WO2023190465A1

WO2023190465A1 - Human anti-sema7a antibody

Info

Publication number: WO2023190465A1
Application number: PCT/JP2023/012409
Authority: WO
Inventors: 耕太郎山本
Original assignee: 株式会社カイオム・バイオサイエンス
Priority date: 2022-03-29
Filing date: 2023-03-28
Publication date: 2023-10-05

Abstract

Provided is a functional human anti-SEMA7A antibody. This invention relates to an anti-SEMA7A antibody in human having a specific CDR sequence, or an antibody fragment of this antibody.

Description

Anti-human SEMA7A antibody

The present invention relates to antibodies against human SEMA7A or fragments thereof, and uses thereof.

Human SEMA7A (Semaphorin 7A) is a GPI (glycosylphosphatidylinositol)-anchored protein with a total length of 604 amino acids and a sema domain and an Ig-like domain. SEMA7A is involved in immune and nervous system regulation. Plexin C1 and integrin β1 are known receptors for SEMA7A, and binding to integrin β1 exerts a significant effect on axonal outgrowth and nerve sprouting via the mitogen-activated protein (MAP) kinase pathway. Furthermore, SEMA7A expressed on activated T cells is known to induce inflammatory cytokine production from monocytes and macrophages via α1β1 integrin (see, for example, Non-Patent Document 1). Furthermore, SEMA7A knockout mice show resistance to the development of contact hypersensitivity reactions and experimental autoimmune encephalomyelitis, suggesting that local inflammatory responses are promoted by activating macrophages via α1β1 integrin. It has been suggested that.

Additionally, SEMA7A has been reported to be involved in various diseases. For example, fibrosis such as idiopathic pulmonary fibrosis (IPF) and non-alcoholic steatohepatitis (NASH), inflammatory diseases such as Crohn's disease and allergic dermatitis, rheumatoid arthritis, multiple sclerosis (MS), Expression of SEMA7A mRNA and protein has been reported in autoimmune diseases such as systemic scleroderma and Sjögren's syndrome, and in various cancers such as lung cancer and breast cancer (see, for example, Non-Patent Document 2).

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are excellent molecular-targeted drugs for the treatment of lung cancer, but EGFR activating gene mutations such as exon 19 deletion mutations (Del19) cause treatment resistance in 30% to 40% of patients. Displaying gender has become an issue. For example, in Non-Patent Document 3, studies using mouse fibroblasts (NIH3T3) and lung cancer patient specimens have shown that SEMA7A expression is induced downstream of EGFR-activating gene mutations, and this SEMA7A is induced by EGFR-TKI. has been identified as a molecule involved in treatment resistance. This has been proven by transplanting a SEMA7A knockout lung adenocarcinoma cell line created using the CRISPR-Cas9 system into immunodeficient mice, and the loss of SEMA7A expression increases sensitivity to EGFR-TKIs.

It has been reported that cell proliferation is enhanced by SEMA7A expression in breast cancer cells (see, for example, Non-Patent Document 4). This is evidenced by a significant reduction in the rate of tumor formation in a xenograft model implanted with a breast cancer cell line in which SEMA7A was knocked down using short hairpin RNA (shRNA).

Generally, when producing antibodies using rodents such as mice, it is difficult to obtain antibodies against molecules that are highly homologous between mammals. In other words, when attempting to produce antibodies using rodents targeting such molecules (human-derived molecules), there is a problem in that antibodies are difficult to produce.

Plexin C1 and integrin β1, which are receptors for SEMA7A, exhibit physiological activity by binding to the sema domain of SEMA7A. That is, in order for the anti-SEMA7A antibody to have functionality, it is preferable to bind to the sema domain and inhibit or suppress interaction with Plexin C1 and integrin β1. Looking at the homology to the human sema domain, the homology is high at 99.1% in cynomolgus monkeys, 90.1% in mice, and 91.1% in rats. Therefore, as mentioned above, it is not easy to obtain antibodies against SEMA7A, and it is also considered difficult to produce antibodies that have the function of inhibiting or suppressing the interaction. In fact, the present inventors confirmed the functionality of commercially available monoclonal antibodies against SEMA7A, but no antibodies with clear functionality existed.

Under these circumstances, it has been desired to obtain and develop functional anti-human SEMA7A antibodies.

The present invention has been made in consideration of the above circumstances, and provides the following anti-human SEMA7A antibodies, antibody fragments thereof, and uses thereof (pharmaceutical compositions, etc.).

[1] An antibody against human SEMA7A,
The amino acid sequences of complementarity determining region (CDR) 1, CDR2, and CDR3 of the heavy chain variable region (VH) are, in order,
Amino acid sequences shown in SEQ ID NOs: 10, 11 and 12,
Amino acid sequences shown in SEQ ID NOs: 18, 19 and 20,
Amino acid sequences shown in SEQ ID NOs: 22, 23 and 24,
Amino acid sequences shown in SEQ ID NO: 26, 27 and 28,
Amino acid sequences shown in SEQ ID NO: 30, 31 and 32,
Amino acid sequences shown in SEQ ID NOs: 34, 35 and 36,
consisting of the amino acid sequences shown in SEQ ID NOs: 38, 39 and 40, or the amino acid sequences shown in SEQ ID NOs: 42, 43 and 44, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region (VL), respectively, in that order:
Consisting of the amino acid sequences shown in SEQ ID NO: 14, 15 and 16,
The antibody.

[2] An antibody against human SEMA7A,
The amino acid sequence of the heavy chain variable region (VH) consists of the amino acid sequence shown in SEQ ID NO: 9, 17, 21, 25, 29, 33, 37 or 41, and
The amino acid sequence of the light chain variable region (VL) consists of the amino acid sequence shown in SEQ ID NO: 13,
The antibody.
[3] The antibody according to [1] or [2] above, which is an antibody that binds to the sema domain of human SEMA7A.
[4] The antibody according to any one of [1] to [3] above, wherein the antibody is a human antibody.

[5] The antibody according to any one of [1] to [4] above, which has the activity of inhibiting or suppressing the interaction between human SEMA7A and Plexin C1 and/or integrin β1.
[6] The antibody according to any one of [1] to [5] above, wherein the antibody has antitumor activity.
[7] The antibody according to any one of [1] to [6] above, which is used for tumor treatment or prevention.

[8] The antibody according to [6] or [7] above, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.
[9] The antibody according to any one of [1] to [5] above, which is used for the treatment or prevention of inflammatory diseases and/or autoimmune diseases.
[10] The inflammatory disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). At least one type of
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
The antibody according to [9] above.

[11] An antibody fragment derived from the antibody according to any one of [1] to [10] above.
[12] An antibody or antibody fragment-drug conjugate, which is a drug conjugated to the antibody according to any one of [1] to [10] above, or the antibody fragment according to [11] above. body.
[13] The complex according to [12] above, wherein the drug is a compound having antitumor activity and/or cell killing activity.

[14] Comprising the antibody according to any one of [1] to [10] above, the antibody fragment according to [11] above, and/or the complex according to [12] or [13] above, Pharmaceutical composition.
[15] The pharmaceutical composition according to [14] above, which is used for the treatment or prevention of tumors.
[16] The pharmaceutical composition according to [15] above, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.

[17] The pharmaceutical composition according to [14] above, which is used for the treatment or prevention of inflammatory diseases and/or autoimmune diseases.
[18] The inflammatory disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). At least one type of
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
The pharmaceutical composition according to [17] above.
[19] The pharmaceutical composition according to [14] above, which is used for inhibiting or suppressing the interaction between human SEMA7A and Plexin C1 and/or integrin β1.

[20] A method for treating or preventing a tumor, which comprises administering to a subject the pharmaceutical composition according to any one of [14] to [16].
[21] The method according to [20] above, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.
[22] A method for treating or preventing inflammatory diseases and/or autoimmune diseases, comprising administering to a subject the pharmaceutical composition according to any one of [14], [17], and [18]. .
[23] The inflammatory disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). At least one type of
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
The method described in [22] above.

[24] A method for inhibiting or suppressing the interaction between human SEMA7A and Plexin C1 and/or integrin β1, which comprises administering to a subject the pharmaceutical composition according to [14] or [19] above.
[25] Contains the antibody according to any one of [1] to [10] above, the antibody fragment according to [11] above, and/or the complex according to [12] or [13] above. A kit for treating, preventing or diagnosing tumors.
[26] The kit according to [25] above, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.

[27] Comprising the antibody according to any one of [1] to [10] above, the antibody fragment according to [11] above, and/or the complex according to [12] or [13] above, A kit for treating, preventing or diagnosing inflammatory diseases and/or autoimmune diseases.
[28] The inflammatory disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). At least one type of
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
The kit according to [27] above.

According to the present invention, a highly active functional antibody that binds to human SEMA7A, preferably its sema domain, can be provided. The anti-human SEMA7A antibody according to the present invention can inhibit or suppress the interaction between human SEMA7A and Plexin C1 or integrin β1, and can be used to treat, for example, idiopathic pulmonary fibrosis (IPF) and non-alcoholic steatohepatitis (NASH). Organ fibrosis, inflammatory diseases such as Crohn's disease, allergic dermatitis, and interstitial pneumonia; autoimmune diseases such as rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome , and can be effective as a therapeutic and preventive drug for various cancers such as lung cancer, breast cancer, oral cancer, osteosarcoma, and chondrosarcoma.

FIG. 2 is a diagram showing the locations where CDR mutations were introduced when producing the heavy chain of anti-SEMA7A antibody. FIG. 2 is a diagram showing the locations where CDR mutations were introduced when producing the light chain of anti-SEMA7A antibody. FIG. 2 is a diagram showing combinations of CDR mutation introduction in the heavy chain of anti-SEMA7A antibody. FIG. 2 is a diagram showing the observation results of the SEMA7A protein expression level on the cell surface of resting HSCs and active HSCs by flow cytometry. FIG. 3 is a diagram showing the ADCC activity of anti-SEMA7A antibody against primary cultured human HSCs. FIG. 3 is a diagram showing the cytotoxic activity of anti-SEMA7A antibody using a second immunotoxin. FIG. 2 is a diagram showing the results of a comparison of the binding affinity between the novel anti-SEMA7A antibody of the present invention and a commercially available anti-SEMA7A antibody. FIG. 3 is a diagram showing the results of comparing the Plexin C1-SEMA7A binding inhibitory activity of anti-SEMA7A antibodies. FIG. 3 is a diagram showing the results of comparing the internalization activities of anti-SEMA7A antibodies. FIG. 2 is a diagram showing the results of comparing the reactivity of anti-SEMA7A antibodies to the semaphorin family (SEMA4F, SEMA5A, SEMA6C, SEMA7A). FIG. 3 is a diagram showing the isoelectric point (pI) of anti-SEMA7A antibody. FIG. 3 is a diagram showing Tm values of Fab in anti-SEMA7A antibodies. The amount of antibody produced by anti-SEMA7A antibody is shown. FIG. 2 is a diagram schematically showing a method for calculating internalization activity. In the figure, "MFI" means Mean Fluorescence Intensity. FIG. 3 is a diagram showing changes over time in internalization activity of anti-SEMA7A antibodies. FIG. 3 is a diagram showing the results of SEMA7A expression analysis in each cancer type. FIG. 3 is a diagram showing changes in SEMA7A expression level due to EGFR mutations in NSCLC. FIG. 3 is a diagram showing the results of SEMA7A expression analysis in each cancer cell line. FIG. 3 is a diagram showing the cytotoxic activity of HC2 second immunotoxin in each cancer type. It is a figure showing the cytotoxic activity of HC1-PBD and HC2-PBD in each cancer cell line.

