Anti-CD37 antibody-drug conjugate

Abstract

The present invention provides: an antibody that specifically binds to CD37-positive tumor cells such as B-cell malignant lymphoma; an antibody-drug conjugate that contains said antibody; a pharmaceutical composition that employs said antibody and that exhibits a therapeutic effect on a tumor; a tumor treatment method employing said pharmaceutical composition; a method for producing said antibody; a method for producing said antibody-drug conjugate; and so forth.　Provided is an anti-CD37 antibody-drug conjugate in which a drug linker, which is represented by the formula below, and an antibody are bonded by means of a thioether bond (in the formula, A indicates the position at which the linker is bonded with the antibody). Specifically, provided are: a humanized anti-CD37 antibody that exhibits an internalization activity; and an antibody-drug conjugate that contains said antibody.

Description

Technical Field

[0001] The present invention relates to an anti-CD37 antibody that binds to CD37, a method for producing the antibody, an antibody-drug conjugate containing the antibody, an antitumor agent containing the antibody-drug conjugate, and the like.

Background Art

[0002] Cancer ranks among the leading causes of death, and its incidence is expected to increase with the aging of the population. However, the treatment needs have not yet been fully met. Conventional chemotherapeutic agents have problems such as side effects due to their low selectivity and damage to not only tumor cells but also normal cells, and the inability to obtain sufficient drug effects due to the inability to administer a sufficient drug dose. Therefore, in recent years, more selective molecular target drugs and antibody drugs targeting molecules showing characteristic mutations or high expression in cancer cells, or specific molecules involved in canceration of cells, have been developed.

[0003] Rituximab is an antibody drug targeting CD20 and was approved by the FDA in 1997 as a therapeutic agent for B-cell non-Hodgkin lymphoma (NHL) (Non-Patent Document 1). The main mechanisms of action of Rituximab are direct apoptosis induction, ADCC (Antibody-dependent cellular cytotoxicity), and CDC (Complement-dependent cytotoxicity). It shows dramatic therapeutic effects against diffuse large B-cell lymphoma (DLBCL) expressing the target CD20 and other B-cell NHLs, and is still widely used. However, in addition to the existence of a certain number of Rituximab non-responsive patients, even Rituximab-responsive patients acquire mechanisms of resistance to the reduction of the expression of the target CD20 and each mechanism of action, and ultimately relapse in many patients, which is a problem in the treatment of B-cell NHL in clinical practice (Non-Patent Document 2). Against this background, antibody drugs targeting other than CD20 or antibody drugs having a mechanism of action different from Rituximab are currently being developed clinically.

[0004] Antibodies are expected to reduce side effects due to their high stability in the bloodstream and their specific binding to target antigens, and numerous antibody drugs targeting molecules highly expressed on the surface of cancer cells have been developed. One technology that utilizes the antigen-specific binding ability of antibodies is antibody-drug conjugate (ADC). An ADC is an antibody that can bind to an antigen expressed on the surface of cancer cells and internalize the antigen into the cell through this binding, and a drug with cytotoxic activity is attached to it. By efficiently delivering drugs to cancer cells, ADCs are expected to accumulate drugs within cancer cells and kill them (Non-Patent Document 3, Patent Documents 1 and 2). As examples of ADCs, Mylotarg® (gemtuzumab ozogamicin), which targets CD33 and conjugates a calicheamicin derivative to a monoclonal antibody, is approved as a treatment for acute myeloid leukemia, while Besponsa® (inotuzumab ozogamicin), which targets CD22, is approved as a treatment for relapsed or refractory precursor B-cell acute lymphoblastic leukemia. In addition, Adcetris® (brentuximab vedotin), which targets CD30 by conjugating monomethyl auristatin E to a monoclonal antibody, has been approved as a treatment for Hodgkin lymphoma and anaplastic large cell lymphoma; Polivy® (polatuzumab vedotin), which targets CD79b, has been approved as a treatment for diffuse large B-cell lymphoma; PADCEV® (enfortumab vedotin), which targets nectin-4, has been approved as a treatment for locally advanced and metastatic urothelial carcinoma; and BLENREP® (verantamab mafodotin), which conjugates monomethyl auristatin F to an anti-B cell maturation antigen monoclonal antibody, has been approved as a treatment for relapsed and refractory multiple myeloma. Furthermore, Kadcyla® (trastuzumab emtansine), which is an anti-HER2 monoclonal antibody conjugated with emtansine, is used to treat HER2-positive advanced and recurrent breast cancer, and Trodelvy® (sacituzumab govitecan), which is conjugated with SN-38, the active metabolite of the anti-TROP2 monoclonal antibody irinotecan, is used to treat advanced triple-negative breast cancer.

[0005] Characteristics of target antigens suitable for ADCs (antitumor drugs) include specific high expression on the surface of cancer cells, low or no expression in normal cells, the ability to be internalized within cells, and the absence of secretion from the cell surface. Furthermore, important characteristics of antibodies suitable for ADCs include specific binding to the target antigen and high internalization ability. Antibody internalization ability depends on the properties of both the target antigen and the antibody; it is difficult to predict suitable antigen-binding sites from the molecular structure of the target, or to easily infer antibodies with high internalization ability from the binding strength and physical properties of the antibody. Therefore, obtaining antibodies with high internalization ability against target antigens is a crucial challenge in developing highly effective ADCs (Non-Patent Literature 4).

[0006] CD37 is a four-transmembrane protein belonging to the tetraspanin superfamily (Non-Patent Literature 5). Previous studies have reported that CD37 regulates cell survival through activation of the PI3K / Akt pathway, and that CD37-deficient mice exhibit decreased IgG1 production, but its exact physiological function remains unclear (Non-Patent Literature 6, 7). CD37 is widely expressed during the differentiation stages from precursor B cells to mature B cells, but not in plasma cells. It is also expressed in T cells, NK cells, and monocytes, but at low levels, and is not expressed in hematological cells such as erythrocytes and platelets. In tumor cells, it is highly expressed in B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and this expression profile suggests that it may be a promising therapeutic target in B-cell malignant lymphomas, leading to the development of several antibody drugs targeting CD37 in clinical trials (Non-Patent Literature 8). Of these, CD37 is considered promising as an ADC target due to its high internalization activity, and IMGN529, an anti-CD37 antibody conjugated with DM1, is currently undergoing clinical trials (Non-Patent Document 9, Patent Document 3). However, in a Phase I trial targeting patients with relapsed / refractory B-cell non-Hodgkin lymphoma (NHL), IMGN529 showed efficacy only in specific patients, with an overall response rate of 12.8% (Non-Patent Document 10). Currently, several CD37-targeted drugs are undergoing clinical trials, including Betalutin (Lutetium-177-labeled anti-CD37 antibody), GEN3009 (anti-CD37 biparatopic antibody), and CAR37 T cells (CD37-targeted CAR T cells) (Non-Patent Documents 11, 12, 13).

[0007] Enhertu® (trastuzumab deruxtecan), a camptothecin derivative conjugated to an anti-HER2 monoclonal antibody, is used to treat HER2-positive advanced and recurrent breast cancer (Non-Patent Literature 14). In addition, deruxtecan-conjugated ADCs such as HER3-DXd (Non-Patent Literature 15) and Trop2-DXd (Non-Patent Literature 16) are currently undergoing clinical trials. However, there have been no reports to date of ADCs conjugated to an anti-CD37 antibody with deruxtecan, nor of deruxtecan-conjugated ADCs targeting B-cell non-Hodgkin lymphoma. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2014 / 057687 [Patent Document 2] U.S. Patent Application Publication No. 2016 / 0297890 [Patent Document 3] International Publication No. 2011 / 112978 [Non-Patent Document 1] Gilles S.,et al.,Adv Ther, 2232-2273,34,2017 [Non-Patent Document 2] Andrew R.,et al.,Best Pract Res Clin Haematol, 203-216,24,2011 [Non-Patent Document 3] Polakis P.,Pharmacological Reviews,3-19,68,2016 [Non-Patent Document 4] Peters C.,et al.,Bioscience Reports,1-20,35,2015 [Non-Patent Document 5] Charrin S., et al., J Cell Sci. 3641-3648,127,2014 [Non-Patent Document 6] Magdalena.,et al.,Expert Opin Investig Drug.171-177,27,2018

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Summary of the Invention

Problems to be Solved by the Invention

[0009] The object of the present invention is to provide an antibody that specifically binds to CD37-positive tumor cells such as B-cell malignant lymphoma, an antibody-drug conjugate containing the antibody, a pharmaceutical composition having a therapeutic effect on tumors using the antibody-drug conjugate, a method for treating tumors using the pharmaceutical composition, a method for producing the antibody, and a method for producing the antibody-drug conjugate, etc. [Means for solving the problem]

[0010] The inventors of this invention diligently studied to achieve the above objectives and discovered that an anti-CD37 antibody-drug conjugate, in which an intracellularly toxic drug is conjugated to an anti-CD37 antibody via a linker of a specific structure, exhibits an antitumor effect against CD37-positive malignant tumors such as B-cell lymphoma, thereby completing the present invention. That is, the present invention encompasses the following inventions. [1] Anti-CD37 antibody is expressed by the following formulas (a)~(f): (a)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (b)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (d)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), An antibody-drug conjugate bound to a drug-linker structure represented by any one formula selected from the group consisting of the following.

