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

Abstract

Provided are a humanized antibody that bonds to an EVA1 protein derived from a human or a functional fragment thereof, an antibody-drug conjugate comprising the humanized antibody or functional fragment thereof, and a chimeric antigen receptor comprising an antibody that bonds to an EVA1 protein derived from a human or a functional fragment thereof.

Description

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

[0001] The present invention relates to a humanized antibody or a functional fragment thereof that binds to a 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.

[0002] Along with coronary artery disease, cancer is a leading cause of death in developed countries, and its incidence rate is steadily increasing. Therefore, the rapid development of cancer eradication therapies is urgently needed. In recent years, the existence of cancer stem cells has become increasingly important in the development of curative cancer therapies. Although cancer stem cells exist only in a small fraction of cancer tissue, they are thought to generate the majority of cancer cells through repeated self-replication and further differentiation. Despite their strong tumorigenic potential, cancer stem cells have also been shown to be highly resistant to chemotherapy and radiotherapy. Therefore, even if chemotherapy and radiotherapy can eradicate the majority of cancer cells in cancer tissue, residual cancer stem cells can lead to cancer recurrence and metastasis. Therefore, targeting and eradicating cancer stem cells is expected to lead to the development of treatments that are effective in preventing cancer metastasis and recurrence.

[0003] In light of this situation, the present inventors have succeeded in identifying the Eva1 protein as a protein that is highly expressed in gliomas and their cancer stem cells. Furthermore, they have demonstrated that the expression of this Eva1 protein correlates with the malignancy of gliomas, and that there is a strong correlation between the survival rate of glioma patients and the expression of the Eva1 gene in gliomas derived from those patients. They have also found that suppressing the function of the Eva1 gene is effective in suppressing the proliferation, tumorigenicity, and tissue invasiveness of glioma cells, as well as the tumor mass formation ability of glioma stem cells (Patent Document 1).

[0004] Furthermore, in cancer, treatment for glioma has remained largely unchanged for the past several decades, primarily consisting of surgery with radiotherapy and chemotherapy as adjuvant therapy. However, no effective treatment has yet been found for glioblastoma multiforme (GBM), the most malignant type of malignant glioma in central nervous system tissue. Furthermore, while temozolomide is used as the standard treatment for malignant glioma, the inventors have confirmed that glioma stem cells are insensitive to temozolomide even at concentrations several times higher than the effective blood concentration. Furthermore, cancer cells exposed to such chemotherapeutic agents develop resistance to them, not just glioma cells, and often develop cross-resistance to multiple other chemotherapeutic agents as well. Furthermore, chemotherapeutic agents often cause cytotoxicity to normal cells, and the dosage or administration of chemotherapeutic agents is often restricted to reduce such side effects.

[0005] Given this situation, the use of antibodies as anticancer agents has recently attracted attention, and their importance is being recognized. For example, if an antibody targets a cancer-specific antigen, it is expected that the administered antibody will accumulate in cancer tissue, and therefore attack cancer cells via the immune system through antibody-dependent cellular cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity.

[0006] Furthermore, by binding a drug such as a cytotoxic substance or a radionuclide to an antibody (i.e., forming a so-called antibody-drug conjugate (ADC)), it becomes possible to efficiently deliver the bound drug to the tumor site, thereby reducing the amount of drug reaching other tissues and, in turn, reducing side effects.

[0007] Furthermore, if a cancer-specific antigen has the activity of inducing cell death, administering an antibody with agonistic activity can be expected to arrest or regress cancer growth through the accumulation of tumor-specific antibodies and the activity of the antibodies. Alternatively, if a cancer-specific antigen is involved in cell proliferation and survival, administering an antibody with neutralizing activity can be expected to arrest or regress cancer growth through the accumulation of tumor-specific antibodies and the activity of the antibodies.

[0008] Furthermore, it is expected that by administering T cells (CAR-T) expressing a chimeric antigen receptor (CAR) incorporating the variable region of an antibody, the cells will be activated against target antigen-positive cancer cells in an HLA-independent manner and induce cell death.

[0009] Due to these properties, antibodies are thought to be suitable for use as anticancer drugs. The present inventors have also produced a high-affinity anti-human Eva1 mouse antibody with the aim of creating an antibody drug targeting the above-mentioned Eva1, and have demonstrated its effectiveness (Patent Document 2).

[0010] International Publication No. 2012 / 043747 International Publication No. 2017 / 022668

[0011] An object of the present invention is to provide an antibody or a functional fragment thereof, or a complex containing the antibody or a functional fragment thereof, which is useful for the treatment of cancer or the like.

[0012] The present inventors attempted to humanize an anti-human Eva1 mouse antibody (the B2E5-48 mouse antibody described in Patent Document 2), and produced 16 types of humanized antibodies by combining four types of humanized heavy chain variable regions (H1 to H4) and four types of humanized light chain variable regions (L1 to L4). As a result, they succeeded in obtaining humanized antibodies (combinations of H2-L2, H2-L4, H3-L4, etc.) that have high binding affinity to the Eva1 protein and high productivity (antibody titer).

[0013] Furthermore, a chimeric antigen receptor (CAR) incorporating the variable region of an anti-human Eva1 antibody was prepared and its efficacy was evaluated. As a result, T cells expressing such a CAR (CAR-T) exhibited activation (cytokine production) and cell proliferation in response to human Eva1. Furthermore, it was revealed that the T cells exhibited cytotoxic activity against Eva1 protein-expressing tumor cells.

[0014] Furthermore, a CAR incorporating the variable region of the B2E5-48 mouse antibody was evaluated by varying the spacer length between the antigen-binding region containing the variable region and the transmembrane region. As a result, it was revealed that when T cells expressing such a CAR were cultured in the absence of IL-2, a CAR-T with a spacer length of 232 amino acids was most superior in terms of cell proliferation and cytokine production. On the other hand, when cultured in the presence of IL-2, the CAR-T with a spacer consisting of 15 amino acids showed the best cell proliferation.

[0015] Furthermore, the researchers also found that, for CARs incorporating the variable regions of the above-mentioned humanized antibodies, the combination of L2-H2 is most useful from the standpoint of activation and cell proliferation in response to human Eva1. Furthermore, for CAR-Ts with L2-H2 as the antigen recognition site, various intracellular domains were tested, and the differences in reactivity due to these differences were evaluated. As a result, it was revealed that CARs having the intracellular domains of 4-1BB and CD79A / CD40 are superior in terms of cell proliferation. Furthermore, the CAR-Ts (L2-H2 hEva1-CAR-Ts) were administered to a cancer-bearing model, and their antitumor activity and safety were confirmed.

[0016] Furthermore, antibody-drug conjugates (ADCs) containing the humanized antibodies were prepared and their efficacy was evaluated. The ADCs demonstrated antitumor activity against various tumors, including glioma, pancreatic cancer, and gastric cancer. However, no adverse effects (such as weight loss or shortened survival time) were observed in the recipients.

[0017] The present invention is based on the above-mentioned findings, and more specifically provides the following aspects.

[0018] <1> A humanized antibody or a functional fragment thereof, which binds to human-derived Eva1 protein, and which has a light chain variable region comprising complementarity-determining regions 1 to 3 each comprising the amino acid sequence of SEQ ID NO: 1, an amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising complementarity-determining regions 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 4 to 6.

[0019] <2> The humanized antibody or functional fragment thereof according to <1>, wherein the light chain variable region comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 7 to 10, amino acid sequences showing 80% or more homology to the amino acid sequences of SEQ ID NOs: 7 to 10, and amino acid sequences in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences of SEQ ID NOs: 7 to 10; and the heavy chain variable region comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 13 to 16, amino acid sequences showing 80% or more homology to the amino acid sequences of SEQ ID NOs: 13 to 16, and amino acid sequences in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences of SEQ ID NOs: 13 to 16.

[0020] <3> The humanized antibody or functional fragment thereof according to <1>, wherein the light chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 7, and at least any one of the amino acid sequences of SEQ ID NO: 7, in which one or several amino acids have been substituted, deleted, added, and / or inserted; and the heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and at least any one of the amino acid sequences of SEQ ID NO: 13, in which one or several amino acids have been substituted, deleted, added, and / or inserted.

[0021] <4> An antibody-drug conjugate comprising the antibody or functional fragment thereof according to any one of <1> to <3> and a drug.

[0022] <5> A pharmaceutical composition comprising the antibody-drug conjugate according to <4>.

[0023] <6> A chimeric antigen receptor comprising a region that binds to a human-derived Eva1 protein, a transmembrane region, and an intracellular signal region.

[0024] <7> The chimeric antigen receptor according to <6>, wherein the region that binds to the human Eva1 protein is a region comprising the antibody or functional fragment thereof according to any one of <1> to <3>.

[0025] <8> The region that binds to the human Eva1 protein comprises a single-chain variable region fragment composed of, in order from the amino terminus, a light-chain variable region, a linker, and a heavy-chain variable region, wherein the light-chain variable region comprises complementarity determining regions 1 to 3 each comprising the amino acid sequence of SEQ ID NO: 1, an amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence of SEQ ID NO: 3, and comprises one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 7, and an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences of SEQ ID NO: 7, and the heavy-chain variable region comprises complementarity determining regions 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 4 to 6, and The chimeric antigen receptor according to <6>, comprising one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any one of the amino acid sequences of SEQ ID NO: 13.

[0026] <9> The chimeric antigen receptor according to any one of <6> to <8>, wherein the region that binds to the human-derived Eva1 protein and the transmembrane region are linked via a spacer consisting of 10 to 300 amino acids.

[0027] <10> The chimeric antigen receptor according to any one of <6> to <9>, wherein the intracellular signal region comprises a CD79A intracellular region and a CD40 intracellular region.

[0028] <11> A nucleotide encoding the chimeric antigen receptor according to any one of <6> to <10>.

[0029] <12> A vector comprising the nucleotide according to <11>.

[0030] <13> A cell expressing the chimeric antigen receptor according to any one of <6> to <10>.

[0031] <14> The cell according to <13>, which is an immune cell.

[0032] <15> A pharmaceutical composition comprising the cell according to <13> or <14>.

[0033] The sequences described in each SEQ ID NO: are as follows:

[0034]

[0035]

[0036]

[0037] As shown in Table 1, SEQ ID NO: 2 is intended to represent the amino acid sequence of CDR2 of Light Chain (Humanized B2E5-48) (an amino acid sequence consisting of valine-threonine-serine in order from the N-terminus) (see also Table 7). However, since this sequence is a sequence of less than four amino acids (three), it is treated as an "intentionally skipped sequence" in the sequence listing.

[0038] According to the present invention, by targeting human Eva1, it is possible to treat or prevent cancer and the like.

[0039] A histogram showing the results of analyzing the expression of human Eva1 in normal human blood cells. A histogram showing the results of analyzing the expression of human Eva1 in various tumor cells. A histogram showing the results of analyzing the expression of human Eva1 in intrahepatic cholangiocarcinoma cases (specimens 1 to 3). A diagram showing an overview of a chimeric antigen receptor (Eva1-CAR) using an anti-Eva1 antibody sequence. A diagram showing an overview of the production schedule of Eva1-CAR-expressing T cells (Eva1-CAR-T). A dot plot showing the results of analyzing the transfection efficiency of the Eva1-CAR gene before selection using tEGFR expression (tEGFR selection). A dot plot showing the results of analyzing the transfection efficiency of the Eva1-CAR gene before tEGFR selection. In the figure, the dots each indicate the origin (5 healthy individuals) of the cells transfected with the Eva1-CAR gene. The thick line indicates the mean value, and the whiskers indicate the standard error. A graph showing the results of analyzing cytokine (IFN-γ) production of Eva1-CAR-T against various tumor cells. In the figure, on the horizontal axis, for each transfected Eva1-CAR, the results for CFPAC-1, NCI-H1975, HuCCT-1, MCF7, HepG2, Huh7, OVTOKO, T98G, PA1 are shown in order from the left. A diagram showing an overview of six different lengths of spacers in Eva1-CAR. Note that the notations "H00" to "H232" shown in Fig. 3F correspond to those in Figs. 3G to 3M. A dot plot showing the results of analyzing the transfection efficiency of the Eva1-CAR gene before tEGFR selection. In the figure, the dots each indicate the origin (5 healthy individuals) of the cells transfected with the Eva1-CAR gene. The thick line indicates the mean value, and the whiskers indicate the standard error. A dot plot showing the results of analyzing the transfection efficiency of the Eva1-CAR gene after tEGFR selection. In the figure, the dots each indicate the origin (5 healthy individuals) of the cells transfected with the Eva1-CAR gene. The thick line indicates the mean value, and the whiskers indicate the standard error. A graph showing the results of analyzing cytokine production of Eva1-CAR-T against various tumor cells. In the figure, the left graph shows the results of analyzing IFN-γ production, and the right graph shows the results of analyzing IL-2 production.1 shows, from left to right, the results for HuCCT-1, CFPAC-1, HepG2, and OVTOKO for each introduced Eva1-CAR. 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 since the start of antigen stimulation (co-culture with Eva1-positive HuCCT-1). The vertical axis shows the number of Eva1-CAR-T cells. 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 type of introduced CAR. The vertical axis shows the number of Eva1-CAR-T cells. Graph showing the expansion efficiency of Eva1-CAR-T after antigen stimulation in the presence of IL-2. In the figure, the horizontal axis indicates the number of days since the start of antigen stimulation (co-culture with Eva1-positive HuCCT-1). The vertical axis indicates the number of Eva1-CAR-T cells. 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 indicates the type of CAR introduced. The vertical axis indicates the number of Eva1-CAR-T cells. This is a dot plot diagram showing the results of analyzing the efficiency of introduction of the Eva1-CAR (hEva1-CAR) gene using an anti-Eva1 humanized antibody sequence before tEGFR selection. In the figure, each dot indicates the origin (four healthy individuals) of the cells into which the hEva1-CAR gene was introduced. The whiskers indicate the standard error, and the middle line between the whiskers indicates the mean value.

