molecule (the construction of the retrovirus was carried out in the same manner as described in Mol Ther 2021Sep 1; 29(9): 2677-2690.). Specifically, retrovirus was added dropwise to a plate coated with retronectin and rotated at 2100 rpm for 120 min. After the plate was then washed with PBS, stimulated CD8+T cells were inoculated and rotated at 800 rpm for 2 min. On the 4th day, it was moved from the plate coated with retronection to a normal culture dish. On the 7th day, the cells were recovered, biotinylated EGFR antibodies (cetuximab) were added, and tEGFR positive cells were purified using MACS anti-biotin beads. The purified cells were continuously cultured from day 7 to day 10. On day 10, the cells were counted and stored. A portion of the cells were stimulated again with CD3 / CD28 beads on day 10 and the experiment was performed on days 17-20.

[0324] In addition, the same method was used for the gene transfer protocol below. In addition, 50 U / ml of human / recombinant IL-2 was added and cultured on days 1, 4, 7, 10, 13, 16, (19).

[0325] (Eva1-CAR-T gene transfer efficiency using anti-human Eva1 mouse antibody sequence)

[0326] The gene transfer efficiency on day 7 performed in this manner is shown in FIG. Figure 3C and 3D .exist Figure 3C In the figure, representative plots are shown for LH-S (CAR using VL-VH with a shorter spacer), HL-S (CAR using VH-VL with a shorter spacer), LH-L (CAR using VL-VH with a shorter spacer), and HL-L (CAR using VH-VL with a longer spacer). (EGFR on the vertical axis represents cells into which gene has been introduced, which are stained once with biotinylated cetuximab and then stained twice with streptavidin-PE.) In addition, Figure 3D The results of the same experiment using cells from 5 healthy human donors are shown in Figure 2. Figure 3C and 3D As shown, the gene introduction efficiency is higher when the spacer is longer.

[0327] (Cytokine production ability of Eva1-CAR-T using anti-human Eva1 mouse antibody sequence)

[0328] Next, the cytokine production capacity of Eva1-CAR-T using the anti-human Eva1 mouse antibody sequence was analyzed. Specifically, the CAR-T prepared as described above was cultured until day 17, and the cells were co-cultured with various major cell lines at a 1:1 ratio. After 6 hours of co-culture, fixation / intracellular staining (BD cytofix / cytoperm buffer) and staining with IFN-gamma (IFN-γ) were performed. The results are shown in Figure 3E .

[0329] like Figure 3E As shown, IFN-gamma production was not observed in the imitation T cells into which only the EGFR gene was introduced, but IFN-gamma production was observed in Eva1-CAR-T. In any Eva1-CAR-T, IFN-gamma production was not observed for Eva1-negative cell lines, but IFN-gamma production was observed for Eva1-positive cell lines, confirming the antigen specificity of Eva1-CAR-T. Stronger IFN-gamma production was observed in the case of longer spacers than in the case of shorter spacers.

[0330] (Optimization of spacer length of Eva1-CAR-T)

[0331] like Figure 3F As shown, mouse antibody-derived Eva1-CAR-T with 6 different lengths of spacer regions was produced. The spacer lengths were 0aa, 15aa (SEQ ID NO: 59), 30aa (SEQ ID NO: 60), 60aa (SEQ ID NO: 61), 125aa (SEQ ID NO: 62), and 232aa (SEQ ID NO: 63). 15aa is the same as the short version in the previous experiment, and 232aa is the same as the long version in the previous experiment. It was integrated into the plasmid, and retrovirus was prepared in the same way, and the gene was introduced into CD8 positive cells in the same way as the above method.

[0332] (Gene transfer efficiency in the preparation of Eva1-CAR-T with 6 different spacer lengths)

[0333] The gene transfer efficiency (EGFR staining) on ​​day 7 performed in this manner is shown in FIG. Figure 3G The results showed that the three CARs with spacer lengths of 30aa, 125aa, and 232aa had higher gene transfer efficiency than the other three (n=5).