[0011] (Here, -(Succinimid-3-yl-N)- is the following equation:

[0012] [ka]

[0013] This is the structure shown, and it binds to the antibody at position 3, and to the methylene group in the linker structure containing it at the nitrogen atom at position 1. GGFG represents an amino acid sequence linked by peptide bonds consisting of glycine-glycine-phenylalanine-glycine. -(NH-DX) is given by the following equation:

[0014] [ka]

[0015] This is a group in which the nitrogen atom of the amino group at position 1 is the bonding site, The anti-CD37 antibody is an antibody that contains a light chain variable region having amino acid sequences 21-128 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence with 90% or more homology to the full-length light chain amino acid sequence shown in SEQ ID NO: 2, and a heavy chain variable region having amino acid sequences 20-138 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence with 90% or more homology to the full-length heavy chain amino acid sequence shown in SEQ ID NO: 4. [2] Antibody-drug conjugates of an anti-CD37 antibody that includes a heavy chain variable region and a light chain variable region as described in any one of the following groups (g) to (j): (g) The light chain variable region consisting of amino acid sequences 21-128 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and the heavy chain variable region consisting of amino acid sequences 20-138 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 4; (h) The light chain variable region consisting of amino acid sequences 21-128 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and the heavy chain variable region consisting of amino acid sequences 20-138 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 6; (i) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (j) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 10. [3] Antibody-drug conjugates of [1] or [2] in which the anti-CD37 antibody is an antibody containing a heavy chain variable region and a light chain variable region selected from any one of the following groups (h) to (j): (h) The light chain variable region consisting of amino acid sequences 21-128 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and the heavy chain variable region consisting of amino acid sequences 20-138 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 6; (i) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (j) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 10. [4] An antibody-drug conjugate from any one of [1] to [3], wherein the anti-CD37 antibody is an antibody comprising the heavy chain and light chain described in any one of the groups (k) to (n) below: (k) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 4; (l) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 6; (m) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (n) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 10. [5] An antibody-drug conjugate from any one of [1] to [4], wherein the anti-CD37 antibody is an antibody comprising the heavy chain and light chain described in any one of the groups (l) to (n) below: (l) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 6; (m) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (n) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 10. [6] Any one antibody-drug conjugate [1] to [5] whose drug-linker structure is represented by any one formula selected from the group consisting of (c), (d), and (e) below: (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (d)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX). [7] One of the antibody-drug conjugates [1] to [6] whose drug-linker structure is represented by formula (c) or (e) below: (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX). [8] The following formula (where A indicates the binding site with the antibody):

[0016] [ka]

[0017] An antibody-drug conjugate, one of [1] to [7], wherein a drug linker, indicated by [1], and an antibody are linked by a thioether bond. [9] The following formula:

[0018] [ka]

[0019] A single antibody-drug conjugate represented by [1] through [8]. (Here, AB represents the antibody, n represents the average number of drug-linker structures bound to the antibody per antibody, and the antibody and linker are bound via sulfhydryl groups derived from the antibody.)

[10] An antibody-drug conjugate, any one of [1] to [9], in which the antibody heavy chain has undergone one or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, amino-terminus processing, carboxyl-terminus processing, deamidation, aspartic acid isomerization, methionine oxidation, tryptophan oxidation, addition of a methionine residue to the amino terminus, amidation of a proline residue, and deletion of one or two amino acids at the carboxyl terminus.

[11] Antibody-drug conjugates in which one or two amino acids are deleted at the carboxyl terminus of the antibody heavy chain.

[10]

[12] An antibody-drug conjugate in which one amino acid is deleted at the carboxyl terminus of both antibody heavy chains

[10] or

[11] .

[13] An antibody-drug conjugate in which the proline residue at the carboxyl terminus of the antibody heavy chain is further amidated.

[10] to

[12]

[14] Any one of the antibody-drug conjugates [1] to

[13] having an average number of drug-linker structures bound per antibody ranging from 1 to 10.

[15] Any one of the antibody-drug conjugates [1] to

[14] in which the average number of drug-linker structures bound per antibody ranges from 2 to 8.

[16] Any one of the antibody-drug conjugates [1] to

[15] in which the average number of drug-linker structures per antibody ranges from 3 to 8.

[17] Any one of the antibody-drug conjugates [1] to

[16] having an average number of drug-linker structures per antibody ranging from 7 to 8.

[18] Any one of the antibody-drug conjugates [1] to

[17] having an average of 8 drug-linker links per antibody. A pharmaceutical composition comprising any one antibody-drug conjugate from [1] to

[18] , a pharmacokinetically acceptable salt thereof, or a hydrate thereof.

[20] A pharmaceutical composition of

[19] characterized by being an antitumor drug.

[21] The pharmaceutical composition of

[20] , characterized in that the tumor is a tumor that expresses CD37.

[22] The pharmaceutical composition of

[20] or

[21] , characterized in that the tumor is one selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal body lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia.

[23] The pharmaceutical composition according to

[20] or

[21] , characterized in that the tumor is one of the tumors selected from the group consisting of peripheral T-cell lymphoma, cutaneous T-cell lymphoma and other T-cell lymphomas, myelodysplastic syndrome and acute myeloid leukemia.

[24] A method for treating a tumor, comprising the step of administering to an individual an antibody-drug conjugate, a pharmacochemically acceptable salt thereof, or a hydrate thereof, from any one of [1] to

[18] .

[25] A treatment method for

[24] characterized in that the tumor is a tumor that expresses CD37.

[26] The treatment method of

[24] or

[25] , characterized in that the tumor is one of the tumors selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal body lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia.

[27] A treatment method for

[24] or

[25] , characterized in that the tumor is one of the tumors selected from the group consisting of peripheral T-cell lymphoma, cutaneous T-cell lymphoma, and other T-cell lymphomas, myelodysplastic syndromes, and acute myeloid leukemia.

[28] A tumor treatment comprising any one antibody-drug conjugate of [1] to

[18] , a pharmacochemically acceptable salt thereof, or a hydrate thereof.

[29] Use of any one antibody-drug conjugate of [1] to

[18] , a pharmacologically acceptable salt thereof, or a hydrate thereof for the treatment of tumors.

[30] Use of any one antibody-drug conjugate, a pharmacochemically acceptable salt thereof, or a hydrate thereof, of any one of [1] to

[18] for the preparation of drugs for the treatment of tumors.

[31] A saline solution preparation containing 0.001 to 100 mg / kg of any one antibody-drug conjugate from [1] to

[18] , a pharmacoagulably acceptable salt thereof, or a hydrate thereof.

[0020] Furthermore, the present invention also encompasses the following inventions. (i) An antibody or functional fragment of said antibody that specifically binds to CD37 and includes a heavy chain variable region and a light chain variable region selected from any one of the following groups (a) to (d): (a) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 4; (b) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 6; (c) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (d) The light chain variable region consisting of amino acid sequences 21 to 128 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and the heavy chain variable region consisting of amino acid sequences 20 to 138 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 10. (ii) The antibody described in (i) or a functional fragment of said antibody that specifically binds to CD37 and comprises a heavy chain and a light chain selected from any one of the following groups (e) to (h): (e) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 4; (f) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 6; (g) A light chain consisting of amino acids 21-234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20-468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (h) A light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acids 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 10. (iii) A polynucleotide encoding the antibody described in (i) or (ii) or a functional fragment of said antibody. An expression vector containing the polynucleotides described in (iv)(iii). Host cells transformed with the expression vectors described in (v)(iv). (vi) The host cell described in (v) is a eukaryotic cell. A method for producing an antibody or a functional fragment of the antibody, comprising the steps of culturing host cells as described in (vii)(v) or (vi), and collecting the antibody of the target from the culture obtained in the said step. An antibody or a functional fragment of the antibody, characterized by being obtained by the manufacturing method described in (viii)(vii). (ix) The antibody or functional fragment of the antibody according to (viii), comprising one or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, amino-terminus processing, carboxyl-terminus processing, deamidation, aspartic acid isomerization, methionine oxidation, tryptophan oxidation, addition of a methionine residue to the amino terminus, amidation of a proline residue, and deletion of one or two amino acids at the carboxyl terminus. (x) The antibody described in (ix) or a functional fragment of said antibody, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (xi) The antibody described in (ix) or (x), or a functional fragment of said antibody, wherein one amino acid is deleted at the carboxyl terminus of both heavy chains. (xii) The antibody according to any one of (ix) to (xi), wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated, or a functional fragment of said antibody. An antibody-drug conjugate in which a drug is bound to an antibody or a functional fragment of the antibody described in any one of (xiii), (viii), to (xii). A method for producing an antibody-drug conjugate, comprising the steps of: culturing host cells as described in (xiv)(v) or (vi); collecting a target antibody or a functional fragment of the antibody from the culture obtained in the step; and reacting the antibody or functional fragment of the antibody obtained in the step with a drug-linker intermediate compound. [Effects of the Invention]

[0021] The anti-CD37 antibody of the present invention is characterized by its specific binding to CD37-positive tumor cells, such as those of B-cell malignant lymphoma. An anti-CD37 antibody-drug conjugate, in which a drug that exhibits intracellular toxicity is conjugated to this antibody via a linker of a specific structure, is expected to achieve excellent antitumor efficacy and safety when administered to patients with cancer cells expressing CD37. In other words, the anti-CD37 antibody-drug conjugate of the present invention is useful as an antitumor agent against B-cell malignant lymphoma and the like. Furthermore, the anti-CD37 antibody-drug conjugate of the present invention has good recovery in physiological saline and can be handled in physiological saline. [Brief explanation of the drawing]