[0033] Figure 1 is a dot plot diagram showing the results of analyzing the amplification efficiency of hEva1-CAR-T after tEGFR selection. In the diagram, each dot indicates the origin (four healthy individuals) of the cells into which the hEva1-CAR gene was introduced. The whiskers indicate the standard error, and the middle line between the whiskers indicates the mean. Figure 2 is a graph showing the results of analyzing IFN-γ production by hEva1-CAR-T in response to various tumor cells. The results for HuCCT-1, CFPAC-1, HepG2, and OVTOKO are shown, from left to right, for each introduced Eva1-CAR. Figure 3 is a graph showing the results of analyzing IL-2 production by hEva1-CAR-T in response to various tumor cells. The results for HuCCT-1, CFPAC-1, HepG2, and OVTOKO are shown, from left to right, for each introduced Eva1-CAR. 1 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 graph, the horizontal axis shows the number of days since the start of antigen stimulation (co-culture with Eva1-positive HuCCT-1). The vertical axis shows the number of hEva1-CAR-T cells. This is a diagram showing an overview of six types of hEva1-CAR-T (antigen recognition site: L2H2) with different intracellular domains and spacer lengths. This is a dot plot diagram showing the results of analyzing the transduction efficiency of the hEva1-CAR gene before tEGFR selection. In the figure, each dot indicates the origin (five healthy individuals) of the cells into which the hEva1-CAR gene was transfected. The middle line indicates the average value. The numbers "28," "41," and "79" on the horizontal axis indicate that the intracellular domains of each CAR are "CD28IC," "4-1BBIC," and "CD79A / CD40IC," respectively. Furthermore, "-S" and "-L" indicate that the spacers are "H15" and "H232" shown in FIG. 4F, respectively. This is a graph showing the results of analyzing IFN-γ production by hEva1-CAR-T in various tumor cells. For each introduced Eva1-CAR, the results for OVTOKO, CFPAC-1, HepG2, HuCCT, and MCF7 are shown, from left to right. The numbers "28," "41," and "79" on the horizontal axis indicate that the intracellular domains of each CAR are "CD28IC," "4-1BBIC," and "CD79A / CD40IC," respectively. 4F 。 Furthermore, "-S" and "-L" indicate that the spacers are "H15" and "H232" shown in FIG. 4F , respectively. This graph shows the results of analyzing IL-2 production by hEva1-CAR-T against various tumor cells. For each introduced Eva1-CAR, the results for OVTOKO, CFPAC-1, HepG2, HuCCT, and MCF7 are shown, from left to right. The numbers "28," "41," and "79" on the horizontal axis indicate that the intracellular domains of each CAR are "CD28IC," "4-1BBIC," and "CD79A / CD40IC," respectively. Furthermore, "-S" and "-L" indicate that the spacers are "H15" and "H232" shown in FIG. 4F . This graph shows 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 graph, the horizontal axis shows the number of days since the start of antigen stimulation (coculture with Eva1-positive HuCCT-1). The vertical axis shows the number of hEva1-CAR-T cells. In addition, "28," "41," and "79" in the legend indicate that the intracellular domains of each CAR are "CD28IC," "4-1BBIC," and "CD79A / CD40IC," respectively. In addition, "-S" and "-L" indicate that the spacers are "H15" and "H232" shown in FIG. 4F, respectively. This is a graph showing the results of a coculture test of hEva1-CAR-T and Eva1-positive myeloma cells (RPMI-8226). In the figure, the graph on the left shows the results for hEva1-CAR-T prepared from the peripheral blood of donor 2, and the graph on the right shows the results for hEva1-CAR-T prepared from the peripheral blood of donor 3. The vertical axis of each graph shows the percentage of tumor cells among all cells. The horizontal axis shows the time from the start of co-culture. In addition, "28," "41," and "79" in the legend indicate that the intracellular domains of each CAR are "CD28IC," "4-1BBIC," and "CD79A / CD40IC," respectively. In addition, "s" and "L" indicate that the spacers are "H15" and "H232," respectively, as shown in Figure 4F. hEva1-CAR-T and ５１ 1 is a graph showing the results of analyzing the cytotoxic activity of cells co-cultured with various Cr-labeled tumor cell lines. ５１ 4F . The graph shows the percentage of damaged cells based on the amount of Cr released. The horizontal axis indicates various tumor cell lines, along with whether they are negative or positive for Eva1 expression. 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" indicate that the spacers are "H15" and "H232," respectively, as shown in FIG. 4F . This graph shows the results of an analysis of 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, based on the results shown in FIG. 4L . In the graph, the vertical axis indicates the amount of Eva1 expressed in the culture supernatant.５１It shows the percentage of damaged cells based on the Cr release amount. On the horizontal axis, it shows the mean fluorescence intensity (MFI) obtained by flow cytometry analysis using anti-human Eva1 antibody in various tumor cell lines, which correlates with the Eva1 expression level. Also, "41" and "79" in the legend indicate that the intracellular domains of each CAR are "4-1BBIC" and "CD79A / CD40IC", respectively. Also, "S" and "L" indicate that the spacers are "H15" and "H232" shown in Fig. 4F, respectively. The figure shows the results of analyzing the anti-tumor activity of L2H2 hEva1-CAR-T prepared from peripheral blood derived from two different donors (Donor A, Donor B) by bioluminescence imaging after introducing it into mice carrying a human lung adenocarcinoma cell line (H1975) into which the firefly luciferase gene has been introduced. As L2H2 hEva1-CAR, the antigen recognition site (L2H2) and the transmembrane / intracellular domain (domain containing 4-1BB or CD79A / CD40) were connected with a short spacer (denoted as "short hinge" in the figure, "H15" shown in Fig. 4F). As a control, an administration example of tEGFR-introduced cells (Mock CAR-T cells transfected only with tEGFR) was also prepared and analyzed. Also, "Day" in the figure indicates the number of days after administering CAR-T. Note that at "Day 14" in the administration example of L2H2 hEva1-CAR-T containing 4-1BB in the transmembrane / intracellular domain, the number of mice decreased by two, which was due to death caused by experimental procedure problems. Also, the emission spectrum is also applicable in Figs. 4P and 4R. The figure shows the results of analyzing the anti-tumor activity of L2H2 hEva1-CAR-T prepared from peripheral blood derived from two different donors (Donor A, Donor B) by bioluminescence imaging after introducing it into mice carrying a human pancreatic cancer cell line (CFPAC1) into which the firefly luciferase gene has been introduced. As L2H2 hEva1-CAR, the antigen recognition site (L2H2) and the transmembrane / intracellular domain (domain containing 4-1BB or CD79A / CD40) were connected with a short hinge. As a control, an administration example of tEGFR-introduced cells was also prepared and analyzed.Furthermore, "Day" in the figure indicates the number of days since administration of CAR-T. The emission spectrum also applies to Figures 4P and 4R. L2H2 hEva1-CAR-T, prepared from peripheral blood derived from two different donors (donor A and donor B), was introduced into mice bearing a human pancreatic cancer cell line (CFPAC1) or a human lung adenocarcinoma cell line (H1975) transfected with a firefly luciferase gene, and the antitumor activity of the CAR-T was analyzed by bioluminescence imaging. The L2H2 hEva1-CAR used consisted of an antigen recognition site (L2H2) and a transmembrane / intracellular domain (a domain containing 4-1BB or CD79A / CD40) linked by a short hinge. An example of administration of tEGFR-transfected cells was also prepared and analyzed as a control. Furthermore, "Day" in the figure indicates the number of days since administration of CAR-T. The luminescence spectrum also applies to Figures 4P and 4R. L2H2 hEva1-CAR-T, prepared from peripheral blood derived from two different donors (donor A and donor B), was introduced into mice bearing a human pancreatic cancer cell line (CFPAC1) or a human lung adenocarcinoma cell line (H1975) transfected with a firefly luciferase gene, and the antitumor activity of the CAR-T was analyzed over time by bioluminescence imaging (luminescence intensity). The L2H2 hEva1-CAR used consisted of an antigen recognition site (L2H2) and a transmembrane / intracellular domain (a domain containing 4-1BB or CD79A / CD40) linked by a short hinge. An example of administration of tEGFR-transfected cells was also prepared and analyzed as a control. This figure shows the results of analyzing the antitumor activity of L2H2 hEva1-CAR-T prepared from peripheral blood derived from donor C, which was introduced into mice bearing a human cholangiocarcinoma cell line (HuCCT cells) into which a firefly luciferase gene had been introduced, by bioluminescence imaging. The L2H2 hEva1-CAR used had an antigen recognition site (L2H2) and a transmembrane / intracellular domain (a domain containing 4-1BB or CD79A / CD40) linked by a short hinge. As a control, an example of administration of tEGFR-introduced cells was also prepared and analyzed. Furthermore, "Day" in the figure indicates the number of days since administration of CAR-T.This figure shows the results of analysis of L2H2 hEva1-CAR-T prepared from peripheral blood derived from donor C, which was introduced into mice bearing a human cholangiocarcinoma cell line (HuCCT cells) into which a firefly luciferase gene had been introduced. The L2H2 hEva1-CAR used had an antigen recognition site (L2H2) and a transmembrane / intracellular domain (a domain containing 4-1BB or CD79A / CD40) connected by a short hinge. As a control, an example of administration of tEGFR-introduced cells was also prepared and analyzed. The graph on the left in the figure shows the results of a time-course analysis of the antitumor activity of the CAR-T by bioluminescence imaging (luminescence intensity). The graph on the right shows the results of a time-course analysis of tumor volume in cancer-bearing mice administered with the CAR-T. 1 is a diagram showing an overview of the manufacturing process for an antibody-drug conjugate (B2E5-ADC) containing an anti-human Eva1 humanized antibody.

[0034] FIG. 1 is a graph showing the results of a cytotoxicity experiment of B2E5-ADC against glioma (hGIC E6, hGIC E16). In the figure, the vertical axis of each graph indicates the viability of tumor cells, and the horizontal axis indicates the concentration of ADC or antibody added to the culture medium.

[0035] FIG. 1 is a graph showing the results of a cytotoxicity experiment of B2E5-ADC against pancreatic cancer (BxPC3). In the figure, the vertical axis of each graph indicates the absorbance, which reflects the degree of tumor cell damage. The horizontal axis indicates the concentration of ADC or drug (DM1) added to the culture medium.

[0036] FIG. 1 is a graph showing the results of a cytotoxicity experiment of B2E5-ADC against gastric cancer (MKN74). In the figure, the vertical axis of each graph indicates the viability of tumor cells. The horizontal axis indicates the concentration of ADC or antibody added to the culture medium.

[0037] FIG. 1 is a graph showing the results of a toxicity test of B2E5-ADC in mice. More specifically, the graph shows the change in body weight over time in wild-type (C57BL / 6) or nude mice (SKN / slc) administered a single dose of B2E5-ADC (100, 33, or 10 μg) via intravenous tail vein injection or intrahecal injection. In the figure, each circle, triangle, and square represents data for one individual mouse.

[0040] <Anti-Eva1 Antibody> The present invention relates to an antibody or a functional fragment thereof that binds to human-derived Eva1 protein (human Eva1).

[0041] In the present invention, "Eva (Epithelial V-like antigen) 1" is a single-pass transmembrane protein, a molecule involved in the cytoskeleton system, also known as MPZL2 (Myelin protein Zero-like 2). If derived from humans, it is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 54 (a protein encoded by the nucleotide sequence set forth in SEQ ID NO: 53). However, the DNA sequence of a gene mutates naturally (i.e., non-artificially) due to mutations and the like, and the amino acid sequence of the protein encoded thereby is also altered accordingly. Therefore, the "Eva1 protein" of the present invention is not limited to proteins consisting of the aforementioned typical amino acid sequence, but also includes such naturally occurring variants.

[0042] As used herein, "antibodies" include all classes and subclasses of immunoglobulins. "Antibodies" include polyclonal and monoclonal antibodies. A "polyclonal antibody" is an antibody preparation containing different antibodies directed against different epitopes. A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. In contrast to polyclonal antibodies, monoclonal antibodies recognize a single determinant on an antigen. The antibodies of the present invention are preferably monoclonal antibodies. The antibodies of the present invention are antibodies that have been separated and / or recovered (i.e., isolated) from components of their natural environment.

[0043] The reactivity of the antibody of the present invention to human Eva1 is 10 in terms of KD (dissociation constant) value. －６ Preferably, it is 6×10 or less. －７It is more preferable that the reactivity is as follows: "Reactivity" refers to the binding affinity and / or specificity for the antigen, human Eva1. Such reactivity can be evaluated by those skilled in the art using immunological techniques, and for example, as shown in the Examples below, it can be determined as a KD value by analysis using surface plasmon resonance. Furthermore, the antibody of the present invention may be an antibody that binds to Eva1 proteins derived from other animals (e.g., mouse Eva1 protein (typically, a protein consisting of the amino acid sequence set forth in SEQ ID NO: 56, a protein encoded by the nucleotide sequence set forth in SEQ ID NO: 55)) in addition to human Eva1 protein.

[0044] 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.

[0045] In the present invention, "ADCC activity (antibody-dependent cellular cytotoxicity)" refers to the activity of an antibody binding to a cell surface antigen on a target cell, whereby effector cells (immune cells such as NK cells and monocytes) are further bound to the Fc region of the antibody, activating the cells and releasing factors to kill the target cells. On the other hand, "CDC activity (complement-dependent cytotoxicity)" refers to the activity of an antibody binding to a target cell, activating the complement system, resulting in lysis of the target cell.

[0046] Furthermore, the antibody of the present invention preferably has ADCC activity at 0.01 μg / mL against target cells expressing human Eva1. Whether or not 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 based on the cell lysis rate, the ADCC activity when using the antibody of the present invention at 0.01 μg / mL preferably results in a cell lysis rate of 30% or more, more preferably 50% or more, and even more preferably 70% or more.

[0047] Furthermore, the antibody of the present invention has CDC activity against target cells expressing human Eva1, preferably at 0.30 μg / mL, more preferably at 0.01 μg / mL. Whether or not the antibody of the present invention has such 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 based on the cell lysis rate, the CDC activity when using 0.30 μg / mL of the anti-Eva1 antibody of the present invention is preferably a cell lysis rate of 20% or more, more preferably 40% or more, even more preferably 60% or more, and more preferably 80% or more.

[0048] The antibody of the present invention may also have antitumor activity. In the present invention, "antitumor activity" refers to at least one of the following activities: the activity of suppressing cancer cell proliferation, the activity of inducing cancer cell death, and the activity of suppressing cancer cell metastasis. Antitumor activity can be evaluated, for example, by analysis using a cancer-bearing model (e.g., a mouse inoculated with B16 melanoma cells) as described in the Examples of Patent Document 2. More specifically, B16 melanoma cells are intravenously administered to mice, and then, starting the next day or several weeks later, an anti-Eva1 antibody is intravenously administered daily or every few days. The number of B16 melanoma cell colonies formed in the lungs can then be counted to evaluate in vivo anticancer activity. As a negative control, a control antibody of the same isotype may be administered, or PBS or the like may be administered. If the number of colonies formed in the anti-Eva1 antibody-administered group is lower than that in the negative control-administered group (preferably 60% or less, more preferably 40% or less, even more preferably 20% or less, and more preferably 10% or less, when the number of colonies formed in the negative control-administered group is taken as 100%), the anti-Eva1 antibody can be determined to have anti-tumor activity.

[0049] The antibody of the present invention is not particularly limited in origin, type, shape, etc., as long as it has reactivity with human Eva1. Specific examples include antibodies derived from non-human animals (e.g., mouse antibodies, rabbit antibodies, rat antibodies, and camel antibodies), antibodies derived from humans, chimeric antibodies, and humanized antibodies.

[0050] In the present invention, examples of "antibodies derived from non-human animals" include antibodies against human Eva1 that possess the following variable regions, as disclosed in Patent Document 2: (a) an antibody that possesses a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; (b) an antibody that possesses a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33; (c) an antibody that possesses a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44.

[0051] Furthermore, the present invention relates to antibodies against human Eva1 that possess, for example, the following CDRs: (a) An antibody that possesses heavy chain CDR1 to 3 determined from a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and light chain CDR1 to 3 determined from a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21. (b) An antibody that possesses heavy chain CDR1 to 3 determined from a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and light chain CDR1 to 3 determined from a light chain variable region comprising the amino acid sequence of SEQ ID NO: 33. (c) An antibody that possesses heavy chain CDR1 to 3 determined from a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and light chain CDR1 to 3 determined from a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44.