[0334] (Expansion efficiency of Eva1-CAR-T with 6 different spacer lengths (CD3 / CD28 bead stimulation))

[0335] Furthermore, the expansion factor when the Eva1-CAR-T cells were continuously stimulated with CD3 / CD28 beads and cultured until day 17 is shown in FIG. Figure 3H The results showed that there was no difference in the expansion efficiency stimulated by CD3 / CD28 beads (n=5).

[0336] (Cytokine production capacity of Eva1-CAR-T with 6 different spacer lengths)

[0337] The experiment was conducted using the Eva1-CAR-T cells at day 17. Specifically, the cells were co-cultured with the tumor cell line at a ratio of 1:1, and the cells were fixed / stained by the same method as above after 6 hours, and intracellular IFN-gamma and IL-2 were evaluated by flow cytometry. The results are shown in Fig. 3I This figure shows the proportion of IFN-gamma and IL-2 positive cells among CD8 positive cells.

[0338] like Fig. 3I As shown, three Eva1CAR-T cells using spacer lengths of 30aa, 125aa, and 232aa showed excellent cytokine production, among which Eva1CAR-T cells with a spacer of 232aa showed the best cytokine production ability.

[0339] (Expansion efficiency of Eva1-CAR-T with 6 different spacer lengths (Eva1 stimulation, no IL-2))

[0340] Eva1-positive HuCCT-1 cells were irradiated with 100 Gy of radiation and co-cultured with CAR-T cells at a 1:1 ratio to analyze their expansion efficiency. The results are shown in Figure 3J and 3K . Figure 3J Amplification efficiency in each time series is shown in . Figure 3K The expansion ratio on day 4 is plotted in FIG. Figure 3J and 3K As shown, cells expanded well when spacer lengths of 15aa, 30aa, and 232aa were used (n=5, CAR-T cells were prepared from peripheral blood of 5 different donors).

[0341] (Expansion efficiency of Eva1-CAR-T with 6 different spacer lengths (Eva1 stimulation, with IL-2))

[0342] Similarly, Eva1-positive HuCCT-1 cells were irradiated with 100 Gy of radiation and co-cultured with CAR-T cells at a ratio of 1:1. Then, IL-2 50 U / ml was added every 3 days on days 1, 4, and 7 for culture. The results are shown in Figure 3L and 3M . Figure 3L The amplification efficiency in each time series is shown in Figure 3M The expansion ratio on day 18 is plotted in Figure . Figure 3L and 3M As shown, a tendency of good cell expansion was confirmed when the spacer lengths were 15aa, 30aa, and 232aa (n=5, CAR-T cells were prepared from peripheral blood from 5 different donors).

[0343] <Example 3> Chimeric Antigen Receptor Using Anti-Human Eva1 Humanized Antibody Sequence

[0344] (Production of humanized Eva1-CAR-T (hEva1CAR-T) using anti-Eva1 humanized antibody sequence)

[0345] Next, a total of 6 types of Eva1CAR-T were produced using the above humanized antibody sequence. Specifically, CARs with a spacer of 232aa were designed for L2-linker-H (L2H2), H2-linker-L2 (H2L2), L4-linker-H (L4H2), H2-linker-L4 (H2L4), L4-linker-H3 (L4H3), and H3-linker-L4 (H3L4). Then, the DNA encoding these CARs was integrated into plasmids, and retroviruses were prepared in the same manner as above, and gene introduction was performed.

[0346] (Gene transfer efficiency in the preparation of hEva1-CAR-T using six types of anti-human Eva1 humanized antibodies)

[0347] The gene transfer efficiency (EGFR staining) on ​​day 7 performed in this manner is shown in FIG. Figure 4A As shown in the figure, it was found that the gene transfer efficiency was higher when L2H2 and L4H3 were used than the other four types (n=4, CAR-T cells were prepared from peripheral blood of four different donors).

[0348] (Expansion efficiency of hEva1-CAR-T using 6 anti-Eva1 humanized antibodies (CD3 / CD28 bead stimulation))

[0349] The expansion factor of hEva1CAR-T cells prepared above was shown in Figure 1 when they were cultured for 17 days under continuous CD3 / CD28 bead stimulation. Figure 4B As shown in the figure, the expansion efficiency using CD3 / CD28 beads stimulation was better when L2H2, H2L2, and H2L4 were used than the other three (n=5, CAR-T cells were prepared from peripheral blood of 5 different donors).