[0022] [Figure 1] This figure shows the nucleotide sequence encoding the hmAb-L11 light chain and the amino acid sequence of the hmAb-L11 light chain. [Figure 2]This figure shows the nucleotide sequence encoding the hmAb-H11 heavy chain and the amino acid sequence of the hmAb-H11 heavy chain. [Figure 3] This figure shows the nucleotide sequence encoding the hmAb-H541 heavy chain and the amino acid sequence of the hmAb-H541 heavy chain. [Figure 4] This figure shows the nucleotide sequence encoding the hmAb-H551 heavy chain and the amino acid sequence of the hmAb-H551 heavy chain. [Figure 5] This figure shows the nucleotide sequence encoding the hmAb-H11a heavy chain and the amino acid sequence of the hmAb-H11a heavy chain. [Figure 6] This figure shows the binding affinity of a humanized anti-CD37 antibody-drug conjugate to the CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7. [Figure 7] This figure shows the in vitro cell proliferation inhibitory activity of a humanized anti-CD37 antibody-drug conjugate against the CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7. [Figure 8] This figure shows the in vivo antitumor effect of a humanized anti-CD37 antibody-drug conjugate on SCID mice transplanted with the CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7. [Figure 9] This figure shows the in vivo antitumor effect of a humanized anti-CD37 antibody-drug conjugate on SCID mice transplanted with the CD37-positive human diffuse large B-cell lymphoma cell line WSU-DLCL2. [Figure 10] This figure shows the in vivo antitumor effect of a humanized anti-CD37 antibody-drug conjugate on SCID mice transplanted with the CD37-positive human diffuse large B-cell lymphoma cell line SU-DHL-8. [Figure 11]This figure shows the in vivo antitumor effect of the humanized anti-CD37 antibody-drug conjugate, POLIVY®, and IMGN529, on SCID mice transplanted with the CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7. [Figure 12] This figure shows the in vivo antitumor effects of the humanized anti-CD37 antibody-drug conjugate, POLIVY, and IMGN529, on SCID mice transplanted with the CD37-positive human diffuse large B-cell lymphoma cell line SU-DHL-8. [Figure 13] This figure shows the in vivo antitumor effects of humanized anti-CD37 antibody-drug conjugates, POLIVY and IMGN529, on SCID mice transplanted with the CD37-positive human diffuse large B-cell lymphoma cell line NU-DUL-1. [Figure 14] This figure shows the in vivo antitumor effect of a humanized anti-CD37 antibody-drug conjugate on SCID mice transplanted with the CD37-positive human diffuse large B-cell lymphoma cell line SU-DHL-4. [Figure 15] This figure shows the in vivo antitumor effects of humanized anti-CD37 antibody-drug conjugates, RITUXAN®, Ibrutinib, Venetoclax, and TREAKISYM®, on SCID mice transplanted with the CD37-positive human chronic lymphocytic leukemia cell line JVM-3. [Figure 16] This figure shows the in vivo antitumor effects of humanized anti-CD37 antibody-drug conjugates, POLIVY and IMGN529, against SCID mice transplanted with the CD37-positive human follicular lymphoma cell line DOHH-2. [Modes for carrying out the invention]

[0023] definition In the present invention, "gene" means a nucleic acid molecule containing a nucleotide sequence that codes for the amino acids of a protein, or its complementary chain. For example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., having a nucleotide sequence that codes for the amino acids of a protein or a nucleotide sequence complementary to that sequence are included in the meaning of "gene." Such genes are single-stranded, double-stranded, or triple-stranded or more nucleotides. The meaning of "gene" also includes aggregates of DNA and RNA strands, those in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on a single nucleotide chain, and double-stranded or triple-stranded or more nucleotides containing such nucleotide chains. Examples of the "CD37 gene" of the present invention include DNA, mRNA, cDNA, cRNA, etc., containing a nucleotide sequence that codes for the amino acid sequence of the CD37 protein.

[0024] In this invention, "nucleotide," "nucleic acid," and "nucleic acid molecule" are synonymous, and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc., are included in these meanings. Such nucleotides are single-stranded, double-stranded, or consist of three or more strands, and the meaning of "nucleotide" also includes aggregates of DNA and RNA strands, those in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on a single nucleotide strand, and aggregates of double-stranded or triple-stranded structures containing such nucleotide strands.

[0025] In this invention, "polypeptide," "peptide," and "protein" are synonymous.

[0026] In the present invention, unless otherwise specified, "protein" refers to "protein" from any vertebrate source, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats).

[0027] In this invention, the term "antigen" may be used to mean "immunogen."

[0028] In this invention, "cells" include various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, and microorganisms.

[0029] In the present invention, the "site" to which an antibody binds, that is, the "site" recognized by the antibody, refers to a partial peptide or partial higher-order structure on an antigen to which the antibody binds or recognizes. In the present invention, such a site is also called an epitope or antibody binding site. Examples of sites on the CD37 protein to which the anti-CD37 antibody of the present invention binds or recognizes include partial peptides or partial higher-order structures on the CD37 protein.

[0030] In this invention, "CDR" refers to the Complemetarity Determining Region, and "FR" refers to the Framework Region. It is known that antibody molecules have three CDRs each in their heavy and light chains. CDRs are also called hypervariable domains and are regions within the variable regions of the antibody's heavy and light chains that exhibit particularly high variability in their primary structure. They are usually separated into three locations on the primary structure of the heavy and light chain polypeptide chains. In this invention, the complementarity determining regions of antibodies are denoted as CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the heavy chain amino acid sequence, and as CDRL1, CDRL2, and CDRL3 from the amino-terminal side of the light chain amino acid sequence. These regions are in close proximity to each other in terms of three-dimensional structure and determine the specificity for the antigen to which they bind. The portion of the heavy chain variable region amino acid sequence other than CDRH1 to CDRH3 is called FR, and the portions from the amino terminus to just before CDRH1, from after CDRH1 to just before CDRH2, from after CDRH2 to just before CDRH3, and from after CDRH3 to the carboxyl terminus are called FRH1 to FRH4, respectively. Similarly, the portion of the light chain variable region amino acid sequence other than CDRL1 to CDRL3 is also FR, and the portions from the amino terminus to just before CDRL1, from after CDRL1 to just before CDRL2, from after CDRL2 to just before CDRL3, and from after CDRL3 to the carboxyl terminus are called FRL1 to FRL4, respectively. In other words, in the variable regions (amino acid sequences) of the heavy and light chains, the amino acids are arranged in the order of FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, moving continuously from the amino terminus towards the carboxyl terminus.

[0031] In the present invention, "functional antibody fragment" means an antibody fragment that performs at least a part of the function performed by the original antibody. Examples of "functional antibody fragments" include, but are not limited to, Fab, F(ab')2, scFv, Fab', single-chain immunoglobulins, etc. Such functional antibody fragments may be obtained by treating the full-length molecule of the antibody protein with an enzyme such as papain or pepsin, or they may be recombinant proteins produced in a suitable host cell using a recombinant gene. Among the "functional antibody fragments," those that have binding activity to an antigen, i.e., human CD37, are called "antibody binding fragments."

[0032] 1. CD37 CD37 is a four-transmembrane protein belonging to the tetraspanin superfamily (Charrin S., et al., J Cell Sci. 3641-3648, 127, 2014). It is a membrane protein consisting of 281 amino acids, and both its amino-terminus and carboxyl-terminus are located inside the cell. It can be referenced by accession numbers such as NM_001774 and NP_001765 (NCBI).

[0033] The CD37 protein used in this invention can be obtained by directly purifying it from CD37-expressing cells of humans or non-human mammals (rats, mice, monkeys, etc.), or by preparing and using the cell membrane fraction of such cells. It can also be obtained by synthesizing CD37 in vitro or by inducing its production in host cells through genetic manipulation. Specifically, in genetic manipulation, the protein can be obtained by incorporating CD37 cDNA into an expressionable vector and then synthesizing it in a solution containing enzymes, substrates, and energy substances necessary for transcription and translation, or by transforming host cells of other prokaryotes or eukaryotes to express CD37. Furthermore, CD37-expressing cells obtained through the aforementioned genetic manipulation, or cell lines expressing CD37, can also be used as the CD37 protein. Additionally, an expression vector incorporating CD37 cDNA can be directly administered to an immunized animal to induce CD37 expression within the animal's body.

[0034] Furthermore, CD37 also includes proteins that have the same biological activity as the above-mentioned CD37 protein, but in which one or more amino acids are substituted, deleted, and / or added to the amino acid sequence. In this specification, "a few" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0035] The human CD37 protein has the amino acid sequence described in SEQ ID NO: 18. The extracellular domain of the human CD37 protein consists of extracellular domain 1 (hereinafter also referred to as EC1), which has amino acid sequences 39 to 59 of the amino acid sequence described in SEQ ID NO: 18, and extracellular domain 2 (hereinafter also referred to as EC2), which has amino acid sequences 112 to 241 of the amino acid sequence described in SEQ ID NO: 18.

[0036] Sequence ID 18 MSAQESCLSL IKYFLFVFNL FFFVLGSLIF CFGIWILIDK TSFVSFVGLA FVPLQIWSKV LAISGIFTMG IALLGCVGAL KELRCLLGLY FGMLLLLFAT QITLGILIST QRAQLERSLR DVVEKTIQKY GTNPEETAAE ESWDYVQFQL RCCGWHYPQD WFQVLILRGN GSEAHRVPCS CYNLSATNDS TILDKVILPQ LSRLGHLARS RHSADICAVP AESHIYREGC AQGLQKWLHN NLISIVGICL GVGLLELGFM TLSIFLCRNL DHVYNRLARY R

[0037] The sequence of the human CD37 protein can also be found by referring to the following description. https: / / www.uniprot.org / uniprot / P11049

[0038] 2. Manufacturing of anti-CD37 antibodies (2-1) Antibodies In this invention, antibodies that bind to CD37 and antibodies that recognize CD37 may both be referred to as "anti-CD37 antibodies" or abbreviated as "CD37 antibodies."