[0052] There are no particular limitations on the method for determining CDRs based on the amino acid sequences of the variable regions of such antibodies, and examples include known numbering schemes such as Chothia, IMGT, Kabat, and Aho. More specifically, examples of CDRs determined by the Chothia numbering method include antibodies against human Eva1 that have the following CDRs: (a-1) An antibody that has the amino acid sequences set forth in SEQ ID NOs: 28 to 30 as heavy chain CDRs 1 to 3, respectively, and the amino acid sequences set forth in SEQ ID NOs: 22 to 24 as light chain CDRs 1 to 3, respectively. (b) An antibody that has the amino acid sequences set forth in SEQ ID NOs: 40 to 42 as heavy chain CDRs 1 to 3, respectively, and the amino acid sequences set forth in SEQ ID NOs: 34 to 36 as light chain CDRs 1 to 3, respectively. (c) An antibody having the amino acid sequences set forth in SEQ ID NOs: 50 to 52 as heavy chain CDRs 1 to 3, respectively, and the amino acid sequences set forth in SEQ ID NOs: 45 to 47 as light chain CDRs 1 to 3, respectively.

[0053] Furthermore, examples of antibodies determined by the IMGT numbering method include antibodies against human Eva1 that have the following CDRs: (a-2) An antibody that has the amino acid sequences set forth in SEQ ID NOs: 4 to 6 as heavy chain CDRs 1 to 3, respectively, and the amino acid sequence set forth in SEQ ID NO: 1, an amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence set forth in SEQ ID NO: 3 as light chain CDRs 1 to 3, respectively.

[0054] Furthermore, the present invention includes not only antibodies that retain variable regions and / or CDRs consisting of such specific amino acid sequences, but also antibodies whose amino acid sequences have been modified without reducing desired activity (such as antigen reactivity). Such amino acid sequence variants can be produced, for example, by introducing mutations into DNA encoding the above-mentioned variable regions, etc., or by peptide synthesis. Such modifications include, for example, substitution, deletion, addition, and / or insertion of residues within the amino acid sequence of the antibody. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy or light chain of the antibody, or may be the variable region (framework region (FR) and CDR) as long as the activity of the antibody is equivalent to that of the antibody before modification. Although modification of amino acids other than those in the CDRs is thought to have a relatively small effect on reactivity with antigens, currently, methods are known in which amino acids in the CDRs are modified to screen for antibodies with increased affinity for antigens (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), WO 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)). Furthermore, currently, by utilizing an integrated computational chemistry system or the like (e.g., Molecular Operating Environment, manufactured by CCG, Canada), it is also possible to model antibodies with enhanced affinity to antigens (see, for example, 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 in which CDR1 of the heavy chain variable region and CDR3 of the light chain variable region are not involved in antigen affinity.Similarly, Molecular Immunology 44:1075-1084 (2007) reports that in most antibodies, CDR2 of the light chain variable region is not involved in antigen affinity. Thus, antibody affinity for an antigen can exhibit equivalent activity even without requiring all of CDR1 to CDR3 of each of the heavy chain variable region and the light chain variable region. In fact, Biochem Biophys Res Commun. 2003 Jul 18;307(1):198-205, J Mol Biol. 2004 Jul 9;340(3):525-42, J Mol Biol. 2003 Aug 29;331(5):1109-20 reports an example in which affinity for an antigen was maintained by retaining at least one CDR of the original antibody.

[0055] Furthermore, with regard to the antibodies of the present invention, the number of amino acids to be altered in the variable region is preferably 10 amino acids or less, more preferably 5 amino acids or less, and even more preferably 3 amino acids or less (e.g., 2 amino acids or less, 1 amino acid or less). Furthermore, from the viewpoint of minimizing the effect on reactivity with the antigen, it is preferable that all such alterations are made in regions other than the CDRs, i.e., in the FRs. Furthermore, the number of amino acids to be altered in the CDRs is preferably 5 amino acids or less, more preferably 3 amino acids or less (e.g., 2 amino acids or less, 1 amino acid or less).

[0056] The amino acid modification is preferably a conservative substitution. In the present invention, "conservative substitution" refers to substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the art to which the present invention pertains. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), and neutral amino acids can be classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxy groups (serine, threonine), sulfur-containing amino acids (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan).

[0057] Furthermore, antibodies containing variable regions containing amino acid sequences that, after modification, have 80% or greater homology at the amino acid sequence level with the variable regions consisting of the specific amino acid sequences described above are also included in the antibodies of the present invention, as long as they have activity equivalent to that of the antibody before modification. Such homology should be at least 80%, but is preferably 85% or greater, more preferably 90% or greater, and even more preferably 95% or greater (e.g., 96% or greater, 97% or greater, 98% or greater, or 99% or greater). Sequence homology can also be determined using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing an amino acid sequence using BLASTP, the parameters are, for example, score = 50 and wordlength = 3. Furthermore, when analyzing an amino acid sequence using the Gapped BLAST program, it can be performed as described in 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. Specific techniques for these analysis methods are known.

[0058] Furthermore, "having equivalent activity" means, for example, that the reactivity to an antigen is equivalent (for example, 70% or more, preferably 80% or more, more preferably 90% or more) to that of a reference antibody (for example, the B2E5-48 mouse antibody described in Patent Document 2).

[0059] Modification of the antibodies of the present invention may also involve modification of the post-translational process of the antibody, such as changing the number or location of glycosylation sites. This can improve, for example, the ADCC activity of the antibody. Antibody glycosylation is typically N-linked or O-linked. Antibody glycosylation largely depends on the host cell used to express the antibody. Glycosylation patterns can be modified by known methods, such as introducing or deleting specific enzymes involved in sugar production (see JP 2008-113663 A, U.S. Pat. Nos. 5,047,335, 5,510,261, 5,278,299, and WO 99 / 54342). Furthermore, in the present invention, deamidation may be suppressed by substituting a deamidated amino acid or an amino acid adjacent to a deamidated amino acid with another amino acid, for purposes such as increasing antibody stability. Furthermore, antibody stability can be increased by substituting glutamic acid with another amino acid. The present invention also provides antibodies stabilized in this manner.

[0060] In the present invention, a "chimeric antibody" refers to an antibody in which the variable region of one antibody is linked to the constant region of a heterologous antibody. Chimeric antibodies can be obtained, for example, by immunizing a mouse with an antigen, excising the antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, ligating it to an antibody constant region (constant region) gene derived from human bone marrow, incorporating it into an expression vector, and introducing it into a host for production (e.g., JP-A-8-280387, U.S. Pat. No. 4,816,397, U.S. Pat. No. 4,816,567, U.S. Pat. No. 5,807,715).

[0061] The constant region of a chimeric antibody is usually derived from a human antibody. For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε can be used as the heavy chain constant region. Furthermore, Cκ and Cλ can be used as the light chain constant region. The amino acid sequences of these constant regions and the nucleotide sequences encoding them are known. Furthermore, to improve the stability of the antibody itself or the stability of antibody production, one or several amino acids in the human antibody constant region can be substituted, deleted, added, and / or inserted.

[0062] In the present invention, a "humanized antibody" refers to an antibody in which the CDR gene sequence of an antibody derived from a non-human animal has been grafted onto a human antibody gene (CDR grafting). Methods for producing such an antibody include known techniques such as overlap extension PCR (see, for example, EP 239400, EP 125023, WO 90 / 07861, and WO 96 / 02576). The variable region of an antibody typically consists of three CDRs sandwiched between four FRs. CDRs are the regions that essentially determine the binding specificity of an antibody. While the amino acid sequences of CDRs are highly diverse, the amino acid sequences constituting the FRs often show high homology even between antibodies with different binding specificities. Therefore, it is generally believed that CDR grafting can transfer the binding specificity of one antibody to another. Furthermore, from the viewpoint of maintaining the function of the CDR, when grafting a non-human CDR onto a human FR, a human FR having a high homology to the non-human animal-derived FR is selected. That is, since the amino acids in the CDR not only recognize the antigen but also coordinate with the amino acids in the FR adjacent to the CDR and are involved in maintaining the loop structure of the CDR, it is preferable to use a human FR having an amino acid sequence that is highly homologous to the amino acid sequence of the FR adjacent to the CDR to be grafted.

[0063] A search for known human FRs highly homologous to non-human animal-derived FRs can be performed, for example, using an antibody-specific search system available on the Internet (http: / / www.bioinfo.org.uk / abysis / ). Mutations can be introduced into sequences other than the CDRs of the non-human antibody so that the sequence matches the human FR sequence obtained in this manner. Alternatively, if a gene (cDNA) encoding the amino acid sequence of the human FR obtained by the search is available, the non-human CDRs can be introduced into that sequence. Introduction of mutations can be performed using techniques known in the art, such as nucleic acid synthesis and site-directed mutagenesis.

[0064] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody thus prepared, it is possible to suitably select FRs of a human-derived antibody that form a good antigen-binding site when linked via the CDRs. Furthermore, if necessary, amino acid residues in the FRs can be substituted so that the CDRs of the humanized antibody form a suitable antigen-binding site, according to the method described in Sato, K. et al., Cancer Res, 1993, 53, 851-856, etc., and mutant FR sequences with desired properties can be selected by measuring and evaluating the antigen-binding activity of mutant antibodies with the amino acid substitutions.

[0065] In the present invention, examples of "humanized antibodies" include humanized antibodies that have a light chain variable region comprising CDRs each comprising the amino acid sequence set forth in SEQ ID NO: 1, an amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising CDR1 to CDR3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6, and that bind to human-derived Eva1 protein.

[0066] More specifically, as shown in the Examples below, examples of humanized antibodies include those in which the light chain variable region comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 7 to 10, amino acid sequences showing 80% or greater homology to the amino acid sequences of SEQ ID NOs: 7 to 10, and amino acid sequences in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences of SEQ ID NOs: 7 to 10, and the heavy chain variable region comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 13 to 16, amino acid sequences showing 80% or greater homology to the amino acid sequences of SEQ ID NOs: 13 to 16, and amino acid sequences in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences of SEQ ID NOs: 13 to 16.

[0067] Modifications and homology of the variable regions are as explained in the section "Antibodies Derived from Non-Human Animals." However, in the humanized antibodies of the present invention, it is preferred that the CDR sequences are not modified, and that the sequences of the light chain variable region CDR1 to CDR3 and the heavy chain variable region CDR1 to CDR3 are specified to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence consisting of Val-Thr-Ser, the amino acid sequence of SEQ ID NO: 3, and the amino acid sequences of SEQ ID NOs: 4 to 6, respectively.

[0068] Furthermore, in such humanized antibodies, from the viewpoint of higher reactivity to antigens, as shown in the Examples below, (H2-L2) a humanized antibody in which the light chain variable region comprises one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 7, and at least one amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 7; and (H1-L4) a humanized antibody in which the heavy chain variable region comprises one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and at least one amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 13. a humanized antibody in which the light chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 8, and at least any one of the amino acid sequences of SEQ ID NO: 8, in which one or several amino acids have been substituted, deleted, added, and / or inserted; and a heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 15, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 15, and at least any one of the amino acid sequences of SEQ ID NO: 15, in which one or several amino acids have been substituted, deleted, added, and / or inserted; (H2-L3) a humanized antibody in which the light chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 10, and at least any one of the amino acid sequences of SEQ ID NO: 10, in which one or several amino acids have been substituted, deleted, added, and / or inserted;Preferred examples include a humanized antibody whose heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and at least one of the amino acid sequences of SEQ ID NO: 13, with one or several amino acids substituted, deleted, added, and / or inserted; and (H2-L4) a humanized antibody whose light chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 8, and at least one of the amino acid sequences of SEQ ID NO: 8, with one or several amino acids substituted, deleted, added, and / or inserted, and whose heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and at least one of the amino acid sequences of SEQ ID NO: 13, with one or several amino acids substituted, deleted, added, and / or inserted.

[0069] Also, taking into consideration high productivity, (H2-L2) a humanized antibody in which the light chain variable region comprises one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 7, and at least one amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 7; and (H2-L4) a humanized antibody in which the heavy chain variable region comprises one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and at least one amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 13. A humanized antibody, wherein the light chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 8, and at least any one of the amino acid sequences of SEQ ID NO: 8, in which one or several amino acids have been substituted, deleted, added, and / or inserted; and the heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and at least any one of the amino acid sequences of SEQ ID NO: 13, in which one or several amino acids have been substituted, deleted, added, and / or inserted; (H3-L4) A humanized antibody, wherein the light chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 8, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 8, and at least any one of the amino acid sequences of SEQ ID NO: 8, in which one or several amino acids have been substituted, deleted, added, and / or inserted;A preferred example is a humanized antibody whose heavy chain variable region comprises one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 16, and an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any one of the amino acid sequences of SEQ ID NO: 16.

[0070] Furthermore, the antibodies of the present invention may also take the form of antibodies that compete with antibodies having variable regions or CDRs containing the above-mentioned specific amino acid sequences or variants thereof in binding to human Eva1. In other words, the antibodies may take the form of antibodies that bind to epitopes reactive with antibodies having variable regions or CDRs containing the above-mentioned specific amino acid sequences or variants thereof.

[0071] In the present invention, the term "epitope" refers to an antigenic determinant present in an antigen, i.e., a site on an antigen to which an antigen-binding domain in an antibody binds. Therefore, an epitope in the present invention may be a polypeptide consisting of multiple consecutive amino acids in the primary amino acid sequence (linear epitope), or a polypeptide formed when non-adjacent amino acids in the primary amino acid sequence are brought into close proximity due to a three-dimensional structure such as peptide or protein folding (discontinuous epitope, structural epitope). Furthermore, such epitopes typically consist of at least three (e.g., four), and most usually at least five (e.g., 6-20, 7-15, 8-10) amino acids.

[0072] Furthermore, once 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 that peptide region. Furthermore, whether two antibodies bind to the same or sterically overlapping epitopes can be determined by a competitive assay.

[0073] In the present invention, the term "functional fragment" of an antibody refers to a portion (partial fragment) of the antibody that exhibits reactivity with human Eva1. Specific examples include Fab, Fab', F(ab')2, variable region fragments (Fv), disulfide-linked Fv, single-chain variable region fragments (single-chain Fv, scFv), sc(Fv)2, diabodies, multispecific antibodies, and polymers thereof.

[0074] As used herein, "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin consisting of one light chain and part of a heavy chain. It can be obtained by papain digestion of an antibody or by recombinant methods. "Fab'" differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the antibody hinge region. "F(ab')2" refers to a bivalent antigen-binding fragment of an immunoglobulin consisting of both light chains and part of both heavy chains.

[0075] A "variable region fragment (Fv)" is the smallest antibody fragment that contains a complete antigen-recognition and binding site. Fv is a dimer in which the heavy and light chain variable regions are tightly linked by non-covalent bonds. A "single-chain variable region fragment (single-chain Fv, scFv)" contains the heavy and light chain variable regions of an antibody, and these regions are present in a single polypeptide chain. An "sc(Fv)2" is a single chain in which two heavy chain variable regions and two light chain variable regions are linked by a linker or other means. A "diabody" is a small antibody fragment that contains two antigen-binding sites. This fragment contains a heavy chain variable region bound to a light chain variable region in the same polypeptide chain, and each region is paired with a complementary region in another chain. A "multispecific antibody" is a monoclonal antibody that has binding specificities for at least two different antigens. For example, it can be prepared by coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities.