The present invention will be explained in detail below. The scope of the present invention is not limited to these explanations, and other than the examples below may be modified and implemented as appropriate without departing from the spirit of the present invention.
This specification is based on the specification of Japanese Patent Application No. 2022-054142 (filed on March 29, 2022) and the specification of Japanese Patent Application No. 2022-173382 (filed on October 28, 2022), which are the basis of the priority claim of this application. ). In addition, all publications cited in this specification, such as prior art documents, publications, patent publications, and other patent documents, are incorporated herein by reference.

1. Antibody against human SEMA7A (anti-human SEMA7A antibody)
(1) Preparation of antigen Information on the amino acid sequence (SEQ ID NO: 46) of human SEMA7A (hereinafter also referred to as hSEMA7A) can be found, for example, on the Uniprot website (https://www.uniprot.org/) under "Primary (citable)". ) accession number: O75326" or on the NCBI (GenBank) website (http://www.ncbi.nlm.nih.gov/) as "Accession number: NP 003603" and "Accession number: O75326". Information on the nucleotide sequence (SEQ ID NO: 45) encoding the amino acid sequence of hSEMA7A is available on the NCBI (GenBank) website under “Accession number: NM 003612".

As the antigen, a polypeptide or peptide (hereinafter also simply referred to as peptide) containing all or part of the amino acid sequence of hSEMA7A can be used. Peptides containing part of the amino acid sequence of hSEMA7A are not limited, but, for example, those containing the extracellular domain of hSEMA7A are preferred, and those containing the sema domain are more preferred. The sema domain is a domain consisting of amino acids 53rd to 490th in the amino acid sequence of hSEMA7A (SEQ ID NO: 46).
The peptide to be used as an antigen may be produced by chemical synthesis or by genetic engineering techniques using Escherichia coli or the like, and methods well known to those skilled in the art can be used.
When chemically synthesizing a peptide, it can be carried out by a well-known method of peptide synthesis. Moreover, both solid phase synthesis method and liquid phase synthesis method can be applied to the synthesis. A commercially available peptide synthesizer (for example, CEM Japan: Parallel automatic peptide synthesizer MultiPep 2, etc.) may be used.

When synthesizing a peptide using genetic engineering, first, DNA encoding the peptide is designed and synthesized. The design and synthesis can be performed, for example, by PCR using a vector containing the full-length hSEMA7A gene as a template and primers designed to synthesize the desired DNA region. Furthermore, based on the amino acid sequence of the peptide, it is also possible to gene synthesize a DNA with a Kozak translation initiation sequence inserted at the 5' end and a translation stop codon at the 3' end (using the services of GENEWIZ, etc.). Then, a recombinant vector for protein expression is obtained by ligating the above DNA to an appropriate vector, and a transformant is obtained by introducing this recombinant vector into a host so that the target gene can be expressed (Molecular cloning 4th Ed. Cold Spring Harbor Laboratory Press (2012)).

A phage or plasmid that can autonomously propagate in a host microorganism is used as a vector. Furthermore, animal virus and insect virus vectors can also be used. To create a recombinant vector, purified DNA may be cut with an appropriate restriction enzyme, inserted into the restriction enzyme site of an appropriate vector DNA, and ligated to the vector. The host used for transformation is not particularly limited as long as it can express the gene of interest. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.), insect cells, or insects. It is also possible to use mammals such as goats as hosts. Methods for introducing recombinant vectors into hosts are known. Then, the transformant is cultured, and a peptide used as an antigen is collected from the culture. "Culture" means any of (a) culture supernatant, (b) cultured cells or cultured bacteria, or a crushed product thereof.

After culturing, if the target peptide is produced within the bacterial body or cells, the peptide is extracted by crushing the bacterial body or cells. Furthermore, when the target peptide is produced outside the bacterial body or cells, the culture solution may be used as is, or the bacterial body or cells may be removed by centrifugation or the like. Thereafter, the target peptide is isolated by using general biochemical methods used for isolation and purification of peptides, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combinations. Can be separated and purified.

Peptides serving as antigens can also be obtained by in vitro translation using a cell-free synthesis system. In this case, two methods can be used: a method using RNA as a template and a method using DNA as a template (transcription/translation). As the cell-free synthesis system, commercially available systems such as Expressway ^TM system (Invitrogen), PURESYSTEM (registered trademark; Post Genome Institute), TNT system (registered trademark; Promega), etc. can be used.
The peptides obtained as described above can also be conjugated to suitable carrier proteins, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), human thyroglobulin, chicken gamma globulin, and the like.

The antigen may also be a peptide consisting of the amino acid sequence of hSEMA7A (SEQ ID NO: 46) or a partial sequence thereof in which one or more amino acids are deleted, substituted, or added. For example, one or more (preferably one or several (for example, 1 to 10, more preferably 1 to 5)) amino acids are deleted from the amino acid sequence of hSEMA7A or a partial sequence thereof. , one or more (preferably one or several (for example, 1 to 10, more preferably 1 to 5)) amino acids are substituted with other amino acids, or one or more (preferably 1 to 10, more preferably 1 to 5)) amino acids are substituted with other amino acids, It is also possible to use a peptide consisting of an amino acid sequence to which one or several (eg, 1 to 10, more preferably 1 to 5) other amino acids are added.

Genes to be introduced into cells include genes encoding hSEMA7A protein, partial fragments thereof, or mutant proteins or fragments thereof. As such a gene, for example, one having the base sequence shown in SEQ ID NO: 45 or a partial sequence thereof can be used.
In addition, as a gene to be introduced into cells, etc., a base sequence that hybridizes under stringent conditions with a sequence complementary to the base sequence shown in SEQ ID NO: 45 and that encodes a protein that has the same activity as hSEMA7A is used. , or a subarray thereof.

"Stringent conditions" are conditions for washing after hybridization, in which the salt (sodium) concentration of the buffer is 10 to 500 mM, and the temperature is 42°C to 72°C, preferably the above salt This refers to conditions where the concentration is 50-300mM and the temperature is 55-68°C.
To introduce a mutation into a gene, a known method such as the Kunkel method or the Gapped duplex method can be used, for example, a mutation introduction kit using site-directed mutagenesis, such as the GeneArt ^TM Site-Directed Mutagenesis System (manufactured by Invitrogen). , TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: manufactured by Takara Bio Inc.).

(2) Preparation of polyclonal antibodies The prepared antigen is administered to mammals for immunization. Mammals are not particularly limited, and include, for example, rats, mice, rabbits, etc., with mice being particularly preferred.
The dose of antigen per animal can be appropriately determined depending on the presence or absence of an adjuvant. Examples of the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant, and the like. Immunization can be mainly performed by injecting intravenously, into the footpad, subcutaneously, intraperitoneally, etc. Furthermore, the interval between immunizations is not particularly limited, and immunizations are performed 1 to 10 times, preferably 2 to 3 times, at intervals of several days to several weeks, preferably one week. Then, 3 to 7 days after the final immunization date, the antibody titer is measured using enzyme-linked immunosorbent assay (ELISA or EIA) or radioimmunoassay (RIA), and blood is collected on the day when the desired antibody titer is shown. , antiserum can be obtained. In the above antibody collection method, if the antibody needs to be purified, a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration chromatography, or affinity chromatography may be selected as appropriate; Purification can be achieved by combining them. After that, the reactivity of the polyclonal antibody in the antiserum is measured by ELISA or the like.

(3) Production of monoclonal antibody (3-1) Collection of antibody-producing cells The anti-hSEMA7A antibody of the present invention is preferably, but not limited to, a monoclonal antibody.
The prepared antigen is administered to mammals such as rats, mice, and rabbits for immunization. The dose of antigen per animal can be appropriately determined depending on the presence or absence of an adjuvant. The adjuvant is the same as above. The immunization method is also the same as above. Antibody-producing cells are then collected 1 to 60 days, preferably 1 to 14 days after the final immunization date. Antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, and the like, with lymph node cells and spleen cells being preferred.

(3-2) Cell fusion To obtain a hybridoma (antibody-producing cell line), perform cell fusion between antibody-producing cells and myeloma cells. As myeloma cells to be fused with antibody-producing cells, commonly available cell lines of animals such as mice can be used. The cell line to be used has drug selectivity, cannot survive in HAT selection medium (containing hypoxanthine, aminopterin, and thymidine) in an unfused state, and can only survive in a state fused with antibody-producing cells. It is preferable to have one.
Examples of myeloma cells include P3-X63-Ag8.653, P3-X63-Ag8(X63), P3-X63-Ag8.U1(P3U1), P3/NS I/1-Ag4-1(NS1), and Sp2 Examples include mouse myeloma cell lines such as /0-Ag14 (Sp2/0). Myeloma cells can be selected by appropriately considering compatibility with antibody-producing cells.

Next, the myeloma cells and antibody-producing cells are fused. Cell fusion is carried out in animal cell culture media such as serum-free DMEM and RPMI-1640 medium with 1×10 ⁶ to 1×10 ⁷ antibody-producing cells/mL and 2×10 ⁵ to 2×10 ⁶ cells/mL. /mL of myeloma cells. The cell ratio between antibody-producing cells and myeloma cells (antibody-producing cells: myeloma cells) is usually preferably 1:1 to 10:1, more preferably 3:1, although it is not limited. Next, a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, for example, polyethylene glycol having an average molecular weight of 1,000 to 6,000 Daltons (D) can be used. Furthermore, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation (eg, electroporation).

(3-3) Selection and cloning of hybridomas Target hybridomas are selected from cells after cell fusion treatment. The method is to appropriately dilute the cell suspension in RPMI-1640 medium containing fetal bovine serum, plate it on a microtiter plate, add selective medium to each well, and then replace the selective medium appropriately. Perform culture. As a result, cells that grow about 14 days after the start of culture in the selective medium can be obtained as hybrida.
Next, the culture supernatant of the proliferating hybridoma is screened for the presence of an antibody that reacts with hSEMA7A. Screening for hybridomas may be carried out in accordance with conventional methods and is not particularly limited. For example, a portion of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by ELISA, EIA, RIA, or the like.
Cloning of fused cells can be performed by limiting dilution method or the like. Antibodies that show strong reactivity to hSEMA7A are determined by flow cytometry, etc., and hybridomas that produce these are selected and established as clones.

(3-4) Collection of monoclonal antibodies As a method for culturing established hybridomas and collecting monoclonal antibodies from the resulting culture, a conventional cell culture method, ascites formation method, etc. can be adopted. "Culture" means growing the hybridoma in a culture dish or bottle, or growing the hybridoma intraperitoneally in an animal as described below.
In the cell culture method, hybridomas are grown in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (e.g., 37°C, 5% CO ₂ concentration). Antibodies can be obtained from the culture supernatant after culturing for 7 to 14 days.