[0039] The anti-CD37 antibody of the present invention may be derived from any species, but preferably from humans, rats, mice, and rabbits. If derived from a species other than humans, it is desirable to chimerize or humanize it using known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred.

[0040] The anti-CD37 antibody of the present invention is an antibody that can target tumor cells, that is, it possesses the characteristics of being able to recognize tumor cells, being able to bind to tumor cells, and being taken up and internalized within tumor cells. Therefore, the anti-CD37 antibody of the present invention and a compound having antitumor activity can be conjugated via a linker to form an antibody-drug conjugate.

[0041] The binding affinity of antibodies to tumor cells can be confirmed using flow cytometry. Antibody uptake into tumor cells can be confirmed using (1) an assay that visualizes antibodies taken up into cells using a fluorescence microscope with a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay that measures the amount of fluorescence when taken up into cells using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay (Bio Techniques 28:162-165, January 2000), which uses an immunotoxin that binds to the therapeutic antibody and suppresses cell proliferation by releasing a toxin upon uptake into cells. Recombinant complex proteins of the catalytic domain of diphtheria toxin and protein G can also be used as immunotoxins.

[0042] As used herein, "high internalization ability" means that the viability of CD37-expressing cells administered with the antibody and saporin-labeled anti-rat IgG antibody (expressed as a relative rate with the cell viability without antibody added being 100%) is preferably 70% or less, more preferably 60% or less.

[0043] Since the antibody-drug conjugate of the present invention has a compound that exerts an antitumor effect attached to it, it is preferable, but not essential, that the antibody itself has an antitumor effect. For the purpose of specifically and selectively exerting the cytotoxic effect of the antitumor compound in tumor cells, it is important and preferable that the antibody has the property of being internalized and migrated to tumor cells.

[0044] Anti-CD37 antibodies can be obtained by immunizing animals with antigenic polypeptides using methods employed in this field, and then collecting and purifying the antibodies produced in vivo. However, since CD37 is a four-transmembrane protein, it is preferable to use CD37 with its three-dimensional structure preserved as the antigen. DNA immunization is one such method.

[0045] Antigens are not limited to human origins; animals can also be immunized with antigens derived from non-human animals such as mice and rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity between the acquired heterologous antigen and the human antigen.

[0046] Furthermore, according to known methods (for example, Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R.ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)), a hybridoma can be established by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells, thereby obtaining monoclonal antibodies. The following explains in detail how to obtain antibodies against CD37.

[0047] (1) Preparation of antigen Antigens can be obtained by genetically modifying host cells to produce the gene encoding the antigen protein. Specifically, a vector capable of expressing the antigen gene is created, introduced into host cells to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing animals with antigen-expressing cells or cell lines expressing the antigen, which have been obtained through the above genetic modification.

[0048] Furthermore, antibodies can also be obtained by incorporating the cDNA of an antigen protein into an expression vector and administering it to an immunized animal, thereby causing the immunized animal to express the antigen protein and produce antibodies against the antigen protein, without using the antigen protein itself. (2) Production of anti-CD37 monoclonal antibodies The anti-CD37 antibody used in the present invention is not particularly limited, but for example, an antibody identified by the amino acid sequence shown in the sequence listing of this application can be suitably used. The anti-CD37 antibody used in the present invention is preferably one that has the following characteristics. (1) An antibody characterized by having the following properties; (a) It specifically binds to CD37.

[0049] (b) It has the activity to be internalized into CD37-expressing cells by binding to CD37. (2) The antibody described in (1) above, wherein CD37 is human CD37. (3) The antibody described in (1) or (2) above that recognizes the higher-order structure of CD37.

[0050] The method for obtaining antibodies against CD37 according to the present invention is not particularly limited as long as anti-CD37 antibodies can be obtained. However, since CD37 is a transmembrane protein, it is preferable to use CD37 that retains its higher-order structure as the antigen.

[0051] DNA immunoassay is one example of a preferred method for obtaining antibodies. DNA immunoassay is a technique that induces immunity to an antigen by introducing an antigen expression plasmid into an animal, such as a mouse or rat, and then expressing the antigen within the animal. Methods of gene introduction include directly injecting the plasmid into the muscle, intravenously injecting introduction reagents such as liposomes or polyethyleneimine, using viral vectors, injecting gold particles with the plasmid attached using a Gene Gun, and the Hydrodynamic method, which involves rapidly injecting a large amount of plasmid solution intravenously. Regarding gene transfer methods using intramuscular injection of expression plasmids, a technique called in vivo electroporation, which involves injecting the plasmid intramuscularly and then applying electroporation to the same site, is known as a method to improve expression levels (Aihara H, Miyazaki J. Nat Biotechnol. 1998 Sep;16(9):867-70 or Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D. Proc Natl Acad Sci US A. 1999 Apr 13;96(8):4262-7). This method can be further improved by treating the muscle with hyaluronidase before plasmid injection (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ. Gene Ther. 2001 Aug;8(16):1264-70). Furthermore, hybridomas can be produced by known methods, for example, using the Hybridoma Production System (Cyto Pulse Sciences).

[0052] Specific examples of obtaining monoclonal antibodies include the following: (a) By incorporating CD37 cDNA into an expression vector and directly administering the vector to an immune animal using methods such as electroporation or a gene gun, an immune response can be induced by expressing CD37 in the animal's body. The vector may be administered once or multiple times, preferably multiple times, if necessary to increase the antibody titer. (b) From the aforementioned animals in which an immune response has been induced, collect tissues containing antibody-producing cells (e.g., lymph nodes), (c) Preparation of myeloma cells (hereinafter referred to as "myeloma") (d) Cell fusion between antibody-producing cells and myeloma, (e) Selection of hybridoma groups that produce the target antibody, (f) Divide into single-cell clones (cloning), (g) Hybridoma culture for the mass production of monoclonal antibodies, or rearing of animals transplanted with hybridomas.

[0053] The resulting monoclonal antibody exhibits high antigen specificity for CD37. While there are no particular restrictions on the monoclonal antibody used, an example is the anti-CD37 mouse monoclonal antibody HH1 (Smeland E, et al., Scand J Immunol, 21(3), 205-214(1985)). (h) Examination of the physiological activity (internalization activity) and binding specificity of the monoclonal antibody produced in this manner, or testing of its properties as a labeling reagent. Examples of antibody titer measurement methods used here include, but are not limited to, flow cytometry or Cell-ELISA. Furthermore, even if the monoclonal antibody is obtained separately and independently by repeating the specific monoclonal antibody acquisition examples (a) to (h) described in "2. Production of Anti-CD37 Antibodies," or by obtaining the monoclonal antibody separately by other methods, it is possible to obtain an antibody having the same cytotoxic activity as the anti-CD37 antibody obtained in step (g). An example of such an antibody is an antibody that binds to the same epitope as the anti-CD37 antibody obtained in step (g). If the newly produced monoclonal antibody binds to the partial peptide or partial three-dimensional structure to which the anti-CD37 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope. In addition, by confirming that the monoclonal antibody competes for the binding of the anti-CD37 antibody to CD37 (i.e., the monoclonal antibody prevents the binding of the anti-CD37 antibody to CD37), it can be determined that the monoclonal antibody binds to the same epitope as the anti-CD37 antibody, even if the specific epitope sequence or structure has not been determined. If the epitopes are confirmed to be identical, it is strongly expected that the monoclonal antibody will have equivalent antigen-binding ability or biological activity to the anti-CD37 antibody.

[0054] (3) Other antibodies The antibodies of the present invention include not only monoclonal antibodies against CD37 as described above, but also genetically modified antibodies, such as chimeric antibodies, humanized antibodies, and human antibodies, which are artificially modified for purposes such as reducing heterologous antigen activity against humans and improving the physical properties of antibody-drug conjugates. These antibodies can be manufactured using known methods.

[0055] Chimeric antibodies include antibodies in which the variable region and constant region are heterogeneous, such as chimeric antibodies in which the variable region of a mouse or rat-derived antibody is conjugated to the constant region of a human-derived antibody (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0056] Examples of humanized antibodies include antibodies in which only the CDR is incorporated into a human-derived antibody (see Nature (1986) 321, pp. 522-525), antibodies in which the CDR sequence and some amino acid residues of the framework are transplanted into a human antibody using the CDR transplantation method (International Publication No. 90 / 07861), and antibodies in which the amino acid sequence of some CDRs is modified while maintaining the ability to bind to the antigen.

[0057] Examples of humanized antibodies of the anti-CD37 mouse monoclonal antibody HH1 include antibodies comprising the light chain variable region of hmAb-L11 and the heavy chain variable region of one of hmAb-H11, hmAb-H541, hmAb-H551, or hmAb-H11a. The amino acid sequence of hmAb-L11 is shown in SEQ ID NO: 2, and the amino acid sequences of hmAb-H11, hmAb-H541, hmAb-H551, or hmAb-H11a are shown in SEQ ID NOs: 4, 6, 8, or 10, respectively. In hmAb, the light chain variable region consists of the sequence shown at amino acid numbers 21 to 128 of the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain variable region consists of the sequence shown at amino acid numbers 20 to 138 of the amino acid sequence shown in each respective SEQ ID NO: 2. Furthermore, the antibody of the present invention includes an antibody comprising the full-length light chain of hmAb-L11 and the full-length heavy chain of any of hmAb-H11, hmAb-H541, hmAb-H551, or hmAb-H11a. The full-length light chain amino acid sequence of hmAb-L11 comprises the sequence shown at amino acid numbers 21 to 234 of the amino acid sequence shown in SEQ ID NO: 2, and the full-length heavy chain amino acid sequences of hmAb-H11, hmAb-H541, hmAb-H551, or hmAb-H11a each comprise the sequence shown at amino acid numbers 20 to 468 of the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10. Specifically, examples include hmAb-H11L11, hmAb-H541L11, hmAb-H551L11, or hmAb-H11aL11.