[0076] The antibodies of the present invention can be produced by the hybridoma method or by recombinant DNA technology. A representative example of the hybridoma method is the method of Kohler and Milstein (Nature, 256:495 (1975)). 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 an antigen (human Eva1, its partial peptides, proteins fused with Fc protein, etc., or cells expressing these). Antibody-producing cells obtained by reacting the antigen in a culture medium with the above cells or lymphocytes, etc., previously isolated from an unimmunized animal can also be used. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be derived from different animal species, as long as they are fusible, but are preferably derived from the same animal species. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and then screening can be performed to obtain hybridomas that produce monoclonal antibodies specific to human Eva1. Monoclonal antibodies against human Eva1 can be obtained by culturing the hybridomas or from the ascites fluid of a mammal to which the hybridomas have been administered.

[0077] The recombinant DNA method involves cloning DNA encoding the antibody of the present invention from hybridomas, B cells, or the like, incorporating it into an appropriate vector, and then introducing it into host cells (e.g., mammalian cell lines such as HEK cells, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme A. M. et al., Eur. J. Biochem. 192:767-775 (1990)). In expressing DNA encoding the antibody of the present invention, DNA encoding the heavy chain and DNA encoding the light chain may be separately incorporated into expression vectors and used to transform host cells, or DNA encoding the heavy chain and DNA encoding the light chain may be incorporated into a single expression vector and used to transform host cells (see WO 94 / 11523). The antibody of the present invention can be obtained in a substantially pure and homogeneous form by culturing the host cells and isolating and purifying it from within the host cells or from the culture medium. Antibody isolation and purification can be performed using methods commonly used for purifying polypeptides. By using transgenic animal production techniques to produce transgenic animals (such as cows, goats, sheep, and pigs) incorporating antibody genes, it is possible to obtain large quantities of monoclonal antibodies derived from the antibody genes from the milk of the transgenic animals.

[0078] The present invention can also provide DNA encoding the antibody of the present invention, a vector containing the DNA, a host cell harboring the DNA, and a method for producing the antibody, comprising culturing the host cell and recovering the antibody.

[0079] <Antibody-drug conjugate> As shown in the Examples below, human Eva1, the target of the antibody of the present invention, is expressed in various cancers. Furthermore, the antibody of the present invention (particularly the humanized antibody) is also useful as an ADC. Therefore, the present invention can also take the form of an antibody-drug conjugate (ADC) comprising the antibody of the present invention (particularly the humanized antibody) or a functional fragment thereof conjugated to a drug.

[0080] In the ADC of the present invention, the "drug" that can be conjugated to the antibody is not particularly limited, and examples include cytotoxic substances known in the art. Examples of "cytotoxic substances" include anticancer agents, for example, irinotecan (CPT-11), irinotecan metabolite SN-38 (10-hydroxy-7-ethylcamptothecin), alkylating agents such as adriamycin, taxol, 5-fluorouracil, nimustine, laministine, and temozolomide, antimetabolites such as gemcitabine and hydroxycarbamide, plant alkaloids such as etoposide and vincristine, anticancer antibiotics such as mitomycin and bleomycin, platinum preparations such as cisplatin, molecular targeted agents such as sorafenib and erlotinib, methotrexate, cytosine arabinoside, 6-thioguanine, 6-mercaptopurine, cyclophosphamide, ifosfamide, busulfan, MMAE (monomethylauristatin E), DM1 (mertansine), and calicheamicin. Cytotoxic agents include radioisotopes, e.g., ３２ P. １４ C. １２５ I, ３ H. １３１ I, １８６ Re, １８８ Re, １０ B. １１１ In, ９０Y can also be mentioned. Cytotoxic substances may be photosensitive substances that exert cytotoxic activity, more specifically, substances that are activated by light irradiation and change into a form that exhibits cytotoxicity (cytotoxicity) themselves, or substances that produce cytotoxic substances (cytotoxic substances). Examples include chlorins, chlorin e6, porfimer sodium, talaporfin sodium, verteporfin, and precursors and derivatives thereof. Cytotoxic substances also include toxic peptides, such as ribosome-inactivating proteins (RIPs) such as saporin, ricin, and Shiga poison.

[0081] Furthermore, the antibody and the cytotoxic substance can be bound by methods known in the art, and may be bound directly or indirectly. For example, a covalent bond can be used as a direct bond, and a bond via a linker can be used as an indirect bond.

[0082] In the present invention, it is preferred that the antibody and the cytotoxic substance are linked via a linker. Linking two molecules via a linker can attenuate the antigenicity of the cytotoxic substance, making it preferable for administration to a subject. General techniques for linkers are described, for example, in Hermanson, G. T., Bioconjugate Techniques, Academic Press, 1996; Harris, J. M. and Zalipsky, S. (eds.), Poly(ethylene glycol), Chemistry and Biological Applications, ACS Symposium Series, 1997; Veronese, F. and Harris, J. M. This is described in "Peptide and protein PEGylation," edited by J. M., Advanced Drug Delivery Review 54(4), 2002.

[0083] Furthermore, linkers include not only linear linkers (divalent linkers) but also branched linkers (trivalent or higher linkers). A linear 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 such linkers include N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(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-hydroxysuccinimide ester (M- ... Examples of peptide linkers include succinimidyl ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), 6-maleimidocaproyl (MC), maleimidopropanoyl (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).

[0084] Techniques for linking an antibody to a cytotoxic substance via a linker are known in the art. For example, the bond between the antibody and the linker may be a covalent bond or a non-covalent bond (e.g., an ionic bond, a hydrophobic bond), preferably a covalent bond. This bond is preferably one that does not easily release the cytotoxic substance in the blood when the conjugate of the present invention is administered to a subject. Examples of such bonds include a bond between a maleimide group and a thiol group, a bond obtained by reacting a haloester with a thiol, an amide bond between a carboxyl group and an amino group, a disulfide bond between a thiol group and another thiol group, a Schiff base formed by an amino group and an aldehyde group, a thioester bond between a thiol group and a carboxylic acid, an ester bond between a hydroxyl group and a carboxyl group, a bond between an amino group and a squaric acid derivative (e.g., dimethylsquaric acid), and a bond between a dienylaldehyde group and an amino group. Specific examples of such bonds include a bond between a maleimide group present at one end of the linker and a thiol group contained in a cysteine ​​residue on the antibody, a dehydration substitution bond between a succinimide group present at one end of the linker and an amino group contained in a lysine residue on the antibody (e.g., WO 2008 / 096760), a dehydration condensation bond between an amino group present at one end of the linker and a carboxylic acid contained in an aspartic acid or glutamic acid on the antibody (e.g., using WSCDI), etc. For specific methods of bonding an antibody to a linker, see, for example, WO 2010 / 055950.

[0085] On the other hand, the bond between the antibody or linker and the cytotoxic substance is a covalent bond or a non-covalent bond (such as an ionic bond or a hydrophobic bond), preferably a covalent bond. In particular, the bond is preferably one that makes it difficult for the cytotoxic substance to be released in the blood when the conjugate of the present invention is administered to a subject. From this perspective, the bond between the antibody or linker and the cytotoxic substance is not limited, but is preferably an ester bond, a carbamate bond, a carbonate bond, or a thiocarbamate bond, more preferably an ester bond.

[0086] In the ADC of the present invention, the number of cytotoxic substances bound per antibody molecule (number of drug conjugations) is determined by specifying the reaction conditions, such as the amounts of raw materials and reagents used, so that the number is a certain number, taking into consideration efficacy and safety. However, unlike chemical reactions of low molecular weight compounds, a mixture of different numbers of drugs is usually obtained. The number of drugs bound to one antibody molecule is specified and expressed as an average value, i.e., the average number of drugs bound. Therefore, in the present specification, unless otherwise specified, the number of drugs bound refers to the average value.

[0087] The number of drugs bound per antibody molecule can be controlled by adjusting the type and concentration of the reducing agent used, the concentrations of the linker and drug relative to the antibody (e.g., molar excess), and the temperature or time of the binding reaction. The "reducing agent" is not particularly limited as long as it can reductively cleave disulfide bonds in the antibody to form bonds via the thiol groups, and examples thereof include dithiothreitol (DTT, DL-DTT), 2-mercaptoethylamine, mercaptoethanol, and TCEP.

[0088] In the present invention, the average number of drugs bound per antibody is not particularly limited, but is, for example, 1 to 10, preferably 3 to 8, and more preferably 3 to 5. As shown in the Examples below, the number of drugs bound per antibody molecule can be determined by measuring the absorbance at the absorption peak of the cytotoxic substance (e.g., DM1) and the absorbance at 280 nm, which is the absorption peak of the antibody, and calculating the ratio of the number of molecules by formulating simultaneous equations taking into account the influence of each on the other's absorption peak. Alternatively, the number of drugs bound per antibody molecule can be determined using a reagent for colorimetric quantitation of thiol groups, such as DTNB.

[0089] Furthermore, the ADC of the present invention may absorb moisture when left in the air or when recrystallized, resulting in adsorbed water or becoming a hydrate, and such water-containing compounds and salts are also included in the present invention.

[0090] One or more atoms constituting the ADC of the present invention may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include: ２ H. ３ H. １２５ I, １４ C. Furthermore, the ADC of the present invention can be detected by bioimaging techniques and the like. Therefore, a labeling substance may be bound to the ADC. The labeling substance is appropriately selected depending on the type of detection method to be used, and examples thereof include radioactive labeling substances, fluorescent labeling substances, paramagnetic labeling substances, superparamagnetic labeling substances, and enzyme labeling substances. Furthermore, the binding of such a labeling substance to the ADC may be direct or indirect. Examples of indirect binding include binding via a secondary antibody bound to a labeling substance or a polymer bound to a labeling substance (protein A, protein B, etc.).

[0091] More specifically, a positron-emitting nuclide can be used as a radioisotope to label an ADC. For example, ６４ Cu, １８ F. ５５ Co, ６６ Ga, ６８ Ga, ７６ Br, ８９ Zr and １２４ An antibody can be labeled with a positron-emitting nuclide such as I. Known methods (Nucl Med Biol. 1999; 26(8):943-50., J Nucl Med. 2013; 54(11):1869-75., etc.) can be used to label an ADC with these positron-emitting nuclides. Furthermore, after an ADC labeled with a positron-emitting nuclide is administered to a subject, radiation emitted from the radioactive nuclide is measured from outside the body using a PET (positron emission tomography) device and converted into an image using a computer tomography technique. In this way, the arrival and accumulation of the ADC at the target site administered to the subject can be tracked.

[0092] <Chimeric Antigen Receptor> As shown in the Examples below, human Eva1, the target of the antibody of the present invention, is expressed in various cancers. Furthermore, the variable regions of the antibody of the present invention are useful in chimeric antigen receptors (CARs) and T cells expressing the same (so-called CAR-Ts). Thus, the present invention may also take the form of a CAR comprising a region that binds to human-derived Eva1 protein, a transmembrane region, and an intracellular signal region.

[0093] In the present invention, the term "chimeric antigen receptor (CAR)" refers to a chimeric protein in which a domain that binds to human-derived Eva1 protein, which serves as an extracellular domain, a transmembrane domain, and an intracellular signal domain are arranged in this order from the N-terminus and linked directly or indirectly.

[0094] In the present invention, the "region that binds to human-derived Eva1 protein (antigen-binding region)" can be, for example, the above-mentioned antibody or a functional fragment thereof, but typically, an scFv is used for CAR.

[0095] As described above, an "scFv" is a structure in which the light chain variable region and heavy chain variable region of a monoclonal antibody (immunoglobulin) are linked via a linker, and retains antigen-binding ability. The order of the N-terminal end of the scFv may be light chain variable region, linker, heavy chain variable region, or heavy chain variable region, linker, light chain variable region, but the former is preferred.

[0096] As the "linker" in scFv, for example, a peptide linker can be used. The length of the linker is not particularly limited. For example, a linker containing 5 to 25 amino acids can be used. The length of the linker is preferably 8 to 25 amino acids, more preferably 15 to 20 amino acids. Suitable examples of peptide linkers include those consisting of glycine or glycine and serine (GGS linker, GS linker, GGG linker, etc.). Another suitable example is a linker consisting of 18 amino acids at positions 134 to 151 of SEQ ID NO: 58. The amino acids glycine and serine that make up these linkers have the advantage that they are small in size and are less likely to form higher-order structures within the linker.

[0097] The monoclonal antibody that serves as the basis for the scFv is not particularly limited, and examples thereof include the above-mentioned antibodies derived from non-human animals, human antibodies, and humanized antibodies. Furthermore, while the preferred examples are as described above, from the viewpoint of activation and cell proliferation in response to human Eva1, the above-mentioned (H2-L2) combination is preferred, as shown in the Examples below. Furthermore, an scFv in which L2, a linker, and H2 are linked in this order from the N-terminus is preferred.

[0098] In the present invention, the "transmembrane region" is not particularly limited as long as it is a polypeptide that has the function of penetrating a cell membrane and can anchor a CAR to the cell membrane. Examples of proteins from which polypeptides carrying a transmembrane region are derived include 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 chain and β chain of a T cell receptor.

[0099] In the present invention, the "intracellular signaling region" refers to a region that is located intracellularly when the CAR is located on the cell membrane, and that is capable of transmitting a signal required for immune cell effector function, i.e., a signal required for immune cell activation (a so-called primary signal) when the antigen-binding region binds to an antigen (human Eva1). Such intracellular signaling regions generally include those that have only an activation signaling domain such as a T cell receptor (TCR) and a CD3 complex (first-generation CARs), those that have a costimulatory signaling domain derived from a costimulatory molecule (e.g., the intracellular domain of CD28 or 4-IBB) in addition to the activation signaling domain (second-generation CARs), and those that have multiple costimulatory signaling domains (e.g., the intracellular domains of CD28 and 4-IBB) in addition to the activation signaling domain (third-generation CARs). Further, examples include those containing the CD79A intracellular region and the CD40 intracellular region previously developed by the present inventors (see Mol Ther 2021 Sep 1; 29 (9): 2677-2690).

[0100] As described above, the "intracellular signaling region" may be any region capable of transmitting a primary signal, and an activation signaling domain of a protein involved in the signal can be used. Primary signaling is known to involve immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of proteins having ITAMs include CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

[0101] Furthermore, as described above and as shown in the Examples below, "a molecule comprising a CD79A intracellular region and a CD40 intracellular region" is also suitable for use as an intracellular signaling region of the present invention. In such cases, the positional relationship between the CD79A intracellular domain and the CD40 intracellular domain is not particularly limited, and either the CD79A intracellular region on the N-terminal side or the CD40 intracellular region on the N-terminal side can be employed. The former positional relationship is preferred. The CD79A intracellular region and the CD40 intracellular region are preferably linked directly or via a linker. The linker is not particularly limited and can be any linker as long as it does not significantly impair the NFκB signal activation activity. The linker is preferably glycine or a linker composed of glycine and serine (e.g., a GGS linker, a GS linker, a GGG linker, etc.). The length of the linker is not particularly limited, but may be, for example, 1 to 20 amino acids, preferably 1 to 10 amino acids, and more preferably 1 to 5 amino acids.

[0102] In the present invention, the number of intracellular signal regions contained in a CAR is not limited to one, and the CAR may contain multiple intracellular signal regions (for example, 1 to 5, 1 to 3, or 1 or 2 signal transduction factors). In this case, the multiple intracellular signal regions contained may be the same or different.

[0103] The CAR of the present invention may comprise other domains in addition to the above-mentioned antigen-binding domain, transmembrane domain, and intracellular signal domain. Examples of other domains include, but are not limited to, a costimulatory signaling domain, a spacer sequence, a signal peptide, etc.