[0350] (Cytokine production capacity of hEva1-CAR-T using 6 anti-Eva1 humanized antibodies)

[0351] The experiment was conducted using hEva1-CAR-T cells at day 17. Specifically, the cells were co-cultured with tumor cell lines at a ratio of 1:1, and 6 hours later, the cells were fixed / stained in the same manner as above, and intracellular IFN-gamma and IL-2 were evaluated by flow cytometry. The results are shown in Figure 4C and 4D These figures show the proportion of CD8-positive cells that are positive for each cytokine. Figure 4C and 4D As shown, there was no significant difference between the hEva1-CAR-T cells, but the CAR-T cells using L2H2 showed the lowest cytokine production against the Eva1-negative cell line OVTOKO.

[0352] (Expansion efficiency of hEva1-CAR-T using 6 anti-Eva1 humanized antibodies (Eva1 stimulation))

[0353] In the same manner as above, Eva1-positive HuCCT-1 cells were irradiated with 100 Gy of radiation and co-cultured with hEva1CAR-T cells at a ratio of 1:1. The results obtained are shown in Figure 4E In this figure, the left side shows the results of culture without IL-2, and the right side shows the results of culture with the addition of IL-2 50 U / ml every 3 days on days 1, 4, and 7. Figure 4E As shown in the figures, CAR-T cells using L2H2 showed the best expansion after stimulation (n=5, CAR-T cells were prepared from peripheral blood of 5 different donors).

[0354] (Study on the intracellular domain of L2H2 hEva1-CAR-T)

[0355] As described above, regarding CAR using humanized antibodies, CAR-T using L2H2 is excellent in antigen specificity and cell proliferation. In addition, as shown in Example 2, regarding CAR using mouse-derived antibodies, in culture in the absence of IL-2, CAR-T with a spacer length of 232aa is the best in terms of cell proliferation and cytokine production. On the other hand, in culture in the presence of IL-2, CAR-T with a spacer of 15aa has the best cell proliferation.

[0356] So then, if Figure 4FAs shown in the figure, six types of CAR-T were produced by connecting three intracellular domains (CD28IC, 4-1BBIC, CD79A / CD40IC) via a single-chain variable region (scFv) composed of L2H2 and a spacer of two spacer lengths (S: short spacer, L: long spacer).

[0357] (Gene transfer efficiency in the production of 6 types of hEva1-CAR-T)

[0358] In the above 6 CAR-T preparations, the gene transfer efficiency (EGFR staining) on ​​day 7 is shown in Figure 4G As shown in the figure, the gene introduction efficiency is high in the case of 4-1BB-L and CD79A / CD40-L. In addition, similarly to the above, the gene introduction efficiency of CAR with a long spacer (L) is higher than that of a short spacer (S), which is also reproduced here (n=5, CAR-T was made from peripheral blood from 5 different donors).

[0359] (Cytokine production capacity of 6 L2H2 hEva1-CAR-T cells)

[0360] The experiment was conducted using the hEva1-CAR-T cells on day 17. Specifically, the cells were co-cultured with the tumor cell line at a ratio of 1:1, and after 4 hours, the cells were fixed / stained in the same manner as above, and intracellular IFN-gamma and IL-2 were evaluated by flow cytometry. The results are shown in Figure 4H and 4I These figures show the proportion of CD8-positive cells that are positive for each cytokine. Figure 4H and 4I As shown, there was no significant difference between the individual hEva1-CAR-Ts, but the CAR with a long spacer had a higher cytokine production ability than the short spacer, which was also reproduced here (n=5, CAR-Ts were prepared from peripheral blood from 5 different donors).

[0361] (Post-stimulation amplification efficiency of 6 types of L2H2 Eva1-CAR-T (Eva1 stimulation))

[0362] In the same manner as above, Eva1-positive HuCCT-1 cells were irradiated with 100 Gy of radiation and co-cultured with hEva1-CAR-T cells at a ratio of 1:1. The results are shown in Figure 4J In this figure, the left side shows the results of culture without IL-2, and the right side shows the results of culture with IL-2 50 U / ml added every 3 days on days 1, 4, and 7. Figure 4JAs shown, both short and long versions of 4-1BB and CD79A / CD40 showed basically equally good results (n=5, CAR-T cells were made from peripheral blood from 5 different donors).