[0058] In Sequence ID No. 2, the sequence consisting of amino acid residues 44 to 54 (KASQDVSTAVD: Sequence ID No. 19) represents CDRL1, the sequence consisting of amino acid residues 70 to 76 (WASTRHT: Sequence ID No. 20) represents CDRL2, and the sequence consisting of amino acid residues 109 to 117 (RQHYSTPFT: Sequence ID No. 21) represents CDRL3. In Sequence IDs No. 4, 6, 8, and 10, the sequence consisting of amino acid residues 45 to 54 (GYSFTDYNMY: Sequence ID No. 22) represents CDRH1, the sequence consisting of amino acid residues 69 to 78 (YIDPYNGDTT: Sequence ID No. 23) represents CDRH2, and the sequence consisting of amino acid residues 118 to 127 (SPYGHYAMDY: Sequence ID No. 24) represents CDRH3. The CDR sequences described are based on the AbM definition (Handbook of Therapeutic Antibodies, Chapter 5, Bioinformatics Tools for Antibody Engineering, Andrew CR Martin, James Allen, 2007).

[0059] Furthermore, as amino acid substitutions in this specification, conservative amino acid substitutions are preferred. A conservative amino acid substitution is a substitution that occurs within an amino acid group related to the amino acid side chain. Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar family = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxyl group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine and isoleucine; and aromatic group = phenylalanine, tryptophan and tyrosine. Such amino acid substitutions are preferably carried out in a manner that does not degrade the properties of the substance having the original amino acid sequence.

[0060] By combining sequences that show high homology to the heavy-chain amino acid sequences and light-chain amino acid sequences described above, it is possible to select antibodies that have equivalent biological activity to each of the antibodies described above. Such homology is generally 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more. Furthermore, by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added to the heavy-chain or light-chain amino acid sequence, it is also possible to select antibodies that have equivalent biological activity to each of the antibodies described above.

[0061] The homology between two amino acid sequences can be determined by using the default parameters of the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can also be accessed via the internet at www.ncbi.nlm.nih.gov / blast.

[0062] In the light chain amino acid sequence shown in Sequence ID No. 2 in the sequence listing, the amino acid sequence consisting of amino acid residues 1 through 20 is the signal sequence, the amino acid sequence consisting of amino acid residues 21 through 128 is the variable region, and the amino acid sequence consisting of amino acid residues 129 through 234 is the constant region. The sequence of Sequence ID No. 2 is shown in Figure 1.

[0063] Furthermore, in the heavy chain amino acid sequences shown in SEQ ID NOs: 4, 6, 8, or 10, the amino acid sequence consisting of amino acids 1 through 19 is the signal sequence, the amino acid sequence consisting of amino acid residues 20 through 138 is the variable region, and the amino acid sequence consisting of amino acid residues 139 through 468 is the constant region. The sequences of SEQ ID NOs: 3, 5, 7, or 9 are shown in Figures 2, 3, 4, or 5.

[0064] The antibodies of the present invention can further include human antibodies that bind to CD37. An anti-CD37 human antibody refers to a human antibody that possesses only the gene sequence of an antibody derived from a human chromosome. Anti-CD37 human antibodies can be obtained by methods using human antibody-producing mice that possess human chromosome fragments containing the genes for the heavy and light chains of human antibodies (see Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p. 722-727, etc.).

[0065] Specifically, these human antibody-producing mice can be created as genetically modified animals in which the endogenous immunoglobulin heavy and light chain gene loci are disrupted and replaced with human immunoglobulin heavy and light chain gene loci via a yeast artificial chromosome (YAC) vector or the like. These can be produced by creating knockout animals and transgenic animals, and then crossbreeding these animals.

[0066] Furthermore, by using genetic engineering technology to transform eukaryotic cells with cDNA encoding the heavy and light chains of such human antibodies, preferably a vector containing the cDNA, and culturing the transformed cells that produce the recombinant human monoclonal antibody, this antibody can also be obtained from the culture supernatant.

[0067] Here, as the host, for example, eukaryotic cells, preferably CHO cells, or mammalian cells such as lymphocytes or myeloma can be used.

[0068] Furthermore, methods for obtaining human antibodies derived from phage displays selected from human antibody libraries are also known (see Wormstone, I et. al., Investigative Ophthalmology & Visual Science. (2002) 43(7), p.2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1(2), p.189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109(3), p.427-431, etc.).

[0069] For example, a phage display method can be used to select phages that bind to an antigen by expressing the variable region of a human antibody as a single-chain antibody (scFv) on the phage surface (Nature Biotechnology (2005), 23, (9), pp. 1105-1116).

[0070] By analyzing the genes of phages selected through antigen binding, it is possible to determine the DNA sequence encoding the variable region of human antibodies that bind to the antigen.

[0071] Once the DNA sequence of the scFv that binds to the antigen is identified, an expression vector containing that sequence can be constructed and introduced into a suitable host to express it, thereby obtaining human antibodies (International Publication Nos. 92 / 01047, 92 / 20791, 93 / 06213, 93 / 11236, 93 / 19172, 95 / 01438, 95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0072] If a newly produced human antibody binds to the partial peptide or partial three-dimensional structure to which the CD37 antibody described herein binds, it can be determined that the human antibody binds to the same epitope. Furthermore, by confirming that the human antibody competes for the binding of the CD37 antibody described herein to CD37 (i.e., the human antibody prevents the binding of the CD37 antibody described herein to CD37), it can be determined that the human antibody binds to the same epitope as the CD37 antibody described herein, even if the specific epitope sequence or structure has not been determined. If the epitopes are confirmed to be the same, it is strongly expected that the human antibody has equivalent antigen-binding ability or biological activity to the CD37 antibody described herein.

[0073] The chimeric antibodies, humanized antibodies, or human antibodies obtained by the above method can be evaluated for their binding affinity to antigens using known methods, and suitable antibodies can be selected.

[0074] Another indicator to consider when comparing the properties of antibodies is their stability. Differential scanning calorimetry (DSC) is a device that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is a good indicator of the relative structural stability of proteins. By measuring Tm values ​​using DSC and comparing these values, differences in thermal stability can be compared. It is known that the storage stability of antibodies correlates to their thermal stability to some extent (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), and suitable antibodies can be selected using thermal stability as an indicator. Other indicators for selecting antibodies include high yield in appropriate host cells and low aggregation in aqueous solutions. For example, the antibody with the highest yield does not necessarily exhibit the highest thermal stability, so it is necessary to make a comprehensive judgment based on the indicators mentioned above to select the antibody most suitable for administration to humans.

[0075] The antibodies of the present invention also include modified antibodies. Such modified antibodies are those that have undergone chemical or biological modifications. Chemical modifications include the attachment of chemical moieties to the amino acid backbone, and chemical modifications of N-linked or O-linked carbohydrate chains. Biological modifications include those that have undergone post-translational modifications (e.g., N-linked or O-linked glycosylation, amino-terminus or carboxyl-terminus processing, deamidation, aspartic acid isomerization, methionine oxidation, tryptophan oxidation), and those that have had a methionine residue added to the amino terminus by expression using prokaryotic host cells. Furthermore, modified antibodies that have been labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescently labeled, or affinity-labeled antibodies, are also included in the definition of such modifications. Such modified antibodies of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and for detection or isolation of antibodies or antigens.

[0076] Furthermore, antibody-dependent cytotoxic activity can be enhanced by regulating the glycosylation (glycosylation, defucoseation, etc.) of the antibodies of the present invention. Known techniques for regulating antibody glycosylation include, but are not limited to, International Publication Nos. 1999 / 54342, 2000 / 61739, 2002 / 31140, 2007 / 133855, and 2013 / 120066. The antibodies of the present invention also include antibodies in which such glycosylation has been regulated.

[0077] (2-2) Method for producing antibodies When antibody genes are isolated and then introduced into a suitable host to produce antibodies, a suitable host and expression vector combination can be used. Specific examples of antibody genes include combinations of genes encoding the heavy chain sequence and light chain sequence of the antibody described herein. When transforming host cells, the heavy chain sequence gene and light chain sequence gene can be inserted into the same expression vector, or they can be inserted into separate expression vectors.

[0078] When using eukaryotic cells as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, animal cells include mammalian cells, such as monkey cells (COS cells, Gluzman, Y. Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast cells NIH3T3 (ATCC No. CRL-1658), dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LAProc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220), and FreeStyle 293F cells (Invitrogen).

[0079] Examples of prokaryotic cells that can be used include E. coli and Bacillus subtilis.

[0080] Antibodies can be obtained by introducing the target antibody gene into these cells by transformation and culturing the transformed cells in vitro. In this culture, the yield may differ depending on the antibody sequence, and it is possible to select antibodies that are easy to produce as pharmaceuticals from among antibodies with equivalent binding activity, using yield as an indicator. Therefore, the antibodies of the present invention also include antibodies obtained by a method for producing antibodies, which is characterized by including the steps of culturing the transformed host cells and collecting the target antibody or a functional fragment of the antibody from the culture obtained in the step.