[0104] It is known that costimulatory molecules expressed on T cells transmit costimulatory signals intracellularly upon binding to ligands specific to each costimulatory molecule expressed on antigen-presenting cells, thereby assisting T cell activation (secondary signaling). In the present invention, a "costimulatory transmission region" refers to an intracellular domain involved in the costimulatory signal transmission of the above-mentioned costimulatory molecules. Examples of costimulatory molecules include CD28, 4-1BB (CD137), OX40 (CD134), ICOS, CD2, CD4, CD5, CD8α, CD8β, CD154, and the like. The CAR of the present invention can comprise the costimulatory transmission region of these costimulatory molecules.

[0105] The number of costimulatory signaling regions that the CAR of the present invention can comprise is not limited to one, and may be more than one. The CAR of the present invention can comprise, for example, 1 to 5, 1 to 3, or 1 or 2 costimulatory signaling regions. When the CAR of the present invention comprises more than one costimulatory signaling region, the regions may be the same or different.

[0106] In the present invention, the term "spacer" refers to a sequence that links each region, etc., and is not particularly limited as long as it does not inhibit the function of each region. The number of amino acids therein is also not particularly limited, but it is usually 1 to 500 amino acids, preferably 5 to 300 amino acids, and more preferably 10 to 100 amino acids.

[0107] In particular, in the CAR of the present invention, the antigen-binding region and the transmembrane region are preferably linked directly or via a relatively short spacer. The number of amino acids constituting the spacer between the antigen-binding region and the transmembrane region is, for example, 1 to 400, and preferably 10 to 300. Furthermore, as shown in the Examples below, when cells expressing the CAR of the present invention are cultured in the absence of IL-2, the number of amino acids constituting the spacer is more preferably 150 to 300, and even more preferably 200 to 250, from the viewpoints of cell proliferation and cytokine production. On the other hand, when cells expressing the CAR of the present invention are cultured in the presence of IL-2, the number of amino acids constituting the spacer is more preferably 10 to 30.

[0108] The spacer sequence is not particularly limited, and examples of spacer sequences that can be used include the hinge region or a portion thereof of IgG, preferably human IgG (e.g., subtypes IgG1 and IgG4), the hinge region and a portion of CH2, or a portion of a factor used in a transmembrane domain (e.g., CD28). By using the sequence of the hinge region or a portion thereof, it is expected that a highly flexible spacer can be constructed. Specific examples of such spacers include spacers consisting of the amino acid sequence set forth in SEQ ID NO: 59, 60, 61, 62, or 62 (positions 268 to 499 of SEQ ID NO: 58).

[0109] In the present invention, the term "signal peptide" refers to a peptide that promotes the secretion of a CAR or directs its localization to the cell membrane, and is also referred to as a leader sequence. The signal peptide can usually be directly or indirectly bound to the N-terminus of the antigen-binding region. Optionally, the signal peptide may be cleaved from the antigen-binding region during cellular processing and localization of the CAR to the cell membrane. Examples of such signal peptides include signal peptides of 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, etc.

[0110] Examples of each region, etc. in the CAR of the present invention have been described above. However, those skilled in the art can appropriately obtain specific amino acid sequences of these regions, etc. by searching known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). Furthermore, as shown in Table 3 above, specific sequences are also disclosed in the sequence listing of the present application. Furthermore, the regions, etc. of the present invention are not limited to such typical amino acid sequences (e.g., NCBI reference sequences), and may also include variants and homologs of these typical amino acid sequences, so long as the function of the region is maintained. Furthermore, such variants and homologs usually have high homology to the typical amino acid sequence. High homology is usually 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0111] The CAR of the present invention may be a molecule consisting of one type of polypeptide or a molecule consisting of a complex of two or more types of polypeptides.The CAR of the present invention may be a molecule consisting of a polypeptide or a complex thereof, or may be a molecule formed by linking another substance (e.g., a fluorescent substance, a radioactive substance, an inorganic particle, etc.) to a polypeptide or a complex thereof.

[0112] The CAR of the present invention may be chemically modified. The polypeptide constituting the CAR of the present invention may have a C-terminus that is a carboxyl group (—COOH), a carboxylate (—COO—), an amide (—CONH ２) or ester (—COOR). Here, examples of R in the ester include C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl; C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; C6-12 aryl groups such as phenyl and α-naphthyl; C1-2 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; and pivaloyloxymethyl. In the polypeptide constituting the CAR of the present invention, a carboxyl group (or carboxylate) other than the C-terminus may be amidated or esterified. In this case, for example, the above-mentioned C-terminal esters, etc. are used. Furthermore, the polypeptide constituting the CAR of the present invention also includes one in which the amino group of the N-terminal amino acid residue is protected with a protecting group (e.g., a C1-6 acyl group such as a formyl group or a C1-6 alkanoyl group such as an acetyl group), one in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated, and one in which a substituent on the side chain of an amino acid in the molecule (e.g., —OH, —SH, an amino group, an imidazole group, an indole group, a guanidino group, etc.) is protected with an appropriate protecting group (e.g., a C1-6 acyl group such as a formyl group or a C1-6 alkanoyl group such as an acetyl group).

[0113] Furthermore, the CAR of the present invention may have another functional protein. The other functional protein is not particularly limited and may be appropriately selected depending on the function to be imparted to the CAR of the present invention. For example, functional proteins used for the purpose of facilitating the purification and detection of the CAR include Myc tag, His tag, HA tag, FLAG tag (registered trademark, Sigma-Aldrich), fluorescent protein tag (GFP, etc.), etc.

[0114] The chimeric antigen receptor of the present invention may 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 both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0115] 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, and acetic acid.

[0116] There have been several reports of 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; Pulé 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 Med365:725-733, 2011; Kalos M, et al. Sci Transl Med 3:95ra73, 2011; Brentjens RJ, et al. Blood 118:4817-4828, 2011; Brentjens RJ, et al. SciTransl Med 5:177 ra38, 2013), the CAR of the present invention can be constructed by referring to these reports. In addition, since specific sequences and the like are also shown in the Examples described later, the CAR of the present invention can also be constructed by referring to these.

[0117] <Nucleotides encoding chimeric antigen receptors> The present invention provides polynucleotides encoding the CARs of the present invention. Information on the nucleotide sequence encoding the antigen-binding region of an scFV or the like can be obtained by producing a monoclonal antibody that recognizes the target antigen, determining the amino acid sequence of the monoclonal antibody by a known method such as the Edman method, and then based on the amino acid sequence. Specific sequences for the above-mentioned humanized antibodies and the like are also disclosed in the sequence listing of the present application. Information can also be obtained by analyzing the nucleotide sequence of a hybridoma that produces the monoclonal antibody.

[0118] Furthermore, specific nucleotide sequences encoding each region, etc., of the CAR of the present invention, including the antigen-binding region, can be appropriately obtained by those skilled in the art by searching known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). Furthermore, the nucleotide sequences encoding the above regions, etc., are not limited to known ones, and any nucleotide sequence encoding the above regions, etc. can be used. Due to the degeneracy of the genetic code, there are multiple codons corresponding to one amino acid. Therefore, there are many nucleotide sequences encoding the same amino acid sequence. The nucleotide sequence of the polynucleotide of the present invention may be any of multiple nucleotide sequences generated by the degeneracy of the genetic code, as long as it encodes the CAR of the present invention. Furthermore, in order to optimize expression in cells expressing a CAR, the codons selected to encode amino acids in the nucleotide sequences encoding each region, etc., of the CAR of the present invention may be modified.

[0119] Based on such nucleotide sequence information, a person skilled in the art can prepare polynucleotides encoding each region, etc., using known techniques such as chemical synthesis (e.g., the phosphoramidite method) or PCR amplification. Furthermore, a polynucleotide encoding the CAR of the present invention can be obtained by ligating the thus obtained polynucleotides encoding each region, etc., directly or via a spacer. Note that ligation of polynucleotides encoding each region, etc. can be carried out using known methods such as overlap extension PCR, as shown in the Examples described later.

[0120] <Vector Comprising a Polynucleotide Encoding a Chimeric Antigen Receptor> The present invention provides a vector comprising the above-described 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.

[0121] Examples of viral vectors include retroviral vectors such as lentivirus, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

[0122] Examples of the plasmid vector include plasmid vectors for expression in animal cells, such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0123] An episomal vector is a vector capable of autonomous replication outside of a chromosome. Specific means for using an episomal vector are known (see Yu et al., Science, 2009, 324, 797-801). Examples of episomal vectors include vectors that contain, as vector elements, sequences necessary for autonomous replication derived from EBV, SV40, etc. Specific examples of vector elements necessary for autonomous replication include a replication origin and a gene encoding a protein that binds to the replication origin and controls replication, such as the replication origin oriP and EBNA-1 gene for EBV, and the replication origin ori and SV40LT gene for SV40.

[0124] Examples of artificial chromosome vectors include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors.

[0125] Of these vectors, retroviral vectors are preferred from the viewpoint that they can easily transfer genes efficiently even into cells that grow slowly, and that stable expression strains can be easily established.

[0126] The vector of the present invention may include, in addition to the above-described polynucleotide encoding a CAR, an expression control sequence such as a promoter, an enhancer, a poly A addition signal, or a terminator, a nucleotide sequence encoding a replication origin or a protein that binds to the replication origin to control replication, nucleotides encoding other proteins, and the like.

[0127] The polynucleotide encoding the above-mentioned CAR and nucleotides encoding other proteins described below can be operably positioned downstream of a promoter to enable efficient transcription of each polynucleotide. Examples of such "promoters" include a CMV (cytomegalovirus) promoter, an SRα promoter, an SV40 early promoter, a retroviral LTR, an Rous sarcoma virus (RSV) promoter, an HSV-TK (herpes simplex virus thymidine kinase) promoter, an EF1α promoter, a metallothionein promoter, and a heat shock promoter.

[0128] Examples of the "nucleotide encoding another protein" include marker genes such as fluorescent protein genes, reporter genes, and drug resistance genes, and genes encoding molecules involved in T cell activation such as cytokines. Furthermore, by using DNA encoding an IRES or a 2A peptide sequence (e.g., 2A peptide (T2A) derived from Thosea asigna), the other protein can be polycistronically expressed together with the above-mentioned CAR.

[0129] Furthermore, in order to appropriately induce apoptosis of the administered CAR-expressing cells in the body of a subject (such as a cancer patient) to which the CAR-expressing cells have been administered, the vector of the present invention may contain a suicide gene as a "nucleotide encoding another protein." Examples of suicide genes include herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 (iCasp9). Examples of drugs that activate the functions of such genes include ganciclovir for HSV-TK and AP1903, a chemical induction of dimerization (CID) compound for iCasp9 (Cooper LJ. et al., Cytotherapy. 2006; 8(2): pp. 105-17; Jensen M.C. et al., Biol Blood Marrow Transplant. 2010 Sep; 16(9): pp. 1245-56; Jones B.S., 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).

[0130] <Cells expressing chimeric antigen receptors> The present invention provides cells expressing the CAR of the present invention. The cells of the present invention can be obtained by introducing into a cell a polynucleotide or vector encoding the above-described CAR of the present invention.

[0131] The "cells" into which the polynucleotide or vector of the present invention is introduced are preferably mammalian-derived cells, and for example, human-derived cells or cells derived from non-human mammals such as rodents (mouse, rat, etc.), cattle, sheep, horses, dogs, pigs, and monkeys can be used. The type of cells is not particularly limited, and cells collected from body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, tumors, cancerous ascites, etc. can be used. A preferred example of a CAR-expressing cell is an immune cell.

[0132] Examples of "immune cells" include 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, and the like. Various cell populations can be used as long as they contain lymphocytes or progenitor cells as described above. PBMCs (peripheral blood mononuclear cells) collected from peripheral blood are one of the preferred immune cells. That is, in a preferred embodiment, gene transfer is performed on PBMCs. PBMCs can be prepared according to or in accordance with a conventional method.

[0133] It is preferable to activate the CAR-expressing cells before introducing the polynucleotide of the present invention. For example, the CAR-expressing cells can be activated by stimulating them with an anti-CD3 antibody and an anti-CD28 antibody. More specifically, stimulation with the anti-CD3 antibody and the anti-CD28 antibody can be added by culturing the cells in a culture vessel (e.g., a culture dish) whose culture surface has been coated with the anti-CD3 antibody and the anti-CD28 antibody. Such stimulation can also be performed using magnetic beads coated with the anti-CD3 antibody and the anti-CD28 antibody (e.g., Dynabeads T-Activator CD3 / CD28 provided by VERITAS).

[0134] In order to increase the cell survival rate / proliferation rate, it is preferable to use a culture medium supplemented with a T cell growth factor during activation treatment. IL-2, IL-15, IL-7, etc. can be used as the T cell growth factor. IL-2, IL-15, IL-7, etc. can be prepared according to standard methods. Commercially available products can also be used. While the use of T cell growth factors from animal species other than humans is not excluded, typically, T cell growth factors derived from humans (which may be recombinant) are used.

[0135] Typically, for application (administration to a subject), cells after introduction of the polynucleotide of the present invention (polynucleotide-introduced lymphocytes of the present invention) are grown. For example, the polynucleotide-introduced lymphocytes of the present invention are cultured (subcultured as necessary) using a culture medium supplemented with a T cell growth factor. In addition to this culture, a treatment (reactivation) similar to that used in the activation of cells for expressing CAR may be performed.

[0136] Although a medium containing serum (human serum, fetal bovine serum, etc.) may be used to culture the cells of the present invention, the use of a serum-free medium makes it possible to prepare cells that are highly safe for clinical application and have the advantage of being less susceptible to differences in culture efficiency due to differences between serum lots. When serum is used, it is preferable to use autologous serum, i.e., serum collected from the subject who will receive the cells of the present invention.

[0137] Furthermore, the cells of the present invention may be cells in which the function of a protein (such as an immune checkpoint substance) that suppresses excessive immune responses, etc., is suppressed. Examples of such proteins include Programmed Death 1 (PD1), PD-L1, PD-L2, CTLA4, TIM3, CEACAM (such as CEACAM-1, CEACAM-3, and CEACAM-5), 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, and GAL9.

[0138] Furthermore, the cells of the present invention may be cells in which the functions of endogenous proteins such as TCR and HLA are suppressed in order to facilitate allogeneic transplantation.

[0139] Methods for suppressing the function of a protein include knockout methods that target the gene encoding the protein, methods that use dsRNA (double-stranded RNA, for example, siRNA) complementary to the transcription product of the gene, methods that use antisense RNA complementary to the transcription product, and methods that use RNA with ribozyme activity that specifically cleaves the transcription product. In addition, genome editing methods, which are methods that use site-specific nucleases (e.g., zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), DNA double-strand cleavage enzymes such as CRISPR-Cas9) to modify target genes, are also suitable for suppressing the function of the substance.

[0140] Methods for introducing the polynucleotide or vector of the present invention into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun methods. Furthermore, when the vector of the present invention is a retroviral vector, appropriate packaging cells may be selected based on the LTR sequence and packaging signal sequence contained in the vector, and used to prepare retroviral particles. Examples of packaging cells include PG13, PA317, GP+E-86, GP+envAm-12, and Psi-Crip. Furthermore, 293 cells and 293T cells, which have high transfection efficiency, can also be used as packaging cells.

[0141] Furthermore, after the polynucleotide or vector of the present invention is introduced into a cell, the expression of CAR in the cell can be confirmed by a known method such as flow cytometry, RT-PCR, Northern blotting, Western blotting, ELISA, or fluorescent immunostaining.