In the case of the ascites formation method, about 1×10 ⁷ hybridomas are intraperitoneally administered to an animal of the same species as the mammal derived from myeloma cells, and the hybridomas are grown in large quantities. Then, it is preferable to collect ascitic fluid after 2 to 3 weeks.
In the above antibody collection method, if antibody purification is required, appropriately select known methods such as ammonium sulfate salting out, ion exchange chromatography, gel filtration, affinity chromatography, or a combination of these methods. It can be purified by

(3-5) Selection of functional clones (3-5-1) The anti-hSEMA7A antibody of the present invention inhibits or inhibits the interaction (preferably binding) between hSEMA7A and Plexin C1 and/or integrin β1. Preferably, the antibody has inhibitory activity.
Here, the inhibition or suppressive activity (%) of the interaction between hSEMA7A and integrin β1 can be calculated, for example, by the following formula.
Inhibition or suppressive activity (%) = 100 - (luminescence intensity when antibody is added/luminescence intensity when antibody is not added) x 100
Furthermore, the inhibition or suppressive activity (%) of the interaction between hSEMA7A and Plexin C1 can be calculated, for example, by the following formula.
Inhibition or suppressive activity (%) = 100 - (OD450 when antibody is added/OD450 when antibody is not added) x 100

(3-5-2) The anti-hSEMA7A antibody of the present invention is preferably an antibody having antitumor activity, for example.
Here, "antitumor activity" means an activity that kills tumor cells (cancer cells) or an activity that inhibits tumor growth. Preferable examples of the antitumor activity include cancer cell proliferation inhibitory activity and tumor angiogenesis inhibitory activity. Furthermore, the types of human tumors (tumor cells) in which the antibody of the present invention can exhibit antitumor activity include various known human tumors in which expression of hSEMA7A has been confirmed, and is not particularly limited. Specifically, the human tumors include human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, human chondrosarcoma, human stomach cancer, human pancreatic cancer, human skin cancer, human ovarian cancer, human colon cancer, and human bladder cancer. Preferred examples include one or more of various human tumors such as cancer, human liver cancer, human esophageal cancer, prostate cancer, and human biliary tract cancer, more preferably human breast cancer, human lung cancer, human oral cavity cancer, and human bone cancer. sarcoma, and human chondrosarcoma. Furthermore, examples of human lung cancer include non-small cell lung cancer, more preferably non-small cell lung cancer in which an activating mutation of the EGFR (epidermal growth factor receptor) gene is observed. Furthermore, the type of tumor mentioned above may be recurrent cancer or metastatic cancer, and the antibody of the present invention can exhibit excellent antitumor activity against these tumors as well.

To confirm in vivo antitumor activity, for example, a tumor-bearing mouse (tumor-bearing animal treatment model) in which the desired tumor cells are subcutaneously implanted is used, and the antibody obtained as described above is administered to this mouse. This can be done by In this case, the antibody may be administered immediately after tumor cell transplantation (prevention model), or after confirming that the tumor has reached a predetermined volume for transplantation (treatment model). The administration method is not limited, but may be, for example, intraperitoneal administration at 5 to 20 mg/kg body weight once every 3 days, 1 week, 10 days, or 2 weeks, or once (only once). can. In the case of the prevention model, the presence or absence and level of antitumor activity can be evaluated based on tumor formation frequency and tumor volume. In the case of a treatment model, the presence or absence and level of antitumor activity can be evaluated based on tumor volume.

Tumor growth inhibitory activity in a tumor-bearing animal model is preferably shown at a lower dose, for example, 20 mg/kg body weight or less (preferably 10 mg/kg body weight or less, more preferably 5 mg The dosage is preferably 1 mg/kg body weight or less, more preferably 1 mg/kg body weight or less.
Here, the tumor growth inhibitory activity (%) can be calculated, for example, by the following formula.
Tumor growth inhibitory activity (%) = 100 - [(Tumor volume or tumor weight of antibody administration group) ÷ (Tumor volume or tumor weight of control group)] × 100

(3-5-3) The anti-hSEMA7A antibody of the present invention is preferably one that can be used for the treatment or prevention of tumors, or one that can be used for the treatment or prevention of symptomatic diseases and/or autoimmune diseases.
Here, as for the tumor, the explanations for various human tumors described above are similarly applicable.
Inflammatory diseases include, but are not limited to, idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, as well as organ fibrosis, including non-alcoholic steatohepatitis (NASH). At least one selected from the group consisting of is preferably mentioned.
The autoimmune disease is not limited, but preferably includes, for example, at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome.

(3-5-4) The anti-hSEMA7A antibody of the present invention has, for example, excellent internalization activity into cells expressing hSEMA7A. The intracellular internalization activity can be evaluated by fluorescently labeling the antibody with rhodamine or the like and observing the intracellular migration behavior and localization of the antibody using a fluorescence microscope or the like. For the method of calculating the activity, see, for example, the description in FIG. 12A.

(3-6) Epitope of anti-hSEMA7A antibody The epitope (antigenic determinant) of the anti-hSEMA7A antibody in the present invention may be at least a partial region of the antigen hSEMA7A, but preferably at least the extracellular domain of hSEMA7A. A partial region, more preferably at least a partial region of the sema domain. An anti-hSEMA7A antibody that recognizes the region (binds to the region or a portion containing it) is useful because it can, for example, more effectively exhibit the properties of the anti-hSEMA7A antibody as described below.
The present invention also includes antibodies that can bind to the epitope region that is bound (recognized) by the anti-hSEMA7A antibody of the present invention.

The anti-hSEMA7A antibody of the present invention preferably has a dissociation constant (Kd value) of 1.0×10 ^-10 M or less, more preferably 1.0×10 ^-11 M or less, and even more preferably 1.0 ×10 ^-12 M or less. Here, the binding ability (affinity) of the antibody can be determined by, for example, the dissociation constant (Kd value), dissociation rate constant (Kdiss [1/Sec]), binding rate, etc. using Scatchard analysis or a surface plasmon resonance sensor called Biacore. It can be measured as a constant (Kass [1/M.Sec]). Examples of Biacore devices include Biacore 3000, Biacore 2000, Biacore X, Biacore J, and Biacore Q (all manufactured by Biacore). Antibodies are preferable in that the smaller the dissociation constant (Kd value), the higher the binding ability (affinity). The Kd value is determined by two parameters, Kdiss and Kass, and can be expressed, for example, by the formula: Kd[M]=Kdiss/Kass.

(4) Genetically recombinant antibodies and antibody fragments (4-1) Genetically recombinant antibodies One of the preferred embodiments of the anti-hSEMA7A antibody of the present invention is a genetically recombinant antibody. Genetically recombinant antibodies include, but are not limited to, chimeric antibodies, humanized antibodies (humanized antibodies), human antibodies (fully human antibodies), and the like.
A chimeric antibody (i.e., a humanized chimeric antibody) is an antibody in which the variable region of a mouse-derived antibody is linked (conjugated) to a human-derived constant region (Proc. Natl. Acad. Sci. USA 81, 6851-6855, (1984 ), etc.), chimeras can be easily constructed by recombinant genetic techniques to obtain such linked antibodies.

When creating a humanized antibody, the complementarity determining regions (CDRs) from the variable region of a mouse antibody are grafted onto the human variable region, so that the framework regions (FR) are of human origin and the CDRs are of mouse origin. (so-called CDR grafting). These humanized rearranged human variable regions are then linked to human constant regions. Methods for producing such humanized antibodies are well known in the art (Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al. ., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); see Special Publication No. 4-502408 (Patent No. 2828340; Queen et al.), etc.).

A human antibody (fully human antibody) generally has the same structure as a human antibody in the structure of the hypervariable region (Hyper Variable region) which is the antigen binding site of the V region, other parts of the V region, and the constant region. be. However, the hypervariable region may be derived from other animals. Techniques for producing human antibodies are also well known, and methods for producing gene sequences common to humans using genetic engineering techniques have been established. Human antibodies can be produced, for example, by a method using human antibody-producing mice that have human chromosome fragments containing the H chain and L chain genes of human antibodies (Tomizuka, K. et al., Nature Genetics, (1977) 16, 133- 143; Kuroiwa, Y.et.al., Nuc. Acids Res., (1998)26, 3447-3448; Yoshida, H.et.al., Animal Cell Technology: Basic and Applied Aspects, (1999)10, 69 -73 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers; Tomizuka, K.et.al., Proc. Natl. Acad. Sci. USA, (2000)97, 722 -727 etc.) and a method for obtaining human antibodies derived from phage display selected from a human antibody library (Wormstone, I. M. et.al, Investigative Ophthalmology & Visual Science., (2002)43 (7) , 2301-8; Carmen, S. et.al., Briefings in Functional Genomics and Proteomics,(2002)1(2), 189-203; Siriwardena, D. et.al., Opthalmology,(2002)109(3) ), 427-431 etc.).

Human antibodies can also be produced (and purified) from clones that specifically bind to the desired antigen (hSEMA7A in the present invention) using human ADLib technology (library described in WO 2015/167011). (in particular, the amino acid sequence of VH or VL, preferably at least one CDR sequence in VH or VL) (preferably with amino acid substitution). You can also do it.

As the anti-hSEMA7A antibody of the present invention, for example,
The amino acid sequences of CDR1, CDR2 and CDR3 of VH are as follows:
Amino acid sequences shown in SEQ ID NOs: 10, 11 and 12,
Amino acid sequences shown in SEQ ID NOs: 18, 19 and 20,
Amino acid sequences shown in SEQ ID NOs: 22, 23 and 24,
Amino acid sequences shown in SEQ ID NO: 26, 27 and 28,
Amino acid sequences shown in SEQ ID NO: 30, 31 and 32,
Amino acid sequences shown in SEQ ID NOs: 34, 35 and 36,
consisting of the amino acid sequences shown in SEQ ID NOs: 38, 39, and 40, or the amino acid sequences shown in SEQ ID NOs: 42, 43, and 44, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL, respectively, in that order,
Preferable examples include antibodies having the amino acid sequences shown in SEQ ID NOs: 14, 15, and 16.

Further, more preferred embodiments of the anti-hSEMA7A antibody of the present invention include, for example,
The amino acid sequence of VH consists of the amino acid sequence shown in SEQ ID NO: 9, 17, 21, 25, 29, 33, 37 or 41, and
Preferred examples include antibodies whose VL amino acid sequence consists of the amino acid sequence shown in SEQ ID NO: 13.

The above-mentioned chimeric antibodies, humanized antibodies, and human antibodies are those in which the N-glycoside-linked complex sugar chain in the antibody Fc region is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain. is preferable, and more specifically, a genetically recombinant antibody having a sugar chain in the Fc region of the antibody molecule that is not α-linked to the 6th position of N-acetylglucosamine in which the 1st position of the fucose is the reducing end of an N-glycoside-linked complex type sugar chain. Examples include antibodies consisting of molecules. Such antibodies can improve ADCC activity. Note that this point (characteristics of N-glycoside-bonded complex sugar chains in the antibody Fc region) is similarly preferable for the above-mentioned polyclonal antibodies and monoclonal antibodies.