[0081] It is known that antibodies produced in mammalian cultured cells lose a lysine residue at the carboxyl terminus of the heavy chain (Journal of Chromatography A, 705:129-134 (1995)), and that two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain are also lost, with a proline residue newly located at the carboxyl terminus being amidated (Analytical Biochemistry, 360:75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector function (such as complement activation or antibody-dependent cytotoxicity) of the antibody. Therefore, the antibody according to the present invention includes antibodies that have undergone such modifications and functional fragments of such antibodies, as well as deletions in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, and amidated deletions (for example, heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as antigen-binding ability and / or effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody according to the present invention are not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be one of the full-length heavy chains and heavy chains selected from the group consisting of the above-mentioned deletions, or a combination of either two. The quantity ratio of each deletion may be affected by the type of mammalian cultured cell that produces the antibody according to the present invention and the culture conditions, but a possible example of the main component of the antibody according to the present invention is the case in which one amino acid residue at the carboxyl terminal is deleted in both of the two heavy chains.

[0082] Examples of antibody isotypes of the present invention include IgG (IgG1, IgG2, IgG3, IgG4), but IgG1 or IgG2 are preferred.

[0083] The biological activities of antibodies generally include antigen-binding activity, activity that internalizes an antigen into cells expressing that antigen by binding to it, activity that neutralizes the activity of an antigen, activity that enhances the activity of an antigen, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cell-mediated cytotoxicity (CDC) activity, and antibody-dependent cell-mediated phagocytosis (ADCP). However, the function of the antibody according to the present invention is CD37 binding activity, and preferably CD37 binding activity that internalizes an antibody into CD37-expressing cells. Furthermore, the antibody according to the present invention may also possess ADCC activity, CDC activity, and / or ADCP activity in addition to intracellular internalization activity.

[0084] The obtained antibodies can be purified to a uniform degree. Antibodies can be separated and purified using the same separation and purification methods used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, and isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but are not limited to these.

[0085] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography.

[0086] These chromatography processes can be performed using liquid chromatography such as HPLC or FPLC.

[0087] Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, columns using protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0088] Furthermore, it is possible to purify antibodies by utilizing their binding properties to antigens using a carrier on which the antigen is immobilized.

[0089] 3. Anti-CD37 antibody-drug conjugate (1) Drugs The anti-CD37 antibody obtained in "2. Production of Anti-CD37 Antibodies" above can be converted into an anti-CD37 antibody-drug conjugate by binding a drug via the linker structure. The drug is not particularly limited as long as it has substituents or substructures that can bind to the linker structure. The anti-CD37 antibody-drug conjugate can be used for various purposes depending on the drug it binds to. Examples of such drugs include substances with antitumor activity, substances effective against hematological disorders, substances effective against autoimmune diseases, anti-inflammatory substances, antibacterial substances, antifungal substances, antiparasitic substances, antiviral substances, and antianesthetic substances.

[0090] (1)-1 Antitumor compounds Examples of using an antitumor compound as the compound bound to the anti-CD37 antibody-drug conjugate of the present invention are described below. The antitumor compound is not particularly limited as long as it is a compound with antitumor effects and has substituents or substructures that can bind to the linker structure. In the case of an antitumor compound, part or all of the linker is cleaved within the tumor cell, releasing the antitumor compound portion and thus exhibiting its antitumor effect. If the linker is cleaved at the drug binding site, the antitumor compound is released in its original structure, and its original antitumor effect is exerted.

[0091] One example of an antitumor compound used in the present invention is the camptothecin derivative exatecan ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13(9H,15H)-dione; formula below:)

[0092] [ka]

[0093] The compound can be suitably used. This compound can be readily obtained, for example, by the method described in U.S. Patent Publication No. 2016 / 0297890 or by other known methods, and the amino group at position 1 can be suitably used as a binding site to the linker structure. Furthermore, although exatecan may be released into tumor cells with a portion of the linker bound, it is a compound that exhibits excellent antitumor effects even in such a state.

[0094] Since exatecan has a camptothecin structure, it is known that in an acidic aqueous medium (e.g., pH 3), the equilibrium shifts to the structure in which the lactone ring is formed (closed form), while in a basic aqueous medium (e.g., pH 10), the equilibrium shifts to the structure in which the lactone ring is opened (open form). Drug conjugates incorporating exatecan residues corresponding to such closed and open ring structures can be expected to have equivalent antitumor effects, and it goes without saying that both are included within the scope of the present invention.

[0095] Other antitumor compounds include, for example, those listed in the literature (Pharmacological Reviews, 68, p3-19, 2016), such as auristatins including doxorubicin, calichemicine, dorastatin 10, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, and pyrrolobenzodiazepines (Py Examples include the dimer SG2000 (SJG-136) of rrolobenzodiazepine, duocarmycins such as SN-38 and CC-1065 which are derivatives of camptothecin, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin or its derivatives), taxol or its derivatives, etc.

[0096] In antibody-drug conjugates, the number of drugs bound to a single antibody molecule is a crucial factor affecting their efficacy and safety. The production of antibody-drug conjugates is carried out by specifying reaction conditions, such as the amount of raw materials and reagents used, to ensure a constant number of drug bindings. However, unlike the chemical reactions of low-molecular-weight compounds, the resulting product is typically a mixture with varying numbers of drugs bound. The number of drugs bound to a single antibody molecule is specified and expressed as an average value, i.e., the average number of drug bindings. In this invention, unless otherwise specified, i.e., when referring to an antibody-drug conjugate with a specific number of drug bindings contained within a mixture of antibody-drug conjugates with different drug binding numbers, the number of drug bindings refers to the average value. The number of exatecans bound to the antibody molecule is controllable. An average of 1 to 10 exatecans can be bound per antibody, preferably 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8. More preferably 5 to 8, even more preferably 7 to 8, and even more preferably about 8 or 8. Those skilled in the art can design a reaction to bind the required number of drugs to the antibody based on the examples described in this application, thereby obtaining an antibody-drug conjugate with a controlled number of exatecans.

[0097] (2) Linker structure The present invention describes a linker structure for binding a drug to an anti-CD37 antibody in an anti-CD37 antibody-drug conjugate.

[0098] In the antibody-drug conjugate of the present invention, the linker structure that conjugates the anti-CD37 antibody and the drug is not particularly limited as long as it can be used as an antibody-drug conjugate, and can be appropriately selected and used depending on the purpose of use. Examples of linker structures include those described in publicly available literature (Pharmacol Rev 68:3-19, January 2016, Protein Cell DOI 10.1007 / s13238-016-0323-0, etc.). More specific examples include VC (valine-citrulline), MC (maleimidocaproyl), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate), SS (disulfide), and SPDB (N-succinimidyl Examples include 4-(2-pyridyldithio)butyrate (N-succinimidyl 4-(2-pyridyldithio)butyrate), SS / hydrazone, hydrazone, and carbonate.

[0099] Another example is the linker structure described in U.S. Patent Publication 2016 / 0297890 (for example, the one described in paragraphs

[0260] to

[0289] of the said publication), and the following structure can be suitably used. The left end of the structure shown below is the antibody binding site, and the right end is the drug binding site. In addition, GGFG in the linker structure below represents an amino acid sequence linked by a peptide bond consisting of glycine-glycine-phenylalanine-glycine (GGFG). -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0100] More preferably, the following can be listed: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-. Furthermore, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-. One could list these:

[0101] The antibody binds to the terminal of -(Succinimid-3-yl-N) (for example, in '-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-', the terminal opposite to where (-CH2CH2CH2CH2CH2-) is bound (the left terminal)), and the antitumor compound binds to the carbonyl group of CH2-O-CH2-C(=O)- at the terminal opposite to -(Succinimid-3-yl-N) (in the above example, the right terminal). '-(Succinimid-3-yl-N)-' is represented by the following formula:

[0102] [ka]

[0103] It has the structure shown. The 3-position of this substructure is the binding site for the anti-CD37 antibody. The binding to the antibody at this 3-position is characterized by the formation of a thioether. The nitrogen atom at position 1 of this structural part is bonded to the carbon atom of the methylene group present in the linker containing this structure.

[0104] In the antibody-drug conjugate of the present invention, in which the drug is exatecan, it is preferable that the drug-linker structure portion having the following structure is conjugated to the antibody. The average number of these drug-linker structure portions conjugated per antibody should be 1 to 10, but preferably 2 to 8, more preferably 5 to 8, even more preferably 7 to 8, and even more preferably 8. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0105] More preferably, the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0106] Furthermore, the following are more even more desirable: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0107] Also, -(NH-DX) is expressed by the following formula:

[0108] [ka]

[0109] This is the structure shown, representing a group formed by removing one hydrogen atom from the amino group at position 1 of exatecan.

[0110] (3) Method for producing antibody-drug conjugates The antibodies that can be used in the antibody-drug conjugate of the present invention are not particularly limited as long as they are anti-CD37 antibodies having internalization activity as described in section 2. Production of anti-CD37 antibodies and the examples above, and functional fragments of said antibodies.

[0111] Next, a typical method for producing the antibody-drug conjugate of the present invention will be described. In the following, the compound numbers shown in each reaction formula will be used to indicate the compounds. That is, they will be referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will also be described similarly.

[0112] (3)-1 Manufacturing method 1 Among the antibody-drug conjugates represented by formula (1) below, those in which the linker structure is linked to the anti-CD37 antibody via a thioether can be produced by reacting an antibody AB, obtained by reducing the anti-CD37 antibody to convert the disulfide bond to a sulfhydryl group, with a compound (2) that can be obtained by known methods (for example, the method described in U.S. Patent Publication No. 2016 / 297890 (for example, the method described in paragraphs

[0336] to

[0374] of said publication)). For example, it can be produced by the following method.