[0142] <Pharmaceutical Composition> The present invention also provides a pharmaceutical composition (e.g., an anticancer agent) containing, as an active ingredient, the antibody, ADC, or cell expressing a CAR of the present invention (hereinafter also simply referred to as "the antibody, etc. of the present invention"), as well as a method for treating or preventing a disease associated with the Eva1 protein (e.g., cancer), which comprises the step of administering to a subject a therapeutically or prophylactically effective amount of the antibody, etc. of the present invention.

[0143] The diseases related to the Eva1 protein targeted by the antibodies of the present invention may be any diseases in which the expression of the Eva1 protein is involved in the onset, progression, or worsening of symptoms, and examples thereof include cancer.

[0144] Furthermore, the "cancer" targeted by the antibody of the present invention may be a solid tumor, leukemia, lymphoma, etc., as long as it expresses at least human Eva1. More specific examples include gliomas (tumors arising from neural stem cells, neural progenitor cells, and glial cells, such as glioblastoma multiforme (GBM), astrocytoma, medulloblastoma, epiventricular tumor, oligodendroglioma, and choroid plexus papilloma, particularly anaplastic astrocytoma, anaplastic oligodendroglioma, and anaplastic oligodendroglioma), pancreatic cancer, gastric cancer, esophageal cancer, colon cancer, bile duct cancer, hepatocellular carcinoma, breast cancer, lung cancer, kidney cancer, urothelial cancer, testicular cancer, prostate cancer, head and neck cancer, thyroid cancer, skin cancer, cervical cancer, uterine cancer, ovarian cancer, hemangioma, sarcoma, myeloma, plasma cell carcinoma, and melanoma. Furthermore, such cancers may be primary or metastatic. Furthermore, cancers according to the present invention also include cancer stem cells.

[0145] The "pharmaceutical composition" of the present invention may contain, in addition to the antibody of the present invention, other pharmacologically acceptable components. Examples of such other components include carriers, emulsifiers, humectants, pH buffering agents, culture media, excipients, disintegrants, buffers, isotonicity agents, suspending agents, solubilizers, soothing agents, stabilizers, preservatives, and antiseptics. More specifically, in the case of liquid preparations such as injections, examples of pharmacologically acceptable other components include aqueous solutions (e.g., physiological saline, water for injection, phosphate buffer, aqueous glucose solution, aqueous glycerol solution), and aluminum hydroxide. In the case of lyophilized preparations, examples include, but are not limited to, sugars (e.g., mannitol, lactose, saccharose), albumin, and the like. Furthermore, the pharmaceutical composition of the present invention may be in the form of a kit in which the above components can be mixed prior to administration. Furthermore, when used as an injection, the composition may be in the form of a syringe.

[0146] The pharmaceutical composition of the present invention may contain only the antibody, etc. of the present invention as an active ingredient, or may contain the antibody, etc. and at least one other anticancer agent. The antibody, etc. of the present invention can also be administered together with other anticancer agents, thereby enhancing the antitumor effect. The other anticancer agents used for such purposes may be administered to a subject simultaneously with the antibody, etc. of the present invention, separately, or consecutively, or may be administered at different administration intervals. Examples of such "anticancer agents" include immune checkpoint inhibitors (anti-PD-1 antibodies, etc.), gemcitabine, S-1, erlotinib, 5-FU, temozolomide, leucovorin, irinotecan (CPT-11), oxaliplatin, abraxane, carboplatin, paclitaxel, pemetrexed, sorafenib, vinblastine, LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), and agents described in WO 2003 / 038043, but are not limited thereto as long as they have antitumor activity. Furthermore, in the case of a pharmaceutical composition containing the cells of the present invention, other components may be included, such as dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting the cells, or antibiotics for the purpose of preventing bacterial contamination. Furthermore, for the purpose of activating, proliferating, or inducing differentiation of the cells, the composition may also include T cell activating factors such as cytokines, growth factors, and steroids, vitamins, immunostimulants, and immune checkpoint inhibitors, and the cells of the present invention may also be used in combination with these other components.

[0147] The method of administering a pharmaceutical composition varies depending on the type of substance to be administered, the form of the composition, and the age, weight, sex, and health condition of the subject, but can be administered by either parenteral administration (e.g., intravenous administration, intraarterial administration, local administration) or oral administration. Parenteral administration is preferred, and intravenous administration is more preferred. Local administration may also be used instead of systemic administration. An example of local administration is direct injection into a target tissue, organ, or tissue.

[0148] The dosage of the pharmaceutical composition may vary depending on the subject's age, body weight, sex, health condition, degree of progression of symptoms, and the components of the pharmaceutical composition to be administered. The administration schedule is also adjusted appropriately depending on these conditions, and may be a single administration or multiple continuous or periodic administrations. Generally, when an antibody is administered intravenously, the dosage is 0.1 to 1000 mg, preferably 1 to 100 mg, per kg of body weight per day for an adult. When an ADC is administered, the dosage is 0.001 to 1000 mg, preferably 0.01 to 100 mg, per kg of body weight per day for an adult, calculated in terms of the amount of drug contained. These antibodies or ADCs may be administered once, or multiple times at intervals ranging from once a day to once a year (e.g., weekly, every 10 to 30 days, monthly, every 3 to 6 months, every six months, or yearly).

[0149] The dose of cells administered per kg of body weight to an adult is 1 x 10 ３ ~1x10 １０ pieces (preferably 1x10 ４ ~1x10 ９ pieces, more preferably 1 x 10 ５ ~1x10 ８ The administration interval can be, for example, once a week, every 10 to 30 days, once a month, every 3 to 6 months, or once a year. Furthermore, since the cells of the present invention can grow autonomously in the body of the recipient, they can be administered only once. Furthermore, the number of cells of the present invention in the body can be monitored after administration, and the timing of administration can be determined based on the results.

[0150] When the target of treatment is the brain, the presence of the blood-brain barrier (BBB) ​​often becomes a problem. However, in patients with glioblastoma multiforme (GBM) or the like, the BBB present in normal brains is not formed in the blood vessels within the angiogenic brain tumor, and therefore the antibody can be delivered to the GBM by intravenous injection or the like.

[0151] Furthermore, even when the BBB is functioning, the antibody of the present invention can be administered by bypassing it. For example, a method can be used in which a cannula or the like is inserted using stereotactic brain surgery, and the antibody is then directly administered to a glioma or the like through the cannula. Another method can be used in which a drug delivery system containing the antibody of the present invention is implanted into the brain (e.g., Gill et al., Nature Med. 9:589-595 (2003)). Another method can be used in which neurons across the BBB are transfected with a vector containing a gene encoding the antibody of the present invention (e.g., U.S. Patent Application Publication No. 2003 / 0083299).

[0152] In addition to the above-mentioned methods for bypassing the BBB, the antibody of the present invention can also be administered by incorporating it into or binding it to a brain-barrier permeable substance, such as liposomes bound to antibody-binding fragments that bind to receptors on the vascular endothelium of the BBB (e.g., U.S. Patent Application Publication No. 20020025313), low-density lipoprotein particles (e.g., U.S. Patent Application Publication No. 20040204354), apolipoprotein E (e.g., U.S. Patent Application Publication No. 20040131692), transferrin (e.g., U.S. Patent Application Publication No. 2003 / 0129186), or a 29-amino acid glycoprotein derived from rabies virus (see Kumar et al., Nature, July 5, 2007, Vol. 448, pp. 39-43).

[0153] Furthermore, by regulating the activity of receptors or channels and administering the antibodies of the present invention, the antibodies can be delivered to gliomas etc. via the BBB. Examples of such methods include increasing the permeability of the blood-brain barrier using glucocorticoid blockers (U.S. Patent Application Publication Nos. 2002 / 0065259, 2003 / 0162695, 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 antibodies of the present invention (U.S. Patent No. 5,004,697, etc.).

[0154] The above has been described with reference to brain diseases such as glioma, but the method of administering the antibody of the present invention to such diseases is not limited to these. Furthermore, with regard to other diseases, as with the above-mentioned brain diseases, a person skilled in the art can appropriately select a known method suitable for the disease and allow the antibody of the present invention to act on the affected area.

[0155] In the present invention, "subjects" to which the antibodies etc. of the present invention are administered include, for example, humans suffering from the above-mentioned cancers etc., humans at risk of recurrence of the above-mentioned cancers etc., and humans at risk of contracting the above-mentioned cancers etc. Furthermore, in the present invention, "treatment" includes alleviating (alleviating) symptoms characteristic of the target disease such as cancer or accompanying symptoms, preventing or delaying the worsening of symptoms, etc. "Prevention" means preventing or delaying the onset, manifestation, or recurrence of a disease (disorder) or its symptoms, or reducing the risk of onset, manifestation, or recurrence.

[0156] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0157] Example 1: Anti-human Eva1 humanized antibody An attempt was made to humanize an anti-human Eva1 mouse antibody (the B2E5-48 mouse antibody described in Patent Document 2) using the materials and methods shown 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.

[0158] (Materials and Methods) 1.1 Analysis of Variable Regions and Identification of CDRs Using the IMGT Domain Gap Align tool (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi), CDRs in the variable regions of the B2E5-48 murine antibody were identified and the best matching germline sequences were analyzed.

[0159] 1.2 Molecular Modeling Molecular models were constructed for VL and VH using Absolute Antibody in-house software based on homology with known antibody crystal structures. Images were generated using PyMol.

[0160] 1.3 Risky Sequence Analysis Antibody sequences were analyzed for specific risk based on published protein motifs. The analysis was performed using a system (Absolute Antibody Sequence Liability Tool) built in Microsoft Excel. This software evaluates risk based on the motifs shown in the table below. In the table, X represents any amino acid other than proline.

[0161]

[0162] 1.4 Gene Synthesis and Cloning As shown in Table 6 below, a total of four humanized heavy chains and four humanized light chains were designed. Each was synthesized separately and cloned into a human IgG1 heavy chain expression vector and a human κ light chain expression vector. During transfection, all possible combinations of humanized sequences were used to produce a total of 16 types of humanized antibodies. In addition, a chimeric human IgG1 (in which the constant region of the B2E5-48 mouse antibody was replaced with that of a human IgG1 type) was produced as a control.

[0163] More specifically, the DNA sequences encoding the heavy and light chain variable regions were designed with appropriate restriction enzyme recognition sites at the 5' and 3' ends to allow cloning into cloning vectors and expression vectors manufactured by Absolute Antibody. Furthermore, these DNA sequences were codon-optimized for expression in human cells. Based on these sequences, the DNA was chemically synthesized and then cloned into an Absolute Antibody vector of the appropriate species and type. The accuracy of the sequences was confirmed by Sanger sequencing after analyzing the raw data using DNASTAR Lasergene software. After this confirmation, plasmid DNA preps of the appropriate size were prepared to prepare a sufficient amount of high-quality DNA required for transfection.

[0164] 1.5 Expression and Purification HEK293T cells were passaged until optimal for transient transfection. Heavy and light chain expression vectors were introduced into the cells and cultured for 6 days. The culture was centrifuged at 4000 rpm, and the culture supernatant was collected and filtered through a 0.22 M filter. The first stage of purification was performed by protein A affinity chromatography. The antibody was eluted with a citrate buffer (pH 3.0) and then neutralized with 0.5 M Tris (pH 9.0). The eluted protein was buffer-exchanged into PBS using a desalting column. The antibody concentration was measured by UV spectroscopy and concentrated as necessary. The chimeric human IgG1 was also expressed and prepared in not only HEK293T cells but also CHO cells.

[0165] 1.6 Antibody Analytics Antibody purity was determined using SDS-PAGE and HPLC. SEC-HPLC was performed on an Agilent 1100 series instrument using an appropriate size exclusion column (SEC). Antibody expression titers were measured by Protein A HPLC.

[0166] 1.7 Surface Plasmon Resonance Analysis The dissociation constants and binding constants of the humanized antibodies and the like were analyzed by surface plasmon resonance (SPR, product name: BiaCore T200 (GE Healthcare)).

[0167] Specifically, a human IgG capture antibody was first immobilized on a CM5 chip by the amine coupling method. This immobilization was performed on both the reference cell and the active cell. Then, kinetic analysis of the anti-Eva1 antibody and hEva1 was performed by the single-cycle method under the following measurement conditions.

[0168] Measurement conditions: Sample compartment temperature: 10°C; Running buffer: HBS-EP+ buffer, pH 7.4; Capturing molecule: Human IgG antibody, reference and active cells; Ligand: Various antibodies 5 nM, active cells only; Analyte: hEva1-mouse Fc1, 5, 25, 125, 250 nM, reference and active cells; Regeneration buffer: 3 M MgCl ２ , Reference & Active Cell Blank (Analyte zero concentration) for 3 cycles The cycle conditions are as shown in the table below.

[0169]

[0170] The results obtained using the above materials and methods are shown below.

[0171] 2.1 Sequence Analysis The VH and VL sequences of the B2E5-48 mouse antibody were analyzed using the IGMT Gap Align tool, comparing them with all known antibody germline sequences. As a result, although not shown in the figure, these sequences were closest to mouse sequences, particularly the IGHV1-53 family for VH and the IGKV4-68 family for VL. Additionally, CDRs were assigned using the IMGT definition.

[0172] 2.2 Germline Selection The VH and VL sequences were aligned and analyzed against the Absolute Antibody database of human germline sequences. As a result, the germline sequences selected as frameworks for humanization are shown in Table 6 below. The table also shows the % homology between the heavy and light chain germline sequences selected as frameworks for humanization and the original mouse VH and VL, respectively.

[0173]

[0174] 2.4 CDR Grafting The VH and VL sequences were analyzed using a CDR grafting algorithm, and the CDRs of the B2E5-48 mouse antibody were grafted onto selected human germline sequences. While CDRs are defined as being primarily involved in antigen binding, amino acids outside of framework regions may be directly involved in binding or may play a role in correctly positioning the CDRs. Therefore, to maintain binding, a structure-guiding method was used to determine which amino acids in the framework regions should remain as mouse-derived (original) amino acids. An overview of the resulting sequences is shown in Table 7. In this table, underlined sequences indicate the CDRs in each variable region.

[0175]

[0176] 2.5 Risky Sequence Analysis To ensure that no significant undesirable sequence risks (issues) were introduced into the humanized sequences, the original murine antibody sequences and the humanized sequences were run through the Absolute Antibody Sequence Liability Tool.

[0177] As a result, glycosylation motifs and free cysteines, which are the sequences with the highest risk among those listed in Table 4, were not detected in either the mouse antibody or the humanized antibody. On the other hand, deamidation and isomerization motifs were found to be present in these antibodies. However, although these motifs are considered high-risk as shown in Table 4, they are commonly found in conventional antibodies. Furthermore, although modifications based on these motifs may cause confusion during production, they can often be eliminated through appropriate management methods. Furthermore, if necessary, these motifs can be removed by mutagenesis. Furthermore, although sequences with medium and low risks were detected from the perspective of obtaining more complete antibodies, they have rarely been reported to cause problems during the antibody production process. Therefore, the sequences of the mouse antibody and humanized antibody were determined to be free of any particular problems.

[0178] 3. Antibody Production and Analysis As described above, we attempted to produce 16 types of humanized antibodies. As a result, no antibodies were detected in the supernatant for six of the types (H1-L1, H1-L2, H1-L3, H2-L1, H3-L1, H4-L1), and the antibody concentrations in the supernatant (Titer (mg / L) below) for the other three types (H2-L3, H3-L3, H4-L2) were significantly low. It is believed that these antibodies were not formed normally and were not sufficiently secreted into the culture supernatant.