[0363] (Co-culture experiment using 6 types of L2H2 hEva1-CAR-T)

[0364] L2H2 hEva1-CAR-T cells were co-cultured with non-irradiated RPMI-8226 (Myeloma cell line) at an E:T ratio of 1:16. RPMI-8226 cells were labeled with CellTrace Violet and then cultured to allow for discrimination between RPMI8226 and CAR-T cells. A portion of the cells was collected 48, 72, and 96 hours after the start of culture and analyzed using flow cytometry. The results are shown in Figure 4K This figure shows the proportion of tumor cells in all cells. In addition, since tEGFR is a simulated CAR-T cell into which only the tEGFR gene is introduced, the number of tumor cells continues to increase. No significant differences were observed in the intracellular domains.

[0365] (Cytotoxicity test (chromium release test) using 6 types of L2H2 hEva1CAR-T)

[0366] L2H2 hEva1-CAR-T 51 Various tumor cell lines labeled with Cr were co-cultured for 4 hours, and the Cr released into the supernatant was measured. 51 Cr was used to investigate the proportion of cells damaged (chromium release test). The E:T ratio was 10:1, 3:1, and 1:1. The results are shown in Figure 4L and 4M .like Figure 4L As shown in Figure 2, significant toxic activity by hEva1-CAR-T was confirmed for cell lines considered Eva1-positive. In addition, no clear differences were confirmed for each intracellular domain. Figure 4M As shown, if the horizontal axis takes the Eva1-MFI of each cell line, the correlation with the MFI can be seen.

[0367] As described above, for hEva1-CAR-T with L2H2 as the antigen recognition site, the difference in reactivity caused by the difference in intracellular domains was evaluated. The results of in vitro confirmation of cell proliferation, cytokine release, and cytotoxic activity showed that CARs with intracellular domains of 4-1BB and CD79A / CD40 were superior in terms of cell proliferation.

[0368] (Evaluation of anti-tumor activity of hEva1-CAR-T using a cancer-bearing model)

[0369] The anticancer effect was evaluated using L2H2 hEva1-CAR-T cells prepared from peripheral blood of two different donors. Specifically, the evaluation was performed as follows.

[0370] 1×10 6 CFPAC1 cells (human pancreatic cancer cell line) or H1975 (human lung adenocarcinoma cell line) expressing the firefly luciferase gene were inoculated intraperitoneally. 6 tEGFR-transfected cells (simulated CAR-T cells with only tEGFR gene introduced, control) or L2H2 hEva1-CAR-T were intravenously administered via the tail vein to evaluate the anti-tumor activity of the CAR-T. In this evaluation, bioluminescence imaging (BLI) was performed using the IVIS spectrum system to evaluate the progression of the tumor. The results are shown in Figures 4N to 4Q Body weight was also measured in parallel.

[0371] In addition, the anticancer effect on cholangiocarcinoma was also evaluated using L2H2hEva1-CAR-T cells produced from peripheral blood of two donors different from those used above.

[0372] Specifically, first, 2×10 6 The firefly luciferase gene was introduced into HuCCT cells (human cholangiocarcinoma cell line) and then subcutaneously transplanted. After 2 weeks, the tumor diameter and body weight were measured and 2×10 6 or 5×10 6 tEGFR-introduced cells (control) or L2H2 hEva1-CAR-T were intravenously administered via the tail vein, and the anti-tumor activity of the CAR-T was evaluated using BLI as an indicator in the same way as above. Tumor volume was measured using a vernier caliper to measure the long and short diameters of the subcutaneous tumor and calculated by "long diameter × short diameter × short diameter × 1 / 2 of the tumor". The results are shown in Figure 4R and 4S In addition, body weight was also measured in parallel.