(4-2) Antibody fragment Fragments (partial fragments) of the anti-hSEMA7A antibody of the present invention are also included in the antibody of the present invention. Here, the antibody fragment of the present invention, like the anti-hSEMA7A antibody of the present invention, has binding activity for hSEMA7A (preferably sema domain) (ie, is capable of binding to hSEMA7A). Preferably, it has the above-mentioned activity of inhibiting or suppressing the interaction (preferably binding) between SEMA7A and Plexin C1 and/or integrin β1, or has antitumor activity. More preferred are those that can be used for the treatment or prevention of tumors, and those that can be used for the treatment or prevention of symptomatic diseases and/or autoimmune diseases, as described above.

The fragment of the antibody means a partial region of an anti-hSEMA7A polyclonal antibody or an anti-hSEMA7A monoclonal antibody (i.e., an antibody fragment derived from the anti-hSEMA7A antibody of the present invention), such as Fab, Fab', F(ab' ) ₂ , Fv (variable fragment of antibody), single chain antibody (H chain, L chain, H chain V region, L chain V region, etc.), scFv, diabody (scFv dimer), dsFv (disulfide stabilized V region) and a peptide that includes at least a portion of a complementarity determining region (CDR).

Fab is a fragment obtained by treating an antibody molecule with the proteolytic enzyme papain, and has antigen-binding activity with a molecular weight of approximately 50,000, in which approximately half of the N-terminal side of the H chain and the entire L chain are bonded by disulfide bonds. It is an antibody fragment. Alternatively, Fab can also be produced by inserting the DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and expressing the vector by introducing the vector into the prokaryotic or eukaryotic organism. can.
F(ab') ₂ is an antigen-binding fragment with a molecular weight of approximately 100,000, which is slightly larger than the fragment obtained by treating an antibody molecule with the proteolytic enzyme pepsin, in which Fab is bound via disulfide bonds in the hinge region. It is an active antibody fragment. Moreover, it can also be produced by linking Fab described below with a thioether bond or a disulfide bond.

Fab' is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond in the hinge region of F(ab') ₂ described above. In addition, Fab' can be produced by inserting the DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and expressing the vector by introducing the vector into a prokaryotic or eukaryotic organism. You can also.
scFv is VH-P-VL or VL-P-VH, in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker (P). It is a polypeptide and an antibody fragment with antigen-binding activity. scFv is produced by obtaining cDNA encoding the VH and VL of an antibody, constructing the DNA encoding the scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and converting the expression vector into a prokaryotic expression vector. It can be expressed and produced by introducing it into an organism or eukaryote.

A diabody is an antibody fragment that is a dimerization of scFv and has bivalent antigen-binding activity. The two valent antigen-binding activities can be the same, or one can have a different antigen-binding activity. Diabody obtains cDNA encoding the VH and VL of an antibody, constructs the scFv-encoding DNA so that the amino acid sequence of the peptide linker (P) is 8 residues or less, and converts the DNA into a prokaryote. It can be produced by inserting it into an expression vector for organisms or an expression vector for eukaryotes, and expressing it by introducing the expression vector into prokaryotes or eukaryotes.

dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue, which are linked via a disulfide bond between the cysteine residues. The amino acid residue to be substituted for the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7, 697-704, 1994). dsFv is produced by obtaining cDNA encoding the VH and VL of an antibody, constructing a DNA encoding dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and translating the expression vector into a prokaryotic expression vector. It can be expressed and produced by introducing it into an organism or eukaryote.

Peptides containing CDRs are composed of at least one region of VH CDRs (CDR1 to 3) and VL CDRs (CDR1 to 3), and include those containing all VH CDRs and all VL CDRs. It is more preferable to include all CDRs of VH and VL (six regions in total), and particularly preferable. Preferred amino acid sequences of CDRs include the various amino acid sequences of CDRs 1 to 3 of VH and VL described above. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker. Peptides containing CDRs can be produced by constructing DNA encoding the CDRs of VH and VL of an antibody, inserting the DNA into an expression vector for prokaryotes or an expression vector for eukaryotes, and translating the expression vector into a prokaryotic or eukaryotic expression vector. It can be expressed and produced by introducing it into an organism. Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

The antibody fragment of the present invention may be a part of the antibody Fc region in which the N-glycoside-linked complex sugar chain does not have fucose bound to N-acetylglucosamine at the reducing end of the sugar chain, or Alternatively, an antibody fragment containing the entirety of the antibody fragment and the N-glycoside-linked complex sugar chain may be used in the antibody Fc region, which is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain. It may be a fusion protein with a part or all of it. Such antibody fragments are preferred because they can dramatically improve ADCC activity.
Hereinafter, in the description herein, the above-mentioned antibody fragments are also included in the anti-hSEMA7A antibody of the present invention.

2. Polynucleotide, Recombinant Vector, and Transformant The present invention can also provide a polynucleotide (gene, DNA) encoding the anti-hSEMA7A antibody of the present invention described above or an antibody fragment thereof. Specifically, the polynucleotide is preferably a polynucleotide containing a base sequence encoding each of the amino acid sequences shown as examples of the anti-hSEMA7A antibody and antibody fragment of the present invention described above. Furthermore, the polynucleotide of the present invention may consist only of a polynucleotide encoding the anti-hSEMA7A antibody or antibody fragment of the present invention, or it may contain the polynucleotide as a part and contain other components necessary for gene expression. It may also contain known base sequences (transcription promoter, SD sequence, Kozak sequence, terminator, etc.), and is not limited to such sequences.

In the polynucleotide encoding the anti-hSEMA7A antibody or antibody fragment of the present invention, codons corresponding to individual amino acids after translation are not particularly limited, and codons that are commonly used in mammals such as humans after transcription are used. It may contain nucleotide DNA indicating codons (preferably frequently used codons), or it may contain nucleotide DNA indicating codons (preferably frequently used codons) that are commonly used in microorganisms such as E. coli and yeast, plants, etc. It may also contain nucleotide DNA that exhibits high codons.
The present invention can also provide a recombinant vector containing the polynucleotide of the present invention and a transformant containing the recombinant vector.

The polynucleotide (gene, DNA) to be incorporated into the expression vector used as a recombinant vector has a transcription promoter, an SD sequence (if the host is a prokaryotic cell) and a Kozak sequence (if the host is a eukaryotic cell) as necessary. ) may be linked, a terminator may be linked downstream, and an enhancer, splicing signal, polyA addition signal, selection marker, etc. may also be linked. Each element necessary for gene expression, such as the above-mentioned transcription promoter, may be included in the polynucleotide from the beginning, or if it is originally included in the expression vector, it may be used. The manner of use is not particularly limited.

As a method for integrating the polynucleotide into an expression vector, various methods using known genetic recombination techniques can be employed, such as methods using restriction enzymes and methods using topoisomerase. Expression vectors include, for example, plasmid DNA, bacteriophage DNA, retrotransposon DNA, retrovirus vectors, artificial chromosome DNA, etc. that contain polynucleotides (genes, DNA) encoding the anti-hSEMA7A antibody of the present invention or antibody fragments thereof. There are no limitations as long as the vector can be retained, and a vector suitable for the host cell to be used can be appropriately selected and used.

Next, the constructed recombinant vector is introduced into a host to obtain a transformant, which is then cultured to express the anti-hSEMA7A antibody or antibody fragment thereof of the present invention. The term "transformant" used in the present invention refers to a host into which a foreign gene has been introduced; for example, a transformant into which a foreign gene has been introduced by introducing plasmid DNA or the like into the host (transformation); It also includes those into which foreign genes have been introduced by infecting the host with various viruses and phages (transduction).
The host is not limited as long as it can express the anti-hSEMA7A antibody of the present invention or its antibody fragment after the recombinant vector is introduced, and it can be selected as appropriate. Known hosts include various animal cells such as mice, various plant cells, bacteria, yeast, and plant cells.

When using animal cells as hosts, for example, human fibroblasts, human fetal kidney cells, HEK293 cells, 293F cells, CHO cells, monkey cells COS-7, Vero, mouse L cells, rat GH3, human FL cells, etc. used. Furthermore, insect cells such as Sf9 cells and Sf21 cells can also be used.
When using bacteria as a host, for example, Escherichia coli, Bacillus subtilis, etc. are used.
When using yeast as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc. are used.
When using plant cells as hosts, for example, tobacco BY-2 cells are used.

The method for obtaining the transformant is not limited and can be selected as appropriate, taking into consideration the combination of host and expression vector, but includes, for example, electroporation, lipofection, heat shock, PEG, Preferred examples include the calcium phosphate method, the DEAE dextran method, and methods of infecting various viruses such as DNA viruses and RNA viruses.
In the resulting transformant, the codon type of the polynucleotide contained in the recombinant vector may be the same as or different from the codon type of the host used, and is not limited.

3. Antibody or antibody fragment-drug conjugate The above-mentioned immunoconjugate using the anti-hSEMA7A antibody or antibody fragment of the present invention contains the antibody or antibody fragment and a drug (preferably, the antibody or antibody fragment contains a drug). , a drug conjugated), an antibody or antibody fragment-drug conjugate.
The above-mentioned drug is preferably, but not limited to, a compound having antitumor activity and/or cell-killing activity, and specifically, for example,
- Tubulin inhibitors and/or microtubule polymerization inhibitors (more specifically, Auristatins (MMAE, MMAF, etc.), Maytansines (DM1, DM4, etc.), Tubulysins, cryptophycins, rhizoxin, etc.);
・Antibiotics (more specifically, Calicheamicins, Doxorubicin, anthracyclines, etc.);
・DNA synthesis inhibitors (more specifically, Duocarmycins, PBDs (Benzodiazepines), IGNs (Indolinobenzodiazepines), etc.);
・Topoisomerase I inhibitors (more specifically, camptothecin analogues (SN-38, DXd, etc.), etc.);
・RNA polymerase II inhibitors (more specifically, Amanitins, etc.); and ・RNA spliceosome inhibitors (more specifically, spliceostatins, thailanstatins, etc.)
Examples include. Further, as a drug exhibiting antitumor activity, for example, a compound that is excited by light energy and exhibits toxicity can also be used. In addition, using genetic recombination technology, a protein toxin as a substance with antitumor activity and/or cell killing activity is linked to an antibody gene or antibody fragment gene on the gene and expressed as one protein (fusion protein). The resulting products are generally called immunotoxins.

Examples of substances having antitumor activity include doxorubicin, calicheamicin, mitomycin C, Auristatin E, pyrrolobenzodiazepine (PBD), and radioisotope (RI). Examples of the substance having cell-killing activity include saporin, ricin, Pseudomonas aeruginosa exotoxin, diphtheria toxin, and radioisotope (RI), among which saporin and Pseudomonas aeruginosa exotoxin are preferably used. Note that RI having antitumor activity and/or cell-killing activity includes, but is not limited to, ⁹⁰ Y, ¹¹¹ In, ¹²⁵ I, ³ H, ³⁵ S, ¹⁴ C, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re. , ¹⁷⁷ Lu, ⁶⁷ Cu, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ¹⁹⁸ Au, ²²⁴ Ac, ¹²⁶ I, ¹³³ I, ⁷⁷ Br, ^113m In, ⁹⁵ Ru, ⁹⁷ Ru, ¹⁰³ Ru, ¹⁰⁵ Ru, ¹⁰⁷ Hg, ²⁰³ Hg, ^94m Tc, ^121m Te, ^122m Te, ^125m Te, ¹⁶⁵ Tm, 167 Tm, ¹⁶⁸ Tm, ¹¹¹ Ag, ¹⁹⁷ Pt, ¹⁰⁹ Pd, ³² P, ³³ P, ⁴⁷ Sc, ¹⁵³ ^{Sm, 177} ^Lu , ¹⁰⁵ Rh, ¹⁴² Pr, ¹⁴³ Pr, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁹⁹ Au, ⁵⁷ Co, ⁵⁸ Co, ⁵¹ Cr, ⁵⁹ Fe, ¹⁸ F, ⁷⁵ Se, ²⁰¹ Tl, ²²⁵ Ac, ⁷⁶ Br, ⁸⁶ Y, ¹⁶⁹ Yb, ¹⁶⁶ Dy , ²¹² Pb and ²²³ Ra.