[0113]

number

[0114] [In the formula, AB represents an antibody having a sulfhydryl group.] Here, L 1 teeth, This is represented by the structure -(Succinimid-3-yl-N)-. L 1 ' represents the malemidyl group, as shown in the following formula.

[0115] [ka]

[0116] -L 1 -L X It has one of the structures shown by the following formulas. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0117] Of these, the following are preferable: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0118] Furthermore, the following are preferably included: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0119] Furthermore, in the above reaction equation, the antibody-drug conjugate (1) is described as a structure in which one structural portion from the drug to the linker end is bound to one antibody. However, this is a simplified description for explanatory purposes, and in reality, multiple such structural portions are often bound to a single antibody molecule. This situation is also true in the following description of the manufacturing method.

[0120] That is, an antibody-drug conjugate (1) can be produced by reacting a compound (2) that is available by known methods (for example, available by the method described in U.S. Patent Publication No. 2016 / 297890 (for example, the method described in paragraphs

[0336] to

[0374] )) with an antibody having a sulfhydryl group.

[0121] Antibodies containing sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, methods include, but are not limited to, reacting the amino group of the antibody with Traut's reagent; reacting the amino group of the antibody with N-succinimidyl S-acetylthioalkanoates followed by hydroxylamine; reacting with N-succinimidyl 3-(pyridyldithio)propionate followed by a reducing agent; or reacting the antibody with reducing agents such as dithiothreitol, 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the disulfide bonds in the interchain region of the antibody and generate sulfhydryl groups.

[0122] Specifically, by using TCEP as a reducing agent in an amount of 0.3 to 3 molar equivalents per interchain disulfide of the antibody, and reacting it with the antibody in a buffer containing a chelating agent, an antibody in which the interchain disulfides are partially or completely reduced can be obtained. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). These can be used at concentrations of 1 mM to 20 mM. As a buffer, sodium phosphate, sodium borate, sodium acetate solution, etc., can be used. In a specific example, by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours, an antibody having partially or completely reduced sulfhydryl groups can be obtained.

[0123] Furthermore, by carrying out a reaction in which a sulfhydryl group is added to the drug-linker portion, the drug-linker portion can be bonded via a thioether bond.

[0124] Next, an antibody-drug conjugate (1) can be produced in which 2 to 8 drug molecules are bound to each antibody by using 2 to 20 molar equivalents of compound (2) per antibody having a sulfhydryl group. Specifically, a solution in which compound (2) is dissolved is added to a buffer containing the antibody having a sulfhydryl group and the reaction is carried out. Here, sodium acetate solution, sodium phosphate, sodium borate, etc. can be used as the buffer. The pH during the reaction is 5 to 9, and more preferably the reaction is carried out at around pH 7. Organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyridone (NMP) can be used as the solvent to dissolve compound (2). The organic solvent solution in which compound (2) is dissolved is added to a buffer containing the antibody having a sulfhydryl group at a concentration of 1 to 20% v / v and the reaction is carried out. The reaction temperature is 0°C to 37°C, more preferably 10°C to 25°C, and the reaction time is 0.5 hours to 2 hours. The reaction can be terminated by deactivating the reactivity of the unreacted compound (2) with a thiol-containing reagent. Examples of thiol-containing reagents are cysteine ​​or N-acetyl-L-cysteine ​​(NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of NAC to the compound (2) and incubating at room temperature for 10 to 30 minutes.

[0125] (4) Identification of antibody-drug conjugates The manufactured antibody-drug conjugate (1) can be identified by the following common procedures: concentration, buffer exchange, purification, measurement of antibody concentration and average number of drug conjugates per antibody molecule.

[0126] (4)-1 Common procedure A: Concentration of antibody or antibody-drug conjugate aqueous solution The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (50,000 MWCO, Millipore Corporation) container and concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000G to 3800G for 5 to 20 minutes.

[0127] (4)-2 Common procedure B: Measurement of antibody concentration Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's specified method. At that time, different 280nm extinction coefficients (1.3 mL mg) were used for each antibody. -1 cm -1 ~1.8mLmg -1 cm -1 ) was used.

[0128] (4)-3 Common procedure C: Antibody buffer exchange NAP-25 columns using Sephadex G-25 support (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) were equilibrated with phosphate buffer (50 mM, pH 6.0) containing sodium chloride (50 mM) and EDTA (2 mM) (hereinafter referred to as PBS6.0 / EDTA) according to the manufacturer's specified method. 2.5 mL of antibody aqueous solution was placed on each NAP-25 column, and the fraction (3.5 mL) obtained by eluting with 3.5 mL of PBS6.0 / EDTA was collected. This fraction was concentrated using common procedure A, and the antibody concentration was measured using common procedure B. After that, the antibody concentration was adjusted to 10 mg / mL using PBS6.0 / EDTA.

[0129] (4)-4 Common operation D: Purification of antibody-drug conjugates The NAP-25 column was equilibrated with any of the buffer solutions of acetate buffer (10 mM, pH 5.5; referred to as ABS in this specification) containing commercially available Sorbitol (5%). An aqueous solution of the antibody-drug conjugate reaction (about 2.5 mL) was placed on this NAP-25 column, and the antibody fraction was fractionated by eluting with the amount of buffer specified by the manufacturer. By repeating the gel filtration purification operation of placing this fractionated fraction on the NAP-25 column again and eluting with the buffer solution 2 to 3 times, an antibody-drug conjugate from which unbound drug linker and low molecular weight compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide) were removed was obtained.

[0130] (4)-5 Common Operation E: Measurement of Antibody Concentration and Average Number of Drugs Bound per Antibody Molecule in Antibody-Drug Conjugate (1) The concentration of the bound drug in the antibody-drug conjugate can be calculated by measuring the UV absorbances at two wavelengths of 280 nm and 370 nm of the aqueous solution of the antibody-drug conjugate and then performing the following calculations.

[0131] Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorption chemical species present in the system [additivity of absorbance], assuming that there is no change in the molar absorption coefficients of the antibody and the drug before and after the conjugation of the antibody and the drug, the antibody concentration and the drug concentration in the antibody-drug conjugate are represented by the following relational expressions. A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Equation (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Equation (2) Here, A 280 represents the absorbance of the aqueous solution of the antibody-drug conjugate at 280 nm 、 A370 This shows the absorbance of the antibody-drug conjugate aqueous solution at 370 nm, A A,280 This shows the absorbance of the antibody at 280 nm, A A,370 This shows the absorbance of the antibody at 370 nm, A D,280 This shows the absorbance of the conjugate precursor at 280 nm, A D,370 This shows the absorbance of the conjugate precursor at 370 nm, and ε A,280 This indicates the molar extinction coefficient of the antibody at 280 nm, and ε A,370 This shows the molar extinction coefficient of the antibody at 370 nm, and ε D,280 This shows the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D,370 This shows the molar extinction coefficient of the conjugate precursor at 370 nm, C A This indicates the antibody concentration in the antibody-drug conjugate, C D This indicates the drug concentration in the antibody-drug conjugate.

[0132] Here, ε A,280、 ε A,370、 ε D,280、 ε D,370 For this, pre-prepared values ​​(calculated estimates or measured values ​​obtained from UV measurements of the compound) are used. For example, ε A,280 This can be estimated from the amino acid sequence of the antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). ε A,370 ε is usually zero. D,280 and ε D,370 This can be obtained by measuring the absorbance of a solution in which the conjugate precursor to be used is dissolved at a certain molar concentration, according to the Lambert-Beer law (absorbance = molar concentration × molar extinction coefficient × cell path length). 280 and A 370 By measuring these values ​​and substituting them into equations (1) and (2) to solve the system of equations, we can find C A and C D It is possible to find C. D to C ABy dividing by this, the average number of drugs bound per antibody can be determined.

[0133] (4)-6 Common procedure F: Measurement of the average number of drug conjugates per antibody molecule in antibody-drug conjugates (2) The average number of drug links per antibody molecule in an antibody-drug conjugate can be determined not only by the "(4)-5 Common Operation E" described above, but also by high-performance liquid chromatography (HPLC) analysis using the following method. Below is a method for measuring the average number of drug links by HPLC when the antibody and drug linker are bonded by a disulfide bond. Those skilled in the art may refer to this method and appropriately measure the average number of drug links by HPLC depending on the bonding mode between the antibody and drug linker.

[0134] F-1. Preparation of samples for HPLC analysis (reduction of antibody-drug conjugates) Mix an antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). Incubate the mixture at 37°C for 30 minutes to cleave the disulfide bonds between the light and heavy chains of the antibody-drug conjugate. Use the resulting sample for HPLC analysis.

[0135] F-2.HPLC analysis HPLC analysis will be performed under the following measurement conditions.

[0136] HPLC System: Agilent 1290 HPLC System (Agilent Technologies) Detector: UV absorbance meter (measurement wavelength: 280 nm) Column: ACQUITY UPLC BEH Phenyl (2.1 × 50 mm, 1.7 μm, 130 Å; Waters, P / N 186002884) Column temperature: 80℃ Mobile phase A: Aqueous solution containing 0.10% trifluoroacetic acid (TFA) and 15% 2-propanol. Mobile phase B: Acetonitrile solution containing 0.075% TFA and 15% 2-propanol. Gradient program: 14%-36% (0 min-15 min), 36%-80% (15 min-17 min), 80%-14% (17 min-17.01 min), 14% (17.01 min-25 min) Sample injection volume: 10 μL F-3. Data Analysis F-3-1 Compared to the light chain (L0) and heavy chain (H0) of an antibody that is not bound to a drug, the light chain bound to a drug (light chain with i units of drug bound: L i ) and heavy chain (a heavy chain in which i drug molecules are linked: H i The hydrophobicity of the substance increases proportionally to the number of bound drugs, and the retention time increases accordingly. For example, the substances are eluted in the order of L0, L1, H0, H1, H2, and H3. By comparing the retention times of L0 and H0, the detection peak can be assigned to one of L0, L1, H0, H1, H2, or H3. The number of bound drugs can be defined by those skilled in the art, but is preferably L0, L1, H0, H1, H2, and H3.