[0373] In addition, in the example of administration of any of the above-mentioned cancer-bearing models, as L2H2 hEva1-CAR, an antigen recognition site (L2H2) and a transmembrane / intracellular domain (containing a domain of 4-1BB or CD79A / CD40) were connected with the above-mentioned short spacer. In addition, T cells expressing this CAR were prepared by the same method as above, and Figure 3BOn the 10th day, the culture medium was replaced with a cell storage solution (Cell banker) without restimulation with beads, and stored in liquid nitrogen. The frozen CAR-T was thawed and cultured overnight, and then restimulated with beads. After 1 week of bead stimulation, it was administered to cancer-bearing mice as described above.

[0374] If Figures 4N to 4S As shown in the results, the anticancer effect of L2H2hEva1-CAR-T was confirmed for pancreatic cancer, lung cancer, and bile duct cancer. In addition, although not shown in the figure, there was no abnormality or significant difference in the weight change of each mouse during the administration period, and no adverse effects on the subjects were confirmed.

[0375] <Example 3> Antibody-drug conjugate comprising anti-human Eva1 humanized antibody

[0376] An antibody-drug conjugate (ADC) was produced using the above-mentioned humanized B2E5-48 antibody H2-L2 (hereinafter also referred to as B2E5-H2-L2).

[0377] Specifically, first, B2E5-L2 expression vector TPG986 and B2E5-H2 expression vector TPG987 were prepared using pcDNA3.4, respectively, introduced into E. coli JM109 for expression, and purified using NucleoBond Xtra Midi Kit (MACHEREY-NAGEL).

[0378] Next, B2E5-H2-L2 was temporarily expressed in ExpiCHO cells using the vector and the ExpiFectamine CHO Transfection System. The resulting culture supernatant was then passed through a protein A column to adsorb the antibody. After dissolution at low pH, the antibody was neutralized and purified using Capto adhere, followed by ultrafiltration. In addition, the molecular weight, sequence, and binding ability to Eva1 of the resulting antibody were confirmed.

[0379] Then, if Figure 5 As shown, B2E5-H2-L2 was reacted with a linker (SMCC) to introduce an MCC group into the antibody, and then the linker was reacted with DM1.

[0380] Specifically, first, the solvent buffer of the B2E5-H2-L2 prepared above was exchanged into PBS6.5 / EDTA. Next, a DMSO solution (0.40 mL; approximately 5.1 equivalents per antibody molecule) of SMCC (1.84 mg) was added at room temperature, the antibody concentration of the reaction solution was adjusted to 20 mg / mL and reacted at room temperature for 2 hours using a tube / rotator. The reaction solution was purified to obtain an antibody induced by SMCC.

[0381] Next, PBS6.5 / EDTA (2.56 mL) and N2-deacetyl-N2-(3-mercapto-1-oxopropyl)-maytansine (4.67 mg; DM1, Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14, Item 4392) in DMA (dimethylformamide) solution (0.93 mL; approximately 5.8 equivalents per molecule of the antibody induced by SMCC) were added to the above solution at room temperature, and the antibody concentration of the reaction solution was adjusted to 10 mg / mL and reacted at room temperature for 16.5 hours using a tube / rotator, and purified to obtain a solution containing the target compound. Next, after purification to remove excess components, the solution was stored in a 6% sucrose, 0.02% Tween20, 10 mM sodium succinate solution (pH 5.0) at a concentration of 9.0 g / L.

[0382] Then, the molecular weight, sequence, binding ability to Eva1, drug-antibody ratio (DAR) and the like of the ADC were confirmed. In addition, DAR was calculated by measuring the absorbance at 252nm, which is the absorption peak of DM1, and the absorbance at 280nm, which is the absorption peak of the antibody, and weighting the influence of each on the absorption peak to establish a simultaneous equation, thereby calculating the concentration of the antibody and DM1, and determining the ratio of the number of molecules. As a result, the DAR in the ADC prepared above was 3.8.

[0383] <Example 4> Antitumor activity of antibody drug conjugates

[0384] The anti-tumor activity of the ADC (B2E5-ADC) comprising the anti-human Eva1 humanized antibody prepared above was evaluated as follows.