Methods for producing antibody or antibody fragment-drug complexes include, but are not limited to, methods in which an antibody or antibody fragment is coupled to a drug via a disulfide bond or a hydrazone bond.
As mentioned above, the anti-hSEMA7A antibody and antibody fragments thereof of the present invention have excellent internalization activity into cells expressing hSEMA7A, and therefore are preliminarily treated with substances having antitumor activity and/or cell-killing activity. By conjugating these substances to the antibody or antibody fragment, these substances can be made to act directly and highly selectively on cells (especially tumor cells). The antibody or antibody fragment-drug conjugate of the present invention has an excellent ability to deliver a drug to target tumor cells.

Furthermore, the present invention can also provide an antibody fragment-drug complex using the above-mentioned antibody fragment instead of the antibody. For details of the antibody fragment-drug conjugate, the above explanation of the antibody-drug conjugate can be similarly applied.
Hereinafter, in the description herein, the antibody-drug conjugate of the present invention includes an antibody fragment-drug conjugate.

4. Pharmaceutical compositions, etc. The anti-hSEMA7A antibody and antibody-drug conjugate (hereinafter also referred to as the conjugate of the present invention) of the present invention are useful as active ingredients contained in pharmaceutical compositions.
Since the anti-hSEMA7A antibody of the present invention may have antitumor activity as described above, the anti-hSEMA7A antibody of the present invention and the complex of the present invention are preferably used for the treatment and/or prevention of tumors. . Therefore, the pharmaceutical composition is useful as a pharmaceutical composition for treating and/or preventing tumors, and further for diagnosis. That is, the anti-hSEMA7A antibody of the present invention and the complex of the present invention are useful as active ingredients contained in tumor therapeutic agents and tumor diagnostic agents. In addition, in the present invention, the above-mentioned tumor treatment includes the meaning of tumor growth inhibition and growth suppression, and specifically, for example, in the case of a tumor therapeutic agent, a tumor growth inhibitor and a form of the growth inhibitor are included. shall also be included.
Further, the anti-hSEMA7A antibody of the present invention and the complex of the present invention are preferably used for the treatment and/or prevention of inflammatory diseases and/or autoimmune diseases, as described above. Therefore, the pharmaceutical composition is useful as a pharmaceutical composition for treating and/or preventing inflammatory diseases and/or autoimmune diseases, as well as for diagnosing them. That is, the anti-hSEMA7A antibody of the present invention and the complex of the present invention are useful as active ingredients contained in therapeutic and diagnostic agents for inflammatory diseases and/or autoimmune diseases.
Furthermore, the pharmaceutical composition of the present invention may be a composition for inhibiting or suppressing interaction (preferably binding) between hSEMA7A and Plexin C1 and/or integrin β1.

The pharmaceutical composition of the present invention is preferably provided in the form of a pharmaceutical composition containing the anti-hSEMA7A antibody of the present invention as an active ingredient, and further containing a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition of the present invention can also be used in combination with a known compound having antitumor activity (eg, cisplatin) and/or a compound having cell killing activity. The mode of combined use may be, for example, a mode in which the pharmaceutical composition of the present invention further contains the compound, or a mode in which it is used in combination with the compound, and is not limited. Such a combination provides even higher antitumor effects.

The above explanation can be similarly applied to human tumors to which the pharmaceutical composition of the present invention is applied. The tumor to be applied may be recurrent cancer or metastatic cancer, and the pharmaceutical composition of the present invention (as well as the anti-hSEMA7A antibody of the present invention or the complex of the present invention) is suitable for the treatment of recurrent cancer or metastatic cancer. It can also be effectively used as an agent, preventive agent, and diagnostic agent.
Further, the above explanation can be similarly applied to inflammatory diseases and autoimmune diseases to which the pharmaceutical composition of the present invention is applied.

"Pharmaceutically acceptable carrier" means excipients, diluents, fillers, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavoring agents, etc. agents, solubilizing agents, and other additives. By using one or more of such carriers, pharmaceutical compositions in the form of injections, solutions, capsules, suspensions, emulsions, syrups, etc. can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include injections containing one or more active substances and formulated in a conventional manner. In the case of an injection, it can be produced by dissolving or suspending it in a pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection.

In particular, when administering antibody fragments (especially low molecular weight ones) derived from the anti-hSEMA7A antibody of the present invention into a living body, a colloidal dispersion system can be used in addition to the above. Colloidal dispersion systems are expected to have the effect of increasing the stability of compounds (antibody fragments) in vivo and the effect of efficiently transporting compounds to specific organs, tissues, or cells. Colloidal dispersion systems are not limited as long as they are commonly used, but include polyethylene glycol, polymer complexes, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles. and lipid-based dispersion systems including liposomes, preferably a plurality of liposomes, artificial membrane vesicles, which have the effect of efficiently transporting compounds to specific organs, tissues or cells. (Mannino et al., Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem.et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cull is , Current Op. Biotech., 1995, 6, 698).

The dosage of the pharmaceutical composition of the present invention is determined depending on the patient's age, sex, weight, symptoms, therapeutic effect, administration method, treatment time, or the anti-hSEMA7A antibody of the present invention or the complex of the present invention contained in the pharmaceutical composition. It varies depending on the type etc. Usually, the dose per adult can be administered in the range of 600 μg to 6000 mg at a time, but the dose is not limited to this range.

For example, when administered by injection, the amount of 100 μg to 100 mg per 1 kg body weight may be administered to human patients once to several times per day on average, preferably for 3 days. It is also possible to administer the drug once every 1 week, 10 days, or 2 weeks, or to administer it once (the total number of administrations is 1 time). The form of administration includes intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and intraperitoneal injection, with intravenous injection being preferred. Injections can also be prepared as non-aqueous diluents (for example, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions, or emulsions, depending on the case. Such injections can be sterilized by filter sterilization, addition of a bactericide, and the like. Injections can be manufactured in a ready-to-use form. That is, it can be prepared into a sterile solid composition by freeze-drying or the like, and dissolved in sterile distilled water for injection or other solvent before use.

The present invention also relates to the treatment, prevention, and/or diagnosis of tumors, inflammatory diseases, and/or autoimmune diseases (hereinafter also referred to as tumors), or the interaction between hSEMA7A and Plexin C1 and/or integrin β1 ( The present invention also provides the use of the anti-hSEMA7A antibody of the present invention and/or the conjugate of the present invention for producing a medicament (drug) that inhibits or suppresses hSEMA7A binding.
Furthermore, the present invention provides a method for treating, preventing, and/or diagnosing tumors, etc., or for inhibiting or suppressing the interaction (preferably binding) between hSEMA7A and Plexin C1 and/or integrin β1. Also provided are anti-hSEMA7A antibodies and/or conjugates of the invention.
Furthermore, the present invention provides a method for treating, preventing, and/or diagnosing tumors, etc., which uses the anti-hSEMA7A antibody of the present invention and/or the complex of the present invention (that is, administering it to a subject (patient)); The present invention provides a method for inhibiting or suppressing the interaction (preferably binding) between hSEMA7A and Plexin C1 and/or integrin β1, and also provides a method for inhibiting or suppressing the interaction (preferably binding) between hSEMA7A and Plexin C1 and/or integrin β1, and for treating, preventing and/or diagnosing tumors, etc., or between hSEMA7A and Plexin Also provided is the use of the anti-hSEMA7A antibody of the invention and/or the conjugate of the invention to inhibit or suppress interaction (preferably binding) with C1 and/or integrin β1.

5. The anti-hSEMA7A antibody of the present invention and the complex of the present invention can be used in kits for the treatment and/or prevention of tumors, etc., such as kits for diagnosis or detection of tumors, or for the interaction of hSEMA7A with Plexin C1 and/or integrin β1 (preferably). In addition to the form of a kit for inhibiting or suppressing the binding of the compound, it can also be provided in the form of a kit for diagnosing or detecting tumors, etc. The above explanation can be similarly applied to specific examples of tumors, inflammatory diseases, and autoimmune diseases to be diagnosed or detected.

The diagnosis and detection involves, for example, reacting the anti-hSEMA7A antibody of the present invention and/or the complex of the present invention with a sample collected from a living body (hereinafter referred to as biological sample), and detecting a signal of the reacted antibody, etc. This can be done by Note that since hSEMA7A has been confirmed to be expressed in various tumor cells, hSEMA7A can also be used as a marker for various tumors.
The detected signals of antibodies and the like serve as an indicator of the amount of antigen (ie, the amount of hSEMA7A) in the biological sample. Diagnosis and detection of tumors, etc. using the antibodies, etc. of the present invention involves first applying an antigen-antibody reaction between a biological sample collected from a subject as a specimen, such as a tissue piece or blood to be tested, and the antibodies, etc. of the present invention. Combine by. Next, the amount of the target antigen in the biological sample is measured based on the measurement result of the amount of bound antibody. The measurement may be performed according to a known immunoassay method, for example, immunoprecipitation method, immunoagglutination method, labeled immunoassay method, immunospecific suspension method, Western blotting method, flow cytometry method, etc. can. In labeled immunoassay, the antibody signal may be expressed by the amount of label directly detected using a labeled antibody, or may be expressed relatively using an antibody of known concentration or known antibody titer as a standard solution. That is, a standard solution and a specimen are measured using a measuring meter, and signals of antibodies, etc. in a biological sample can be expressed relatively based on the value of the standard solution. Examples of labeled immunoassay methods include ELISA method, EI method, RIA method, fluorescence immunoassay (FIA) method, and chemiluminescence immunoassay method. Among these, the ELISA method is particularly preferred because it is simple and highly sensitive.

The condition of a tumor, etc. can be evaluated or diagnosed using the detection results obtained as described above as an index. For example, a detection result exceeding a predetermined standard value is considered positive, a detection result below a predetermined reference value is considered negative, and in the case of a positive result, it is determined that there is a possibility that one of the tumors has developed. The condition of tumors etc. can be evaluated. Here, the condition of a tumor, etc. refers to the presence or absence of a tumor, etc., or the degree of progression thereof, and includes the presence or absence of a tumor, etc. onset, degree of progression, malignancy, and presence or absence of recurrence. Examples include presence or absence.