[0179]

[0180] Furthermore, the binding affinity of the antibodies was analyzed by the SPR method, and as shown in the table, the average dissociation constants (K Ｄ ) is 10 －７ In particular, for the humanized antibodies, H2-L2, H1-L4, H2-L3, and H2-L4 were comparable to chimeric antibodies (produced in HEK293T cells). Furthermore, taking the above antibody concentrations into consideration, it was suggested that H2-L2, H2-L4, and H3-L4 would be useful in the antibody applications described below.

[0181] <Reference Example> Expression of human Eva1 protein (Analysis of Eva1 protein expression in normal human blood cells) In order to analyze the usefulness of anti-Eva1 antibodies in pharmaceutical applications, first, to analyze the usefulness in the chimeric antigen receptor (CAR) described below, the expression of Eva1 protein was evaluated in various blood cells, which are the site of CAR expression.

[0182] Specifically, peripheral blood from healthy individuals was hemolyzed (BD FACS Lysing Solution), then stained with CD4, CD8, CD19, neutrophils (gated from scatter plots), and monocytes (gated from scatter plots), and simultaneously stained with mouse anti-human Eva1 antibody and a secondary antibody, followed by analysis by flow cytometry.

[0183] As a result, as shown in Figure 1, no expression of Eva1 protein was observed in any of CD4, CD8 T lymphocytes, CD19 B lymphocytes, neutrophils, and monocytes, suggesting that blood cells expressing a CAR that uses the Eva1 protein as an antigen are highly likely not to target these cells.

[0184] (Analysis of Eva1 protein expression in tumors) Next, the expression of Eva1 protein in various tumors was evaluated. Specifically, various tumor cell lines were first detached from culture flasks to prepare cell suspensions, and then stained for Eva1 protein in the same manner as described above (Analysis of Eva1 protein expression in normal human blood cells).

[0185] As a result, as shown in FIG. 2A, expression of Eva1 protein was observed in various tumor cell lines other than OVTOKO, PA-1, and T98G.

[0186] We also analyzed Eva1 protein expression in cells isolated from surgical specimens of intrahepatic cholangiocarcinoma cases. Specifically, tumor tissue was collected from surgical specimens of intrahepatic cholangiocarcinoma, and a cell suspension was prepared (MACS Tumor Dissociation Kit, human, Miltenyi Biotec). Tumor cells were purified using a tumor cell isolation kit and human, Miltenyi Biotec, and stained for Eva1 protein in the same manner as above, followed by flow cytometry analysis.

[0187] As a result, as shown in FIG. 2B, Eva1 was positive in three out of three cases of intrahepatic bile duct cancer.

[0188] Example 2 Chimeric Antigen Receptor Using Mouse Anti-Eva1 Antibody Sequence (Preparation of Eva1-CAR Using Anti-Human Eva1 Mouse Antibody Sequence) Next, a chimeric antigen receptor (CAR) targeting the Eva1 protein was prepared to analyze its usefulness. Specifically, as shown in Figure 3A, a single-chain antibody was constructed using the VL and VH of the anti-human Eva1 mouse antibody (the above-mentioned B2E5-48 mouse antibody). Then, VL-linker-VH and VH-linker-VL were prepared, and a CD28 transmembrane domain (CD28TM), CD28 intracellular domain (CD28IC), and CD3zeta were linked to each other via a short spacer domain of 15 aa (comprising amino acids at positions 268 to 282 of SEQ ID NO: 58 or the amino acid sequence of SEQ ID NO: 59) or a long spacer domain of 232 aa (comprising amino acids at positions 268 to 499 of SEQ ID NO: 58 or the amino acid sequence of SEQ ID NO: 63), thereby constructing a CAR molecule.

[0189] Furthermore, EGFR without an intracellular domain (truncated EGFR, tEGFR) was attached after the T2A sequence. This is transcribed as a single mRNA, and after translation, it is separated into CAR and tEGFR molecules at the T2A sequence, and they are distributed in a 1:1 ratio. tEGFR can be used as a gene transfer marker and for selecting gene-transfected cells.

[0190] Specifically, Eva1-CAR (where the variable regions from the N-terminus are VL and VH in that order, and which has the long spacer domain) has the configuration shown in Table 9 below.

[0191]

[0192] In Table 9, the numbers in the "cDNA" column indicate the position (bp) in the DNA sequence described in SEQ ID NO: 57, and the numbers in the "Polypeptide" column indicate the position (amino acid) in the sequence described in SEQ ID NO: 58.

[0193] (Preparation of Eva1-CAR-T using anti-human Eva1 mouse antibody sequence) Next, as shown in FIG. 3B , blood was collected from a healthy donor on Day 0 and purified to CD8-positive cells using CD8 microbeads (Miltenyi Biotec). The T cells were then mixed with CD3 / CD28 beads (Invitrogen) at a ratio of T cell:beads = 1:3 to activate them. On Day 3, gene transfer was performed using a retrovirus capable of transferring a sequence encoding the CAR molecule (the retrovirus was constructed in the same manner as the method described in Mol Ther 2021 Sep 1; 29(9): 2677-2690). Specifically, retrovirus was loaded onto a retronectin-coated plate and spun at 2100 rpm for 120 min. The plate was then washed with PBS, and stimulated CD8+ T cells were seeded onto it and spun at 800 rpm for 2 min. On Day 4, the cells were transferred from the retronectin-coated plate to a regular culture dish. On Day 7, the cells were collected, and a biotinylated EGFR antibody (Erbitax) was added. tEGFR-positive cells were then 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 was stimulated again on Day 10 using CD3 / CD28 beads, and the experiment was carried out on Days 17-20.

[0194] In the following, the same gene transfer scheme was used. Human recombinant IL-2 (50 U / ml) was added on days 1, 4, 7, 10, 13, 16, and (19) before culturing.

[0195] (Efficiency of Eva1-CAR-T gene transfer using anti-human Eva1 mouse antibody sequence) The gene transfer efficiency on Day 7, which was performed in this manner, is shown in Figures 3C and 3D. Figure 3C shows a representative plot (EGFR on the vertical axis was primarily stained with biotinylated erbitax, followed by secondary staining with streptavidin-PE; gene-transferred cells are shown) for LH-S (CAR using VL-VH with a short spacer), HL-S (CAR using VH-VL with a short spacer), LH-L (CAR using VL-VH with a short spacer), and HL-L (CAR using VH-VL with a long spacer). Figure 3D also shows the results of a similar experiment using cells derived from five healthy donors. As shown in Figures 3C and 3D, the gene transfer efficiency was higher when the spacer was longer.

[0196] (Cytokine production ability of Eva1-CAR-T using anti-human Eva1 mouse antibody sequence) Next, the cytokine production ability of Eva1-CAR-T using 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 primary cell lines in a 1:1 ratio. After 6 hours of co-culture, the cells were fixed and intracellularly stained (BD cytofix / cytoperm buffer) and stained with IFN-gamma (IFN-γ). The results are shown in Figure 3E.

[0197] As shown in Figure 3E, IFN-gamma production was not observed in mock T cells transfected with only EGFR, but IFN-gamma production was observed in Eva1-CAR-T. None of the Eva1-CAR-Ts produced IFN-gamma against the Eva1-negative cell line, but IFN-gamma production was observed against the Eva1-positive cell line, confirming the antigen specificity of Eva1-CAR-T. Stronger IFN-gamma production was observed with cells with a longer spacer than with cells with a shorter spacer.

[0198] (Optimization of spacer length of Eva1-CAR-T) As shown in Figure 3F, mouse antibody-derived Eva1-CAR-Ts with spacer regions of six different lengths were produced. The spacer lengths were 0 aa, 15 aa (SEQ ID NO: 59), 30 aa (SEQ ID NO: 60), 60 aa (SEQ ID NO: 61), 125 aa (SEQ ID NO: 62), and 232 aa (SEQ ID NO: 63). 15 aa is the same as the short version in the previous experiment, and 232 aa is the same as the long version. This was incorporated into a plasmid to prepare a retrovirus in the same manner, and gene transfer into CD8-positive cells was performed in the same manner as above.

[0199] (Gene transfer efficiency in the preparation of Eva1-CAR-T with six different spacer lengths) The gene transfer efficiency (EGFR staining) on ​​Day 7, which was performed in this manner, is shown in Figure 3G. As a result, it was revealed that the three CARs with spacer lengths of 30 aa, 125 aa, and 232 aa had higher gene transfer efficiency than the other three types (n = 5).

[0200] (Amplification efficiency of Eva1-CAR-T with six different spacer lengths (stimulation with CD3 / CD28 beads)) Furthermore, the Eva1-CAR-T was subsequently stimulated with CD3 / CD28 beads, and the amplification fold when the culture was continued until Day 17 is shown in FIG. 3H. As a result, there was no difference in the amplification efficiency due to stimulation with CD3 / CD28 beads (n=5).

[0201] (Cytokine production ability of Eva1-CAR-T with six different spacer lengths) An experiment was carried out using the Eva1-CAR-T on Day 17. Specifically, the cells were co-cultured with a tumor cell line at a 1:1 ratio, and after 6 hours, the cells were fixed and 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 3I. The figure shows the percentage of positive CD8-positive cells.

[0202] As shown in Figure 3I, the three cells using spacer lengths of 30 aa, 125 aa, and 232 aa showed excellent cytokine production, and among them, Eva1CAR-T cells with a 232 aa spacer showed the most excellent cytokine production ability.

[0203] (Amplification efficiency of Eva1-CAR-T with six different spacer lengths (Eva1 stimulation, without IL-2)) Eva1-positive HuCCT-1 cells were irradiated with 100 Gy and co-cultured with CAR-T at a 1:1 ratio, and their amplification efficiencies were analyzed. The results are shown in Figures 3J and 3K. Figure 3J shows the amplification efficiency over time. Figure 3K plots the amplification fold on Day 4. As shown in Figures 3J and 3K, good cell expansion was achieved when spacer lengths of 15 aa, 30 aa, and 232 aa were used (n = 5, CAR-Ts were produced from peripheral blood from five different donors).

[0204] (Amplification efficiency of Eva1-CAR-T with six different spacer lengths (Eva1 stimulation, with IL-2)) In the same manner as above, Eva1-positive HuCCT-1 cells were irradiated with 100 Gy and co-cultured with CAR-T cells at a 1:1 ratio. IL-2 50 U / ml was then added once every three days on Days 1, 4, and 7, and culture was continued. The results are shown in Figures 3L and 3M. Figure 3L shows the amplification efficiency over time. Figure 3M plots the amplification fold on Day 18. As shown in Figures 3L and 3M, a tendency for good cell expansion was observed when spacer lengths of 15 aa, 30 aa, and 232 aa were used (n = 5, CAR-T cells were prepared from peripheral blood derived from five different donors).

[0205] Example 3 Chimeric Antigen Receptor Using Anti-Human Eva1 Humanized Antibody Sequence (Preparation of Humanized Eva1-CAR-T (hEva1CAR-T) Using Anti-Eva1 Humanized Antibody Sequence) Next, a total of six types of Eva1CAR-T were prepared using the above-described humanized antibody sequence. Specifically, CARs having a 232 aa spacer were designed for each of the six types: 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). DNA encoding these CARs was then incorporated into each plasmid, and retroviruses were prepared in the same manner as above, followed by gene transfer.

[0206] (Gene transfer efficiency in the preparation of hEva1-CAR-T using six types of anti-human Eva1 humanized antibodies) The gene transfer efficiency (EGFR staining) on ​​Day 7, which was performed in this manner, is shown in Figure 4A. As shown in the figure, it was revealed that the gene transfer efficiency was higher when L2H2 and L4H3 were used than the other four types (n = 4, CAR-Ts were prepared from peripheral blood from four different donors).

[0207] (hEva1-CAR-T Amplification Efficiency Using Six Types of Anti-Eva1 Humanized Antibodies (Stimulation with CD3 / CD28 Beads)) The hEva1CAR-T prepared above were continuously cultured up to Day 17 under stimulation with CD3 / CD28 beads, and the amplification fold is shown in FIG. 4B. As shown in the figure, the amplification efficiency by CD3 / CD28 bead stimulation was better when L2H2, H2L2, and H2L4 were used than the other three types (n=5, CAR-Ts were prepared from peripheral blood derived from five different donors).

[0208] (Cytokine production ability of hEva1-CAR-T using six types of anti-Eva1 humanized antibodies) An experiment was performed using hEva1-CAR-T on Day 17. Specifically, the cells were co-cultured with a tumor cell line at a 1:1 ratio, and 6 hours later, the cells were fixed and stained as described above, and intracellular IFN-gamma and IL-2 were evaluated by flow cytometry. The results are shown in Figures 4C and 4D. These figures show the percentage of CD8-positive cells that were positive for each cytokine. As shown in Figures 4C and 4D, no significant differences were observed between the individual hEva1-CAR-Ts, but the CAR-T using L2H2 produced the lowest cytokine against the Eva1-negative cell line, OVTOKO.

[0209] (Expansion efficiency of hEva1-CAR-T using six types of anti-Eva1 humanized antibodies (Eva1 stimulation)) In the same manner as above, Eva1-positive HuCCT-1 cells were irradiated with 100 Gy and co-cultured with hEva1CAR-T cells at a 1:1 ratio. The results are shown in Figure 4E. In the figure, the left side shows the results of culture in the absence of IL-2, and the right side shows the results of culture with the addition of 50 U / ml of IL-2 once every three days on days 1, 4, and 7. As shown in Figure 4E, in all cases, CAR-T using L2H2 showed the best expansion after stimulation (n = 5, CAR-Ts were prepared from peripheral blood from five different donors).

[0210] (Study of the intracellular domain of L2H2 hEva1-CAR-T) As described above, for CARs using humanized antibodies, CAR-T using L2H2 was superior in antigen specificity and cell proliferation. Furthermore, as shown in Example 2, for CARs using mouse-derived antibodies, CAR-T with a spacer length of 232 aa exhibited the best cell proliferation and cytokine production when cultured in the absence of IL-2. On the other hand, CAR-T with a 15 aa spacer exhibited the best cell proliferation when cultured in the presence of IL-2.

[0211] Therefore, as shown in Figure 4F, six types of CAR-T were prepared by linking three types of intracellular domains (CD28IC, 4-1BBIC, CD79A / CD40IC) via a single-chain variable region (scFv) consisting of L2H2 and spacers of two spacer lengths (S: short spacer, L: long spacer).

[0212] (Gene transfer efficiency in the production of six types of hEva1-CAR-T) The gene transfer efficiency (EGFR staining) on ​​Day 7 in the production of the six types of CAR-T is shown in Figure 4G. As shown in the figure, the gene transfer efficiency was high in the cases of 4-1BB-L and CD79A / CD40-L. Furthermore, as mentioned above, it was also reproduced here that the gene transfer efficiency was higher in CARs with a long spacer (L) than in those with a short spacer (S) (n = 5, CAR-Ts were produced from peripheral blood from five different donors).