[0385] (B2E5-ADC cytotoxicity experiment on glioma)

[0386] Human glioma stem cells (hGIC) were seeded at 1000 cells per well in a 96-well plate. Next, 100 μl / well of 2-fold serially diluted B2E5-H2-L2, B2E5-ADC or Kadcyla (trastuzumab emtansine) was added. Culture was carried out in a 37°C CO2 incubator for 3 days. Then, 5 μl of MTT (5 mg / ml, Wako Pure Chemical Industries, Ltd.) was added to each well and cultured in a 37°C CO2 incubator for 2 hours. After discarding the culture medium, 90 μl of DMSO was added, the cells were dissolved, and the absorbance (570 nm) was measured using a Benchmark (Bio-Rad). The results are shown in Fig. 6A .

[0387] In addition, as a positive control (100%), the measured value in the wells without the antibody was used, and as a negative control (0%), the measured value obtained by adding 5 μl of 1% TritonX100 to the wells without the antibody was used.

[0388] (B2E5-ADC cytotoxicity experiment on pancreatic cancer)

[0389] The pancreatic cancer cell line (BxPC3) recovered from the culture dish by trypsin treatment was diluted to 5×10 4 cells / mL, 100 μL was inoculated in each well of a 96-well plate, and cultured in a CO2 incubator (37°C, CO2 concentration 5%) for 24 hours. The culture medium (RPMI-1640, Wako187-02705, 10% FBS) was aspirated with an aspirator, and 900 μL of fresh culture medium was added to each well. DM1 and B2E5-ADC were diluted with PBS (DM1: 3800nM~0.93nM, B2E5-ADC: 1000nM~0.24nM), and 10 μL of each was added to BxPC3. After culturing in a CO2 incubator for 48 hours, the LDH released from the dead cells was measured using the Cytotoxicity LDHAssay Kit-WST (Dojin Chemical). The results are shown in Figure 6B .

[0390] (B2E5-ADC cytotoxicity experiment on gastric cancer)

[0391] For the gastric cancer cell line (MKN74) cultured in a 10 cm cell culture dish, 1 mL of 2.5 g / L-trypsin / 1 mmol / L-EDTA solution [NAKALI TESK 32777-15] containing phenol red was added, and after being placed at 37°C for 5 minutes, 3 mL of culture medium was added and recovered in a 15 mL centrifugal sedimentation tube. Centrifuge at 1200 rpm for 5 minutes, aspirate the culture medium with an aspirator, add new culture medium and resuspend. Use trypan blue to count the number of cells per 1 μL and adjust to 30,000 cells / mL.

[0392] 100 μL (3000 cells / well) was inoculated in each well of a 96-well culture plate and cultured in a CO2 incubator for 24 hours. The culture medium was aspirated with an aspirator, and 90 μL of fresh culture medium was added to each well. B2E5-H2-L2 and B2E5-ADC were serially diluted (6.5 μg / mL~) using PBS, and 10 μL of each was added to each well, and cultured in a CO2 incubator for 72 hours. Then, 10 μL of the live cell assay reagent [NAKALI TESQUE 07553-44] was added to each well, and after culturing in a CO2 incubator for 3 hours, the absorbance at 450 nm was measured. Based on the measurement results, ImageJ was used to fit and plot the graphs, and the cytotoxicity of B2E5-ADC on gastric cancer was evaluated. The results are shown in Figure 6C .

[0393] (B2E5-ADC single administration toxicity test in mice)

[0394] B2E5-ADC (100, 33, 10 μg) was administered once to 9-10 week old C57BL / 6 male mice (Japan Cura) or 10 week old male nude mice (SKN / slc, Japan SLC) via tail vein or lumbar puncture (three individuals were prepared for each administration concentration), and body weight was measured over time. The results are shown in Figure 7 .

[0395] (Summary of experimental results using B2E5-ADC)

[0396] like Fig. 6A As shown in Figure 2, B2E5-ADC showed a concentration-dependent toxicity to hGIC. On the other hand, B2E5-H2-L2 and Kadcyla did not show toxicity to hGIC. Figure 6B and 6C As shown, the antitumor activity of B2E5-ADC was confirmed not only against the glioma but also against pancreatic cancer and gastric cancer.

[0397] On the other hand, the results of the toxicity test on mice of B2E5-ADC were as follows Figure 7As shown, no effect of B2E5-ADC administration on mice was observed, and survival of mice was confirmed for more than one month after administration.