In the above evaluation, one state of the tumor, etc. may be selected, or a combination of multiple states may be selected. The presence or absence of a tumor or the like can be evaluated by determining whether or not the patient is suffering from a tumor or the like using a predetermined reference value as a boundary based on the obtained detection results. The malignancy of a tumor, etc. is an index indicating how far the symptoms of a tumor, etc. have progressed. In the case of tumors, it is also possible to classify and evaluate the disease stage, or to classify and evaluate early cancer or advanced cancer, based on the detection results. For example, it is possible to evaluate whether the cancer is early stage cancer or advanced cancer using the detection result as an indicator. Tumor metastasis can be evaluated by using the detection results as an indicator and determining whether a neoplasm has appeared in a site distant from the primary tumor. Recurrence of a tumor, etc. can be evaluated by determining whether the detection result exceeds a predetermined reference value again after an intermittent period or remission.

In addition to containing the anti-hSEMA7A antibody of the present invention or the complex of the present invention, the kit of the present invention may also contain a labeling substance, or a solid-phase reagent on which the antibody or its label is immobilized. Labeling substances for antibodies refer to those labeled with enzymes, radioactive isotopes, fluorescent compounds, chemiluminescent compounds, and the like. In addition to the above-mentioned components, the kit of the present invention includes other reagents for carrying out the detection of the present invention, such as an enzyme substrate (such as a chromogenic substrate) and an enzyme substrate solution when the labeled substance is an enzyme label. , an enzyme reaction stop solution, or a sample diluent. In addition, various buffers, sterile water, various cell culture vessels, various reaction vessels (Eppendorf tubes, etc.), blocking agents (serum components such as Bovine Serum Albumin (BSA), Skim milk, Goat serum, etc.), detergents, surfactants, It may also include various plates, preservatives such as sodium azide, and experimental operation manuals (instructions).

The present invention will be described below in more detail with reference to Examples, but the present invention is not limited thereto.

1. Expression and purification of recombinant human SEMA7A extracellular domain protein Sequence with a 6×His tag inserted at the C-terminus of amino acids 45 to 643 of the amino acid sequence (SEQ ID NO: 46) of human SEMA7A (hSEMA7A) protein designed. The amino acid sequence designed in this way was converted into a base sequence based on the mammalian codon table, and a DNA sequence with a Kozak translation initiation sequence inserted at the 5' end and a translation stop codon at the 3' end was synthesized by gene synthesis ( GENEWIZ). An expression vector was created by inserting the synthesized DNA between the restriction enzyme KpnI recognition sequence and the XbaI recognition sequence of pEF1/V5-His A (Thermo Fisher Scientific).

The prepared expression vector plasmid was transiently transfected into FreeStyle 293 cells (Thermo Fisher Scientific) using the polyethyleneimine method, and then cultured at 37° C. in a 5% CO ₂ incubator for 5 days. The culture supernatant was collected, filtered through a 0.22 μm filter, and then bound to a HisTrap excel column (GE Healthcare). It was then eluted with a concentration gradient from 20mM to 500mM imidazole in 20mM phosphate buffer/300mM NaCl/pH 7.5 buffer. The elution fraction was separated into 1 mL portions, and the fraction in which a band of approximately 75 kDa was observed by SDS-PAGE was collected. The recovered protein was used as purified recombinant hSEMA7A extracellular domain protein (hereinafter referred to as hSEMA7A-ECD).

2. Culture of the library The human library was cultured using DT40 cells, a cell line derived from chicken B cells, as a host according to the following method. The cells were cultured at 39.5° C. in the presence of 5% CO ₂ , and the medium used was IMDM (Life Technologies) with the addition of 10% FBS (Biosera) and 1% Penicillin-Streptomycin. Hereinafter, unless otherwise specified, CS(-) medium refers to the medium described above. CS/FK506-containing medium refers to CS(-) medium to which 5% avian serum (Life Technologies) and 1 μM FK506 (Cayman) are added.

3. The monoclonal antibody selection library by the ADLib system was a mixture of the human library described in WO 2015/167011 and the Kappa human library (Human bioKI Lib mix). Biotinylated hSEMA7A-ECD antigen was added to the library suspended in CS/FK506-containing medium to a final concentration of 30 nM, and the mixture was allowed to react at 4°C for 30 minutes while being mixed by inversion. After the reaction was completed, the cells were washed twice with CS/FK506-containing medium and resuspended in CS/FK506-containing medium. 1/50 amount of Anti-Biotin MicroBeads UltraPure (Miltenyi Biotec) and 1/2000 amount of Goat Anti-Human IgG-PE (SouthernBiotech) were added, and the mixture was allowed to react while being mixed by inversion at 4°C for 30 minutes. After the reaction was completed, 1/1000 amount of Streptavidin Alexa Fluor 647 Conjugate (Life Technologies) was added, and the mixture was allowed to react at 4°C for 10 minutes while being mixed by inversion. After the reaction was completed, the column was concentrated using an autoMACS Pro Separator (Miltenyi Biotec), and the fraction that was not adsorbed to the column (negative fraction) was measured using a FACS Aria Fusion (Becton Dickinson) to set the sorting range. Thereafter, the entire positive fraction sample was loaded, and the positive cell population was sorted at 1 cell/well in a 96-well plate into which each medium was dispensed.

4. Screening by antigen solid-phase ELISA D-PBS(-) (Nacalai Tesque) diluted hSEMA7A-ECD antigen solution to 2.5 μg/mL and negative control antigen (hHER2-His) solution 20 μL were divided into MAXISORP 384 IMMUNO PLATE (NUNC). The mixture was poured into a solid phase by reacting at 4°C overnight. The solution was removed by centrifugation using a small centrifuge dedicated to microplates (GYRO mini GM-01, MICRONIX), and 45 μL of blocking solution (PBS containing 1% BSA (SIGMA)) was added and reacted at room temperature for 1 hour. The solution was removed by centrifugation using GYRO mini, 25 μL of culture supernatant was added, and the mixture was allowed to react at room temperature for 1 hour. The solution was removed by centrifugation with GYRO mini, 25 μL of Goat anti-Human IgG-Fc HRP-conjugated (BETHYL) diluted 2000 times with blocking solution was added, and the mixture was allowed to react at room temperature for 30 minutes. It was washed five times with a washing solution (PBS containing 0.05% Tween20 (Wako)), and 25 μL of TMB (Nacalai Tesque) was added and reacted for 10 minutes. The reaction was stopped by adding 25 μL of 1 N sulfuric acid (Wako), and the absorbance at 450 nm was measured using a microplate reader (TECAN).

5. Antibody Sequence Analysis Regarding the DT40 cells that produce antibodies that specifically react with hSEMA7A-ECD, as described in Section 4 above, the sequence of the immunoglobulin gene variable region was analyzed as follows.
DT40 cells were expanded and cultured, and genomic DNA was extracted using the Wizard SV Genomic DNA Purification System (Promega). Antibody genes were amplified from the extracted DNA by PCR. Both the heavy chain and light chain were amplified using primers that recognize the upstream of the variable region and the downstream of the constant region. Each primer used and its sequence are as follows.

For heavy chain 5' amplification: SEQ3777F (AAGCAATTAAGTCGAGGCTGACGA (SEQ ID NO: 47))
For heavy chain 3' amplification: CH2a_620R (CCGCAAATGATGGACCGACC (SEQ ID NO: 48))
For κ/λ light chain 5' amplification: Primer05 (CCCACACCTCAGGTACTCGT (SEQ ID NO: 49))
For κ/λ light chain 3' amplification: VL7 SEQ R7 (CGCCAAGTCCAAGAAAACCCCCA (SEQ ID NO: 50))

The obtained DNA fragment was amplified using CH2a 620R and VL7 SEQ R7, and DNA sequence analysis was performed.
For the resulting antibody sequences, CDR regions were determined according to the method of Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth edition, NIH Publication No. 91-3242, US Department of Health and Human Services, 1991). The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the analyzed cloned antibody (antibody name: pre-AM) and the amino acid sequences of their respective CDR regions are shown in Table 1 below. Indicate by number.

6. Improving affinity by affinity maturation (AM) DT40 cells that produce antibodies that specifically react with hSEMA7A-ECD were cultured at 39.5°C in the presence of 5% _CO2 , and the medium was IMDM (Life Technologies) with 10% FBS (Biosera), 1% Penicillin-Streptomycin, and 10 μM A66 (SIGMA) were added and used. After culturing for 2 to 3 weeks, biotinylated hHER2-His antigen was added to the DT40 cells suspended in CS/FK506-containing medium to a final concentration of 10 nM, and the cells were reacted with inversion for 30 minutes at 4°C. . After the reaction was completed, the cells were washed twice with CS/FK506-containing medium and resuspended in CS/FK506-containing medium. Next, 1/500 amount of Streptavidin Alexa Fluor 488 Conjugate (Life Technologies) was added, and the mixture was allowed to react at 4°C for 30 minutes while being mixed by inversion. After the reaction was completed, the cells were washed twice with CS/FK506-containing medium and resuspended in CS/FK506-containing medium. Next, add hSEMA7A-EDC Alexa Fluor 647 Conjugate and 1/2000 amount of Goat Anti-Human IgG-PE (SouthernBiotech) to a final concentration of 1 to 10 nM, and react with inversion for 30 minutes at 4°C. Ta. After the reaction was completed, the cells were washed twice with CS/FK506-containing medium and resuspended in CS/FK506-containing medium. The suspension was measured using FACS Aria Fusion (Becton Dickinson), and the sorting range was set to include the top approximately 0.1% of the population with improved affinity. Thereafter, the positive cell population was sorted to 1 cell/well in a 96-well plate into which the CS/FK506-containing medium was dispensed.

DT40 cells, which were sorted in Section 6 (Improvement of affinity by Affinity maturation (AM)) and were considered to have improved affinity, were screened by flow cytometry.
After the sorted single clones have grown sufficiently, biotinylated hHER2-His antigen was added to the DT40 cells suspended in CS(-) medium to a final concentration of 10 nM, and the mixture was incubated at 4°C for 30 minutes by inversion. Made it react. After the reaction was completed, the cells were washed twice with CS(-) medium and resuspended in CS(-) medium. Next, 1/500 amount of Streptavidin Alexa Fluor 488 Conjugate (Life Technologies) was added, and the mixture was allowed to react at 4°C for 30 minutes while being mixed by inversion. After the reaction was completed, the cells were washed twice with CS(-) medium and resuspended in CS(-) medium. Next, add hSEMA7A-EDC Alexa Fluor 647 Conjugate and 1/2000 amount of Goat Anti-Human IgG-PE (SouthernBiotech) to a final concentration of 1 to 10 nM, and react with inversion for 30 minutes at 4°C. Ta. After the reaction was completed, the cells were washed twice with CS(-) medium and resuspended in CS(-) medium. The suspension was measured using FACS CantoII (Becton Dickinson) to screen for clones with improved staining intensity with hSEMA7A-EDC than the original clone.

After screening, clones with improved affinity were subjected to antibody sequence analysis as described in the previous section, and the site of mutation in the antibody sequence was identified (Figure 1A, Figure 1B, Figure 1C). The amino acid sequences of VH and VL of the obtained cloned antibodies (antibody names: HC1 to HC8) and the amino acid sequences of their respective CDR regions are shown in Table 1 above by each sequence number.