[0213] (Cytokine production ability of six types of L2H2 hEva1-CAR-T) An experiment was performed using the hEva1-CAR-T on Day 17 described above. Specifically, the cells were co-cultured with a tumor cell line at a 1:1 ratio, and after 4 hours, the cells were fixed and stained in the same manner as above, and intracellular IFN-gamma and IL-2 were evaluated by flow cytometry. The results are shown in Figures 4H and 4I. These figures show the percentage of CD8-positive cells that were positive for each cytokine. As shown in Figures 4H and 4I, no significant differences were observed between the individual hEva1-CAR-Ts, but the finding that CARs with long spacers had higher cytokine production ability than CARs with short spacers was also reproduced here (n = 5, CAR-Ts were prepared from peripheral blood from five different donors).

[0214] (Expansion efficiency after stimulation of six types of L2H2 Eva1-CAR-T (Eva1 stimulation)) As described above, Eva1-positive HuCCT-1 were irradiated with 100 Gy and co-cultured with hEva1-CAR-T at a 1:1 ratio. The results are shown in Figure 4J. In the figure, the left side shows the results of culture in the absence of IL-2, and the right side shows the results of culture on days 1, 4, and 7, with the addition of 50 U / ml of IL-2 once every three days. As shown in Figure 4J, both short and long 4-1BB and CD79A / CD40 showed almost equally good results (n = 5, CAR-Ts were produced from peripheral blood from five different donors).

[0215] (Co-culture test using six types of L2H2 hEva1-CAR-T) L2H2 hEva1-CAR-T and non-irradiated RPMI-8226 (myeloma cell line) were co-cultured at an E:T ratio of 1:16. RPMI-8226 was labeled with CellTrace Violet before initiating culture, making it possible to distinguish between RPMI-8226 and CAR-T. Aliquots were collected 48, 72, and 96 hours after the initiation of culture and analyzed using flow cytometry. The results are shown in Figure 4K. The figure shows the percentage of tumor cells among all cells. Note that tEGFR is a mock CAR-T cell transfected with only tEGFR, and therefore tumor cells continue to proliferate. No clear differences were observed between intracellular domains.

[0216] (Cytotoxicity test (chromium release test) using six types of L2H2 hEva1CAR-T) L2H2 hEva1-CAR-T and ５１ The cells were co-cultured with various Cr-labeled tumor cell lines for 4 hours, and the released ５１The percentage of damaged cells was determined by measuring Cr (chromium release test). The E:T ratios were 10:1, 3:1, and 1:1. The results are shown in Figures 4L and 4M. As shown in Figure 4L, significant cytotoxic activity by hEva1-CAR-T was observed against cell lines that appeared to be Eva1-positive. No clear differences were observed between the intracellular domains. Furthermore, as shown in Figure 4M, when the Eva1-MFI of each cell line was plotted on the horizontal axis, a correlation with MFI was observed.

[0217] As described above, the difference in reactivity due to differences in intracellular domains was evaluated for hEva1-CAR-T, which has L2H2 as its antigen recognition site. As a result of confirming cell proliferation, cytokine release, and cytotoxic activity in vitro, it was found that CARs having the intracellular domains of 4-1BB and CD79A / CD40 were superior in terms of cell proliferation.

[0218] (Evaluation of antitumor activity of hEva1-CAR-T using a cancer-bearing model) The anticancer effect was evaluated using L2H2 hEva1-CAR-T prepared from peripheral blood from two different donors. Specifically, the evaluation was performed as follows.

[0219] 1 × 10 ６ CFPAC1 cells (human pancreatic cancer cell line) or H1975 (human lung adenocarcinoma cell line) into which 10 firefly luciferase genes had been introduced were intraperitoneally inoculated. Two weeks later, 2 × 10 ６ The antitumor activity of the CAR-T was evaluated by intravenously administering tEGFR-transduced cells (mock CAR-T cells transduced with tEGFR alone, a control) or L2H2 hEva1-CAR-T via the tail vein. In this evaluation, bioluminescence imaging (BLI) was performed using an IVIS Spectrum system to assess tumor progression. The results are shown in Figures 4N to 4Q. Body weight measurements were also performed in parallel.

[0220] Furthermore, the anticancer effect against bile duct cancer was also evaluated using L2H2 hEva1-CAR-T produced from peripheral blood derived from a donor different from the two used above.

[0221] Specifically, 7-week-old NOG mice were first injected with 2 x 10 ６ Firefly luciferase gene-transduced HuCCT cells (human cholangiocarcinoma cell line) were subcutaneously transplanted. After two weeks, tumor size and body weight were measured, and 2 × 10 ６ pcs or 5 x 10 ６ tEGFR-introduced cells (control) or L2H2 hEva1-CAR-T were intravenously administered via the tail vein, and the antitumor activity of the CAR-T was evaluated using BLI as an indicator, as described above. Tumor volume was calculated by measuring the major and minor axes of the subcutaneous tumor using a caliper and calculating "tumor major axis x minor axis x minor axis x 1 / 2." The results are shown in Figures 4R and 4S. Body weight was also measured in parallel.

[0222] In all of the above administration examples to cancer-bearing models, the L2H2 hEva1-CAR used was one in which the antigen recognition site (L2H2) and the transmembrane / intracellular domain (a domain containing 4-1BB or CD79A / CD40) were connected via the short spacer described above. T cells expressing such CAR were prepared by the same method as described above, and on day 10 in Figure 3B, the medium was replaced with a cell preservation solution (Cell Banker) without restimulation with beads, and the cells were stored in liquid nitrogen. The frozen CAR-T were thawed and cultured overnight, and then restimulated with beads. One week after bead stimulation, the cells were administered to cancer-bearing mice as described above.

[0223] As is clear from the results shown in Figures 4N to 4S, the anticancer effects of L2H2 hEva1-CAR-T were confirmed against pancreatic cancer, lung cancer, and bile duct cancer. Furthermore, although not shown in the figures, there were no abnormalities or significant differences in the weight changes of each mouse during the administration period, and no adverse effects were observed in the administered subjects.

[0224] Example 3 Antibody-drug conjugate containing anti-human Eva1 humanized antibody 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).

[0225] Specifically, first, B2E5-L2 expression vector TPG986 and B2E5-H2 expression vector TPG987 were prepared using pcDNA3.4. Each was introduced into E. coli JM109 for expression, and purified using NucleoBond Xtra Midi Kit (MACHEREY-NAGEL).

[0226] Next, using the vector and the ExpiFectamine CHO Transfection System, B2E5-H2-L2 was transiently expressed in ExpiCHO cells. The resulting culture supernatant was then passed through a Protein A column to adsorb the antibody. After elution at low pH, the antibody was neutralized and subsequently purified with Capto adhere, followed by ultrafiltration. The resulting antibody was then confirmed for its molecular weight, sequence, and binding ability to Eva1.

[0227] Next, as shown in Figure 5, a linker (SMCC) was reacted with B2E5-H2-L2 to introduce an MCC group into the antibody, and then the linker was reacted with DM1.

[0228] Specifically, the solvent of the B2E5-H2-L2 solution prepared above was first buffer exchanged with PBS 6.5 / 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, and the antibody concentration of the reaction solution was adjusted to 20 mg / mL. The reaction was allowed to proceed at room temperature for 2 hours using a tube rotator. This reaction solution was purified to obtain an SMCC-derivatized antibody.

[0229] Next, PBS6.5 / EDTA (2.56 mL) and a DMA (dimethylacetamide) solution (0.93 mL; approximately 5.8 equivalents per SMCC-derivatized antibody molecule) of N2-deacetyl-N2-(3-mercapto-1-oxopropyl)-maytansine (4.67 mg; DM1, Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14, p. 4392) were added to the solution at room temperature, and the antibody concentration in the reaction solution was adjusted to 10 mg / mL. The reaction was then carried out at room temperature for 16.5 hours using a tube rotator, followed by purification to obtain a solution containing the compound of interest. The product was then purified to remove unnecessary components, and then stored at a concentration of 9.0 g / L in a solution of 6% sucrose, 0.02% Tween 20, and 10 mM sodium succinate (pH 5.0).

[0230] The molecular weight, sequence, binding ability to Eva1, drug-antibody ratio (DAR), etc. of the ADC were then confirmed. The DAR was determined by measuring the absorbance at 252 nm, which is the absorption peak of DM1, and the absorbance at 280 nm, which is the absorption peak of the antibody, and calculating the concentration of the antibody and DM1 by formulating simultaneous equations taking into account the influence of each on the other's absorption peak, and determining the ratio of the number of molecules. As a result, the DAR of the ADC prepared above was 3.8.

[0231] Example 4 Antitumor Activity of Antibody-Drug Conjugate The antitumor activity of the ADC (B2E5-ADC) containing the anti-human Eva1 humanized antibody prepared above was evaluated as follows.

[0232] (Cytotoxicity experiment of B2E5-ADC against glioma) Human glioma stem cells (hGIC) were seeded at 1,000 cells per well in a 96-well dish. Then, 100 μl / well of two-fold serially diluted B2E5-H2-L2, B2E5-ADC, or Kadcyla (trastuzumab emtansine) was added. The cells were incubated at 37°C in CO ２ The plates were cultured in an incubator for 3 days. Then, 5 μl of MTT (5 mg / ml, Wako Pure Chemical Industries) was added to each well, and the plates were incubated at 37°C with CO ２The cells were cultured in an incubator for 2 hours. After discarding the culture medium, 90 μl of DMSO was added to lyse the cells, and the absorbance (570 nm) was measured using Benchmark (Bio-Rad). The results are shown in Figure 6A.

[0233] The positive control (100%) was the measured value in a well without antibody, and the negative control (0%) was the measured value obtained by adding 5 μl of 1% Triton X100 to a well without antibody.

[0234] (Cytotoxicity experiment of B2E5-ADC against pancreatic cancer) Pancreatic cancer cell line (BxPC3) recovered from a culture dish by trypsin treatment was diluted to 5 × 10 4 cells / mL, and 100 μL was seeded into each well of a 96-well plate. ２ In an incubator (37°C, CO ２ The cells were cultured for 24 hours in a 5% CO solution. The medium (RPMI-1640, Wako 187-02705, 10% FBS) was aspirated, and 900 μL of fresh medium was added to each well. DM1 and B2E5-ADC were each diluted with PBS (DM1: 3800 nM to 0.93 nM, B2E5-ADC: 1000 nM to 0.24 nM), and 10 μL each was added to BxPC3. ２ After culturing in an incubator for 48 hours, LDH released from dead cells was measured using Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories). The results are shown in Figure 6B.

[0235] (Cytotoxicity experiment of B2E5-ADC against gastric cancer) 1 mL of 2.5 g / L trypsin / 1 mmol / L EDTA solution containing phenol red [Nacalai Tesque 32777-15] was added to a gastric cancer cell line (MKN74) cultured in a 10 cm cell culture dish, and the cell was incubated at 37°C for 5 minutes. 3 mL of medium was then added and the cell was collected in a 15 mL centrifuge tube. The cell was centrifuged at 1200 rpm for 5 minutes, the medium was aspirated using an aspirator, and fresh medium was added for resuspension. The number of cells per μL was counted using trypan blue and adjusted to 30,000 cells / mL.

[0236] 100 μL (3000 cells / well) was seeded in a 96-well culture plate and incubated with CO ２ The cells were cultured in an incubator for 24 hours. The medium was aspirated using an aspirator, and 90 μL of fresh medium was added to each well. B2E5-H2-L2 and B2E5-ADC were serially diluted (from 6.5 μg / mL) using PBS, and 10 μL of each was added to each well. ２ The cells were cultured in an incubator for 72 hours. Then, 10 μL of a viable cell measurement reagent [Nacalai Tesque 07553-44] was added to each well and the cells were incubated in a CO ２ After 3 hours of incubation in an incubator, the absorbance at 450 nm was measured. Based on the measurement results, fitting was performed using ImageJ to create a graph, and the cytotoxicity of B2E5-ADC against gastric cancer was evaluated. The results are shown in Figure 6C.

[0237] (B2E5-ADC single-dose toxicity test in mice) A single dose of B2E5-ADC (100, 33, or 10 μg) was administered via tail vein or lumbar puncture to 9-10 week-old C57BL / 6 male mice (CLEA Japan) or 10 week-old male nude mice (SKN / slc, SLC Japan) (three mice were prepared for each administration concentration), and body weight was measured over time. The results are shown in FIG.

[0238] (Summary of Experimental Results Using B2E5-ADC) As shown in Figure 6A, B2E5-ADC exhibited concentration-dependent cytotoxic activity against hGIC. On the other hand, B2E5-H2-L2 and Kadcyla showed no cytotoxic activity against hGIC. Furthermore, as shown in Figures 6B and 6C, the antitumor activity of B2E5-ADC was observed not only against glioma but also against pancreatic cancer and gastric cancer.

[0239] On the other hand, as a result of a toxicity test of B2E5-ADC in mice, as shown in FIG. 7, no effect of B2E5-ADC administration on the mice was observed, and the mice survived for more than one month after administration.

[0240] As described above, according to the present invention, it is possible to exert high antitumor activity by targeting human Eva1. Therefore, the present invention is useful as a drug for treating or preventing cancer.

Claims

1. A humanized antibody or functional fragment thereof, which binds to human-derived Eva1 protein, and which has a light chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequence set forth in SEQ ID NO: 1, an amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 4 to 6.

2. The humanized antibody or functional fragment thereof according to claim 1, wherein the light chain variable region comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 7 to 10, amino acid sequences with 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 several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences set forth in SEQ ID NOs: 7 to 10; and the heavy chain variable region comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 13 to 16, amino acid sequences with 80% or more homology to the amino acid sequences set forth in SEQ ID NOs: 13 to 16, and amino acid sequences in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences set forth in SEQ ID NOs: 13 to 16.

3. The humanized antibody or functional fragment thereof according to claim 1, wherein the light chain variable region comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 7, and at least one of the amino acid sequences set forth in SEQ ID NO: 7, in which one or several amino acids have been substituted, deleted, added, and / or inserted; and the heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 13, and at least one of the amino acid sequences set forth in SEQ ID NO: 13, in which one or several amino acids have been substituted, deleted, added, and / or inserted.

4. An antibody-drug conjugate comprising the antibody or functional fragment thereof according to any one of claims 1 to 3 and a drug.

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

6. A chimeric antigen receptor comprising a region that binds to human-derived Eva1 protein, a transmembrane region, and an intracellular signal 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 functional fragment thereof according to any one of claims 1 to 3.

8. The region that binds to the human Eva1 protein comprises a single-chain variable region fragment consisting of, in order from the amino terminus, a light chain variable region, a linker, and a heavy chain variable region, wherein the light chain variable region comprises complementarity determining regions 1 to 3 each comprising the amino acid sequence of SEQ ID NO: 1, an amino acid sequence consisting of Val-Thr-Ser, and the amino acid sequence of SEQ ID NO: 3, and comprises one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 7, and an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences of SEQ ID NO: 7, and the heavy chain variable region comprises complementarity determining regions 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 4 to 6, and The chimeric antigen receptor according to claim 6, comprising one amino acid sequence selected from the amino acid sequence of SEQ ID NO: 13, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 13, and an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any one of the amino acid sequences of SEQ ID NO:

13.

9. A chimeric antigen receptor according to any one of claims 6 to 8, wherein the region that binds to the human-derived Eva1 protein and the transmembrane region are linked via a spacer consisting of 10 to 300 amino acids.

10. The chimeric antigen receptor according to any one of claims 6 to 9, wherein the intracellular signal region comprises a CD79A intracellular region and a CD40 intracellular region.

11. A nucleotide encoding a 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 of any one of claims 6 to 10.

14. The cell of claim 13, which is an immune cell.

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