[0398] Industry Utilization Possibility

[0399] As described above, according to the present invention, high antitumor activity can be achieved by targeting human Eva1. Therefore, the present invention is useful as a drug for treating or preventing cancer.

Claims

1. A humanized antibody or a functional fragment thereof that binds to a human-derived Eva1 protein, comprising a light chain variable region and a heavy chain variable region, The light chain variable region comprises: complementary determining regions 1 to 3 respectively comprising the amino acid sequence recorded in sequence number 1, the amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence recorded in sequence number 3, The heavy chain variable region comprises complementarity determining regions 1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

2. The humanized antibody or functional fragment thereof according to claim 1, The light chain variable region comprises: an amino acid sequence selected from the group consisting of an amino acid sequence set forth in SEQ ID NOs: 7 to 10, an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NOs: 7 to 10, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 10, The heavy chain variable region comprises: an amino acid sequence selected from the amino acid sequences recorded in sequence numbers 13 to 16, an amino acid sequence having more than 80% homology with the amino acid sequences recorded in sequence numbers 13 to 16, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or several amino acids in at least any one of the amino acid sequences recorded in sequence numbers 13 to 16.

3. The humanized antibody or functional fragment thereof according to claim 1, The light chain variable region comprises: an amino acid sequence selected from the group consisting of an amino acid sequence recorded in SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence recorded in SEQ ID NO: 7, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or more amino acids in at least any one of the amino acid sequences recorded in SEQ ID NO: 7, The heavy chain variable region comprises: an amino acid sequence selected from the amino acid sequence recorded in sequence number 13, an amino acid sequence having more than 80% homology with the amino acid sequence recorded in sequence number 13, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or several amino acids in at least any one of the amino acid sequences recorded in sequence number 13.

4. An antibody-drug conjugate, which is the antibody or a functional fragment thereof according to any one of claims 1 to 3 combined with a drug.

5. A pharmaceutical composition comprising the antibody-drug conjugate according to claim 4.

6. A chimeric antigen receptor comprising a region that binds to the human-derived Eva1 protein, a transmembrane region, and an intracellular signaling region. 7 . The chimeric antigen receptor according to claim 6 , wherein the region that binds to the human-derived Eva1 protein is a region comprising the antibody or a functional fragment thereof according to any one of claims 1 to 3 .

8. The chimeric antigen receptor according to claim 6, wherein the region binding to the human-derived Eva1 protein comprises a single-chain variable region fragment consisting of a light chain variable region, a linker, and a heavy chain variable region linked in order from the amino terminal side, The light chain variable region comprising: complementary determining regions 1 to 3 respectively comprising the amino acid sequence described in SEQ ID NO: 1, the amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence described in SEQ ID NO: 3, and comprising an amino acid sequence selected from the group consisting of the amino acid sequence described in SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence described in SEQ ID NO: 7, and an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in at least any one of the amino acid sequences described in SEQ ID NO: 7, The heavy chain variable region comprising: complementary determining regions 1 to 3 comprising the amino acid sequences of SEQ ID NOs: 4 to 6, respectively, And it includes an amino acid sequence selected from the amino acid sequence recorded in sequence number 13, an amino acid sequence having more than 80% homology with the amino acid sequence recorded in sequence number 13, and an amino acid sequence obtained by replacing, deleting, adding and / or inserting one or several amino acids in at least any one of the amino acid sequences recorded in sequence number 13. 9 . The chimeric antigen receptor according to claim 6 , wherein the region binding to the human-derived Eva1 protein and the transmembrane region are bound via a spacer consisting of 10 to 300 amino acids. 10 . The chimeric antigen receptor according to claim 6 , wherein the intracellular signaling region comprises a CD79A intracellular region and a CD40 intracellular region.

11. A nucleotide encoding the chimeric antigen receptor according to any one of claims 6 to 10.

12. A vector comprising the nucleotide of claim 11.

13. A cell expressing the chimeric antigen receptor according to any one of claims 6 to 10. The cell according to claim 13 , which is an immune cell.

15. A pharmaceutical composition comprising the cells of claim 13 or 14.

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