7. Antibody Affinity Measurement Culture supernatants were prepared for representative clones of each sequence group, and affinity with the antigen was measured using the SPR method (Biacore T200, GE Healthcare). After anti-human IgG Fc antibodies were immobilized on a CM5 chip (GE Healthcare) using a Human Antibody Capture Kit (GE Healthcare), the antibodies in the culture supernatant were captured. 4-100 nM hSEMA7A-EDC was reacted for 240 seconds and then dissociated for 300 seconds. One cycle was completed by reacting 3 M _MgCl2 as a regeneration solution for 30 seconds. The buffer used was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20 (pH 7.4) (GE Healthcare)), and the flow rate was measured at 30 μL/min.

Fitting was performed using the Langmuir 1:1 binding model for each SPR sensorgram, the binding rate constant kon and dissociation rate constant koff were calculated, and the KD value was determined using the formula KD=koff/kon. The KD values of anti-SEMA7A antibodies (HC1 to HC8) are shown in Figure 5.

8. ADCC activity in primary cultured human HSCs First, we compared the expression level of SEMA7A on the cell surface of normal, resting HSCs and active HSCs, which are thought to be involved in pathological conditions. Activated HSCs were obtained by treating quiescent HSCs with 1 ng/mL hTGF-β1 (R&D) overnight. Next, the isotype control antibody and anti-SEMA7A antibody (HC2) were reacted at 4°C for 30 minutes at a final concentration of 1 μg/mL, and after the reaction was completed, the cells were washed twice with CS(-) medium. Subsequently, 1/2000 amount of Goat Anti-Human IgG-PE (SouthernBiotech) was added, and the mixture was allowed to react at 4°C for 30 minutes while being mixed by inversion. After the reaction was completed, the cells were washed twice with CS(-) medium and resuspended in CS(-) medium. The suspension was measured using FACS CantoII (Becton Dickinson) and the SEMA7A expression levels were compared. As a result, while SEMA7A was hardly expressed in resting HSCs, strong induction of SEMA7A expression was confirmed in active HSCs (Figure 2).

The ADCC assay was performed according to the protocol of the ADCC Reporter Bioassay, Complete Kit (Promega). First, as described above, quiescent HSCs and activated HSCs were seeded at 6 × 10 ³ cells/well in CellBIND 384-well plate (CORNING), and a dilution series of anti-SEMA7A antibody (HC1 to HC8) was diluted to a final concentration of 4 to 1000 ng. /mL. Next, ADCC Bioassay Effector Cell was added at 1.2×10 ⁴ cells/well and reacted at 37° C. for 6 hours. After the reaction was completed, Bio-Glo ^™ Luciferase Assay Buffer was added, and the reaction was allowed to proceed at room temperature for 15 minutes while shielding from light. Luminescence of the reaction solution was measured using an EnspireAlpha plate reader (ParkinElmer) to evaluate the ADCC activity of the anti-SEMA7A antibody. As a result, no ADCC activity was observed in resting HSCs, and significant ADCC activity was observed in all anti-SEMA7A antibodies (HC1 to HC8) against active HSCs (Figure 3). In addition, the strength of the ADCC activity of anti-SEMA7A antibodies against activated HSCs differs depending on the antibody variant, and in descending order of activity, HC6 > HC2 > HC1 ≒ HC3 ≒ HC4 ≒ HC5 > HC7 ≒ HC8. Ta.

9. Cytotoxic activity of anti-SEMA7A antibodies using second immunotoxin Strong internalization activity was observed with anti-SEMA7A antibodies (HC1 to HC8) (Figure 7). Next, we evaluated the potential of anti-SEMA7A antibody as an antibody-drug conjugate (ADC). First, equal volumes of 480 nM IgGs Anti-Mouse IgG Fc-PBD Antibody with Cleavable Linker (Moradec), 160 nM anti-SEMA7A antibody, and isotype control antibody were mixed and incubated at room temperature for 1 hour (PBD is pyrrolobenzodiazepine). ). A dilution series was prepared by repeating 2-fold dilution of the ADC antibody nine times. Next, MDA-MB-231 cells (ATCC) were seeded at 500 cells/well in a CellBIND 384-well plate (CORNING), and an ADC antibody dilution series was added. Thereafter, the cells were cultured at 37° C. in the presence of 0% CO ₂ , and after 7 days, CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added and the number of living cells was measured using an EnspireAlpha plate reader (ParkinElmer). As a result, anti-SEMA7A antibody-specific cytotoxic activity was observed (Figure 4).

10. The clone names and sources of commercially available anti-SEMA7A mouse monoclonal antibodies used for comparison of binding affinity between the novel anti-SEMA7A antibody of the present invention and commercially available antibodies are shown in Table 2 below.

For each commercially available anti-SEMA7A mouse monoclonal antibody, affinity with the antigen was measured using the SPR method (Biacore T200, GE Healthcare). After immobilizing anti-mouse IgG Fc antibodies on a CM5 chip (GE Healthcare) using a Mouse Antibody Capture Kit (GE Healthcare), the antibodies in the culture supernatant were captured. 4-100 nM hSEMA7A-EDC was reacted for 240 seconds and then dissociated for 300 seconds. One cycle was completed by reacting 10 mM Gly-HCl (pH 1.7) as a regeneration solution for 30 seconds. The buffer used was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20 (pH 7.4) (GE Healthcare)), and the flow rate was measured at 30 μL/min.

Each SPR sensorgram was fitted using the Langmuir 1:1 binding model, the binding rate constant kon and the dissociation rate constant koff were calculated, and the KD value was determined using the formula KD=koff/kon. As shown in Figure 5, the affinities other than 3A5G1 were so weak that they could not be detected. Furthermore, the affinity of anti-SEMA7A antibodies (HC1 to HC8) was approximately 100 times stronger than that of 3A5G1 that could be measured.

11. Comparison of Plexin C1-SEMA7A binding inhibitory activity in anti-SEMA7A antibodies Dispense 25 μL of hPlexin C1 solution (R&D) diluted to 1.25 μg/mL with D-PBS(-) (Nacalai Tesque) to MAXISORP 384 IMMUNO PLATE (NUNC). , and solidified by reacting overnight at 4°C. The solution was removed by centrifugation using a small centrifuge dedicated to microplates (GYRO mini GM-01, MICRONIX), and 45 μL of blocking solution (PBS containing 1% BSA (SIGMA)) was added and reacted at room temperature for 1 hour. The solution was removed by centrifugation using GYRO mini, and 25 μL of dilution series of anti-SEMA7A antibody and isotype control antibody and 25 μL of 10 nM biotinylated hSEMA7A-Fc (R&D) were simultaneously added and reacted for 1 hour at room temperature. The solution was removed by centrifugation using GYRO mini, 25 μL of High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific) diluted 5000 times with blocking solution was added, and the mixture was allowed to react at room temperature for 30 minutes. It was washed five times with a washing solution (PBS containing 0.05% Tween20 (Wako)), and 25 μL of TMB (Nacalai Tesque) was added and reacted for 10 minutes. The reaction was stopped by adding 25 μL of 1 N sulfuric acid (Wako), and the absorbance at 450 nm was measured using a microplate reader (TECAN). The results are shown in Figure 6. Among commercially available anti-SEMA7A antibodies, 3A5G1 was the only one that showed Plexin C1-SEMA7A binding inhibitory activity, but the activity was so weak that IC ₅₀ could not be calculated. On the other hand, the novel anti-SEMA7A antibodies of the present invention (particularly HC1, HC2, HC4, HC6, and HC8) have an activity that can virtually 100% inhibit Plexin C1-SEMA7A binding, which is overwhelmingly superior to commercially available antibodies. It had a strong inhibitory activity.

12. Construction of Ba/F3 cells stably expressing full-length hSEMA7A A sequence was designed in which a c-Myc tag was inserted at the C-terminal side of position 640 for the amino acid sequence from positions 1 to 666 of UniProt number O75326 (SEQ ID NO: 46). did. This amino acid sequence was converted into a nucleotide sequence based on the mammalian codon table, and a DNA sequence containing this sequence with a Kozak sequence at the 5' end and a translation stop codon at the 3' end was synthesized by gene synthesis (GENEWIZ). . The synthesized DNA was inserted between the KpnI/XhoI recognition sequences of pCDNA3.1+ (Thermo Fisher Scientific) to create an expression vector.

The plasmid was linearized with the restriction enzyme PvuI, and 2 μg was introduced into 2×10 ⁶ mouse B cell-derived cell line Ba/F3 cells using Amaxa Cell Line Nucleofector Kit V (Lonza). After gene introduction, cells were seeded in a 96-well plate, and G418 (Nacalai Tesque) was added at a final concentration of 1 μg/mL. After 6 days, colonies with G418-resistant growth were collected from the wells, and the expression of hSEMA7A was confirmed by flow cytometry using anti-c-Myc antibody (Santa Cruz Biotechnology). Limit dilution was performed. Proliferated cells were collected, hSEMA7A expression was confirmed by flow cytometry using an anti-hSEMA7A antibody (Abnova), and a monoclonal cell line was finally selected. These cells were used as the Ba/F3-hSEMA7A cell line in the following experiments.

13. Comparison of internalization activity in anti-SEMA7A antibodies First, equal amounts of 480 nM AcidiFluor ORANGE-labeled anti-human/mouse IgG Fc Fab antibody, 160 nM anti-SEMA7A antibody, and isotype control antibody were mixed and incubated at room temperature for 10 minutes. The antibody complex thus prepared and Ba/F3-hSEMA7A cells were reacted at 37°C for 1 hour. After the reaction was completed, the suspension was measured using FACS CantoII (Becton Dickinson) to compare the internalization activities of each antibody. As a result, all of the novel anti-SEMA7A antibodies (HC1 to HC8) of the present invention showed strong internalization activity, while commercially available anti-SEMA7A antibodies showed no internalization activity at all (FIG. 7).

14. Comparison of antigen specificity in anti-SEMA7A antibodies From the viewpoint of side effects, it is very important to ensure the antigen specificity of antibodies. Therefore, the antigen specificities of the novel anti-SEMA7A antibody of the present invention and a commercially available anti-SEMA7A antibody were compared using hSEMA4F, hSEMA5A, and hSEMA6C, which are highly homologous to the sema domain of hSEMA7A.

First, hSEMA7A-ECD, hSEMA4F-GST (Abnova), hSEMA5A-His (SINO BIO), hSEMA6C-GST (Abnova) and negative control antigen (hHER2 -His, GST) solution was dispensed onto MAXISORP 384 IMMUNO PLATE (NUNC) and reacted overnight at 4°C to solidify. The solution was removed by centrifugation using a small centrifuge dedicated to microplates (GYRO mini GM-01, MICRONIX), and 45 μL of blocking solution (PBS containing 1% BSA (SIGMA)) was added and reacted at room temperature for 1 hour. The solution was removed by centrifugation using GYRO mini, and 25 μL of 10 μg/mL anti-SEMA7A antibody was added and reacted for 1 hour at room temperature. The solution was removed by centrifugation using GYRO mini, and 25 μL of Goat anti-Human/Mouse IgG-Fc HRP-conjugated (BETHYL) diluted 2000 times with blocking solution was added, and the mixture was allowed to react at room temperature for 30 minutes. The cells were washed 5 times with a washing solution (PBS containing 0.05% Tween20 (Wako)), and 25 μL of TMB (Nacalai Tesque) was added and reacted for 10 minutes. The reaction was stopped by adding 25 μL of 1 N sulfuric acid (Wako), and the absorbance at 450 nm was measured using a microplate reader (TECAN). As shown in FIG. 8, all of the novel anti-SEMA7A antibodies (HC1 to HC8) of the present invention were confirmed to be hSEMA7A-specific. On the other hand, among commercially available anti-SEMA7A antibodies, C-6, D-4, 1G1, and 1D5G8, which have weak affinity and do not have Plexin C1-SEMA7A binding inhibitory activity, were hSEMA7A-specific, but 3A5G1 was hSEMA4F, hSEMA5A-specific. It was confirmed that 3D3 also intersected with hSEMA6C, and hSEMA4F and hSEMA6C. Therefore, it was revealed that the novel anti-SEMA7A antibodies (HC1 to HC8) of the present invention have superior antigen specificity compared to commercially available anti-SEMA7A antibodies.

15. Measurement of isoelectric point (pI) of anti-SEMA7A antibody pI is an important factor that affects the pharmacokinetics (PK) of antibody drugs. Generally, if the pI is high (9.5 or higher), the PK is expected to be poor, so modification by introducing mutations is necessary. In order to predict the PK of anti-SEMA7A antibodies, the pI of each antibody was calculated using a gradient analysis method using a pH 5.6 buffer and a pH 10.2 buffer. As a result, the pI of HC1 to HC5 was below 9.5, and the PK was expected to be in a good range (Figure 9).

16. Evaluation of thermal stability of anti-SEMA7A antibodies SYPRO Orange fluorescent dye was mixed as appropriate with HC1 to HC8 diluted to 0.5 mg/mL. As a comparison target for anti-SEMA7A antibodies, the same procedure was performed for eight antibody drugs already on the market (Herceptin, Humira, Lucentis, Poteligio, Avastin, Atezolizumab, Nivolumab, Synagis). The Tm value, which is the midpoint of the thermal denaturation curve for the Fab region, was determined using an RT-PCR machine programmed to increase the temperature from 30°C to 99°C at a rate of 3°C/min, and continuously Calculated by reading. The results shown in FIG. 10 show that the anti-SEMA7A antibody has a Tm value comparable to that of commercially available antibodies, and has high overall thermal stability.

17. Evaluation of antibody production amount of anti-SEMA7A antibody In order to estimate the antibody production amount of anti-SEMA7A antibody, small-scale antibody expression and purification was performed using Expi293 Expression System. The transfected Expi293 culture medium was subjected to affinity purification using a Protein A column, and the antibody concentration was measured by the BCA method. As a result, it was found that HC1 and HC2 produced particularly high amounts of antibodies (Figure 11).

18. Time course of internalization activity of anti-SEMA7A antibody In Figure 7, anti-SEMA7A antibody and cells were reacted at 37°C for 1 hour, and only the presence or absence of internalization activity was confirmed. Therefore, this time we evaluated the rate at which SEMA7A on the cell surface disappears over time due to internalization. When evaluated according to the evaluation method and internalization activity calculation method shown in Figure 12A, the internalization activity of anti-SEMA7A antibody (HC2) reached a plateau in 1 hour, and approximately 90% of SEMA7A on the cell surface It was found that % was internalized (Figure 12B). Therefore, HC2 was found to be a class of antibody with high internalization activity.

19. Analysis of SEMA7A expression in tissues of each cancer type SEMA7A expression intensity and To investigate the frequency, tissue microarrays were purchased from US Biomax and TriStar Technology Group and expression analysis was performed. First, the slides were deparaffinized and reacted with mouse monoclonal anti-SEMA7A antibody (clone: C-6). Thereafter, it was reacted with an HRP-labeled secondary antibody, and finally colored with a DAB solution. The staining results were classified from score 0 to score 3+ according to the index in FIG. 13A, and the frequency was calculated. As a result, TNBC, osteosarcoma, and chondrosarcoma tended to have a score of 2+. Regarding the difference in the presence or absence of EGFR mutations in NSCLC, there was a clear tendency for SEMA7A expression intensity to be stronger in patients with EGFR mutations (Figure 13B).

20. Analysis of SEMA7A expression in each cancer cell line For TNBC, osteosarcoma, chondrosarcoma, and NSCLC for which SEMA7A expression was confirmed by immunohistochemistry, we obtained each cancer cell line from ATCC and analyzed SEMA7A expression using FACS. I did it. Specifically, the TNBC cell line is "MDA-MB-231," the osteosarcoma cell line is "U-2 OS" and "143B," the chondrosarcoma cell line is "SW1353," and the NSCLC cell line. "SK-MES-1" (EGFR WT) and "H1975" (EGFR Mut) were used.
The analysis method involved reacting each cancer cell line with anti-SEMA7A antibody (HC2) at 4°C for 30 minutes, followed by detection with PE-labeled anti-hIgG Fc antibody. As a result, SW1353, a chondrosarcoma cell, was most strongly expressed, and the remaining cell lines had moderate expression levels (Figure 14).

21. Cytotoxic activity of HC2 second immunotoxin in each cancer cell line Using cancer cell lines in which SEMA7A expression was confirmed, the second immunotoxin conjugated with PBD was used to verify the potential of anti-SEMA7A antibody (HC2) as an ADC. Cytotoxic activity was investigated using immunotoxins. Equal volumes of 80 nM anti-SEMA7A antibody and 240 nM PBD-conjugated secondary antibody (IgGs Anti-Mouse IgG Fc-PBD Antibody with Cleavable Linker (Moradec)) were mixed and incubated at room temperature for 1 hour. A 3-fold dilution series was prepared with the mixture and administered to each cancer cell line. One week after administration, CellTiter-Glo was added and left at room temperature for 10 minutes, and luminescence was measured using an EnspireAlpha plate reader (ParkinElmer). As shown in Figure 15, HC2 showed significant cytotoxic activity against the four types of cancer cell lines tested this time. From the above, it was demonstrated that HC2 is an antibody suitable for ADC as well.

22. Cytotoxic activity of anti-SEMA7A antibody directly labeled with PBD in each cancer cell line Next, a cytotoxicity assay was performed using an ADC-conjugated anti-SEMA7A antibody directly labeled with PBD. The drug to antibody ratio (DAR) of the antibodies used in the assay was 3.2 for the isotype control antibody, 3.0 for HC1, and 3.1 for HC2. A 3-fold dilution series (0 to 40 nM) of each ADC was administered to a plate seeded with each cancer cell line at 500 cells/well. One week after administration, CellTiter-Glo was added and left at room temperature for 10 minutes, and luminescence was measured using an EnspireAlpha plate reader (ParkinElmer). As a result, significant cytotoxic activity was confirmed in all five types of cancer cell lines (Figure 16).

SEQ ID NO: 1-44: Recombinant peptide SEQ ID NO: 47-50: Synthetic DNA

Claims

An antibody against human SEMA7A,
The amino acid sequences of complementarity determining region (CDR) 1, CDR2, and CDR3 of the heavy chain variable region (VH) are, in order,
Amino acid sequences shown in SEQ ID NOs: 10, 11 and 12,
Amino acid sequences shown in SEQ ID NOs: 18, 19 and 20,
Amino acid sequences shown in SEQ ID NOs: 22, 23 and 24,
Amino acid sequences shown in SEQ ID NO: 26, 27 and 28,
Amino acid sequences shown in SEQ ID NO: 30, 31 and 32,
Amino acid sequences shown in SEQ ID NOs: 34, 35 and 36,
consisting of the amino acid sequences shown in SEQ ID NOs: 38, 39 and 40, or the amino acid sequences shown in SEQ ID NOs: 42, 43 and 44, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region (VL), respectively, in that order:
Consisting of the amino acid sequences shown in SEQ ID NO: 14, 15 and 16,
The antibody.
An antibody against human SEMA7A,
The amino acid sequence of the heavy chain variable region (VH) consists of the amino acid sequence shown in SEQ ID NO: 9, 17, 21, 25, 29, 33, 37 or 41, and
The amino acid sequence of the light chain variable region (VL) consists of the amino acid sequence shown in SEQ ID NO: 13,
The antibody.
The antibody according to claim 1 or 2, which is an antibody that binds to the sema domain of human SEMA7A.
The antibody according to any one of claims 1 to 3, wherein the antibody is a human antibody.
The antibody according to any one of claims 1 to 4, which has the activity of inhibiting or suppressing the interaction between human SEMA7A and Plexin C1 and/or integrin β1.
The antibody according to any one of claims 1 to 5, wherein the antibody has antitumor activity.
The antibody according to any one of claims 1 to 6, which is used for the treatment or prevention of tumors.
The antibody according to claim 6 or 7, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.
The antibody according to any one of claims 1 to 5, which is used for the treatment or prevention of inflammatory diseases and/or autoimmune diseases.
The inflammatory disease is at least one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). is a seed,
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
The antibody according to claim 9.
An antibody fragment derived from the antibody according to any one of claims 1 to 10.
An antibody or antibody fragment-drug conjugate, which is a drug conjugated to the antibody according to any one of claims 1 to 10 or the antibody fragment according to claim 11.
The complex according to claim 12, wherein the drug is a compound having antitumor activity and/or cell killing activity.
A pharmaceutical composition comprising the antibody according to any one of claims 1 to 10, the antibody fragment according to claim 11, and/or the conjugate according to claim 12 or 13.
The pharmaceutical composition according to claim 14, which is used for the treatment or prevention of tumors.
The pharmaceutical composition according to claim 15, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.
The pharmaceutical composition according to claim 14, which is used for the treatment or prevention of inflammatory diseases and/or autoimmune diseases.
The inflammatory disease is at least one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). is a seed,
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
The pharmaceutical composition according to claim 17.
The pharmaceutical composition according to claim 14, which is used for inhibiting or suppressing the interaction between human SEMA7A and Plexin C1 and/or integrin β1.
A method for treating or preventing a tumor, which comprises administering to a subject the pharmaceutical composition according to any one of claims 14 to 16.
The method according to claim 20, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.
A method for treating or preventing inflammatory diseases and/or autoimmune diseases, which comprises administering to a subject the pharmaceutical composition according to any one of claims 14, 17, and 18.
The inflammatory disease is at least one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). is a seed,
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
23. The method according to claim 22.
A method for inhibiting or suppressing the interaction between human SEMA7A and Plexin C1 and/or integrin β1, which comprises administering to a subject the pharmaceutical composition according to claim 14 or 19.
A kit for treating, preventing or diagnosing a tumor, comprising the antibody according to any one of claims 1 to 10, the antibody fragment according to claim 11, and/or the conjugate according to claim 12 or 13.
The kit according to claim 25, wherein the tumor is at least one selected from the group consisting of human breast cancer, human lung cancer, human oral cavity cancer, human osteosarcoma, and human chondrosarcoma.
A method for treating inflammatory diseases and/or autoimmune diseases, comprising the antibody according to any one of claims 1 to 10, the antibody fragment according to claim 11, and/or the conjugate according to claim 12 or 13. Kits for treatment, prevention or diagnosis.
The inflammatory disease is at least one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), Crohn's disease, allergic dermatitis, and interstitial pneumonia, and organ fibrosis including nonalcoholic steatohepatitis (NASH). is a seed,
The autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, multiple sclerosis (MS), systemic sclerosis, and Sjögren's syndrome,
A kit according to claim 27.