[0137] F-3-2 Because drug linkers have UV absorption, the peak area value is corrected according to the following formula using the molar extinction coefficients of the light chain, heavy chain, and drug linker, depending on the number of drug linker bonds.

[0138]

number

[0139]

number

[0140] Here, the molar extinction coefficients (280 nm) of the light and heavy chains of each antibody can be estimated from the amino acid sequences of the light and heavy chains of each antibody using a known calculation method (Protein Science, 1995, vol.4, 2411-2423). In the case of H01L02, 31710 was used as the estimated molar extinction coefficient of the light chain and 79990 as the estimated molar extinction coefficient of the heavy chain, according to its amino acid sequence. Furthermore, the molar extinction coefficient (280 nm) of the drug linker was measured from compounds obtained by reacting each drug linker with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to succinimidothioether. The wavelength at which absorbance is measured can be appropriately set by those skilled in the art, but preferably it is a wavelength at which the antibody peak can be measured, and more preferably it is 280 nm.

[0141] F-3-3 The ratio (%) of each chain's peak area to the total peak area correction value is calculated according to the following formula.

[0142]

number

[0143] F-3-4 The average number of drug conjugates per antibody molecule in an antibody-drug conjugate is calculated according to the following formula.

[0144] Average number of drug bindings = (L0 peak area ratio x0 + L1 peak area ratio x1 + H0 peak area ratio x0 + H1 peak area ratio x1 + H2 peak area ratio x2 + H3 peak area ratio x3) / 100 x 2 To ensure a sufficient quantity of antibody-drug conjugates, multiple antibody-drug conjugates with similar average drug binding counts (e.g., ±1) obtained under similar conditions can be mixed to create a new lot. In this case, the average drug binding count will fall within the range of the average drug binding counts before mixing.

[0145] One specific example of the antibody-drug conjugate of the present invention is as follows:

[0146] [ka]

[0147] Or the following formula:

[0148] [ka]

[0149] Examples of structures having the form shown can be given.

[0150] Here, AB represents the anti-CD37 antibody disclosed herein, which is bound to the linker via a sulfhydryl group derived from the antibody. Here, n is synonymous with the so-called DAR (Drug-to-Antibody Ratio) and represents the drug-to-antibody ratio per antibody. That is, it represents the number of drug molecules bound to one antibody molecule, which is a value that is specified and expressed as an average value, i.e., the average number of drug bindings. In the case of the antibody-drug conjugates shown in Chemical Formulas 9 and 10 of the present invention, in the measurement by common operation F, n may be from 2 to 8, preferably from 5 to 8, more preferably from 7 to 8, and even more preferably 8.

[0151] An example of the antibody-drug conjugate of the present invention is an antibody-drug conjugate or a pharmacologically acceptable salt thereof, wherein the antibody represented by AB in the structure shown in formula 9 or formula 10 above contains heavy-chain and light-chain antibodies or functional fragments thereof selected from any one of the group consisting of (a) to (e) below: (a) An antibody comprising a light chain consisting of amino acid sequences 21-234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acid sequences 20-468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 4; (b) An antibody comprising a light chain consisting of amino acid sequences 21-234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acid sequences 20-468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 6; (c) An antibody comprising a light chain consisting of amino acid sequences 21-234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acid sequences 20-468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; (d) An antibody comprising a light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 10; or (e) An antibody according to any one of the group (a) to (d), wherein the heavy chain or light chain includes one or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, amino-terminus processing, carboxyl-terminus processing, deamidation, aspartic acid isomerization, methionine oxidation, tryptophan oxidation, addition of a methionine residue to the amino terminus, amidation of a proline residue, pyroglutamine oxidation of amino-terminus glutamine or amino-terminus glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

[0152] 4. Pharmaceuticals The anti-CD37 antibody and functional fragment of the present invention described in section 2, "Production of Anti-CD37 Antibody," and the examples above, bind to CD37 on the surface of tumor cells and have internalization activity. Therefore, they can be used as pharmaceuticals to treat B-cell non-Hodgkin lymphoma (NHL), such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), limbic body lymphoma (MZL), Burkitt lymphoma (BL), or chronic lymphocytic leukemia (CLL), as well as T-cell lymphomas (TCL) such as peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL), and further as therapeutic agents for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

[0153] Furthermore, it can be used to detect cells that express CD37.

[0154] Furthermore, since the anti-CD37 antibody and the functional fragment of the antibody of the present invention possess internalization activity, they can be used as antibodies for antibody-drug conjugates.

[0155] Among the anti-CD37 antibody-drug conjugates of the present invention described in section 3. Anti-CD37 antibody-drug conjugates and examples above, those in which a drug having antitumor activity such as cytotoxic activity is used as the drug are conjugates of an anti-CD37 antibody having internalization activity and / or a functional fragment of said antibody and a drug having antitumor activity such as cytotoxic activity. Since they exhibit antitumor activity against cancer cells expressing CD37, they can be used as pharmaceuticals, particularly as therapeutic agents and / or prophylactic agents for cancer.

[0156] The anti-CD37 antibody-drug conjugate of the present invention may become hydrated by absorbing moisture or adsorbing water upon exposure to air or by recrystallization or purification. Compounds containing such water or pharmaceutically acceptable salts are also included in the present invention.

[0157] If the anti-CD37 antibody-drug conjugate of the present invention has a basic group such as an amino group, it can optionally form a pharmacodynamically acceptable acid addition salt. Examples of such acid addition salts include hydrohalides such as hydrofluoric acid, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfonates such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates; allylsulfonates such as benzenesulfonates and p-toluenesulfonates; organic acid salts such as formate, acetate, trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; or amino acid salts such as ornithine, glutamate, and aspartate.

[0158] When the anti-CD37 antibody-drug conjugate of the present invention has an acidic group such as a carboxy group, a pharmacologically acceptable base addition salt can be formed if desired. Examples of such base addition salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt, and the like.

[0159] The present invention may also include an anti-CD37 antibody-drug conjugate in which one or more of the atoms constituting the antibody-drug conjugate are substituted with isotopes of those atoms. There are two types of isotopes: radioactive isotopes and stable isotopes. Examples of isotopes include, for example, isotopes of hydrogen (2H and 3H), isotopes of carbon (11C, 13C, and 14C), isotopes of nitrogen (13N and 15N), isotopes of oxygen (15O, 17O, and 18O), isotopes of fluorine (18F), and the like. Compositions containing an antibody-drug conjugate labeled with an isotope are useful, for example, as therapeutic agents, prophylactic agents, research reagents, assay reagents, diagnostic agents, in vivo imaging diagnostic agents, and the like. Antibody-drug conjugates labeled with an isotope, and mixtures of antibody-drug conjugates labeled with an isotope in any proportion are also all included in the present invention. An antibody-drug conjugate labeled with an isotope can be produced by a method known in the art, for example, by using an isotope-labeled raw material instead of the raw material in the production method of the present invention described later.

[0160] The cytotoxic activity in vitro can be measured, for example, by the cell growth inhibitory activity. For example, a cancer cell line overexpressing CD37 is cultured, and an anti-CD37 antibody-drug conjugate is added to the culture system at various concentrations, and the inhibitory activities against focus activity, colony formation, and spheroid growth can be measured. Here, for example, by using cancer cell lines derived from diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), etc., the cell growth inhibitory activity against diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), etc. can be examined.

[0161] The therapeutic effect on cancer using experimental animals in vivo can be measured, for example, by administering an anti-CD37 antibody-drug conjugate to SCID mice transplanted with a tumor cell line highly expressing CD37 and measuring the changes in cancer cells. Here, for example, by using an animal model in which cells derived from B-cell non-Hodgkin lymphoma (NHL) such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma (BL), or chronic lymphocytic leukemia (CLL), T-cell lymphoma (TCL) such as peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), or myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) are transplanted into immunodeficient mice, the therapeutic effect against diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma (BL), chronic lymphocytic leukemia (CLL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML) can be measured.

[0162] The types of cancer to which the anti-CD37 antibody-drug conjugate of the present invention can be applied are not particularly limited as long as the cancer cells to be treated express CD37. Examples include diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, limbic lymphoma, Burkitt lymphoma, or cells derived from chronic lymphocytic leukemia, but are not limited to these as long as they express CD37. More preferred examples of cancer types to which the anti-CD37 antibody-drug conjugate of the present invention can be applied include diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia.

[0163] The anti-CD37 antibody-drug conjugate of the present invention can be suitably administered to mammals, but more preferably to humans.

[0164] In the pharmaceutical composition containing the anti-CD37 antibody-drug conjugate of the present invention, the substance used can be appropriately selected from pharmaceutical additives and others commonly used in this field, in terms of dosage and concentration.

[0165] The anti-CD37 antibody-drug conjugate of the present invention may be administered as a pharmaceutical composition comprising one or more pharmaceutically compatible components. For example, the pharmaceutical composition typically comprises one or more pharmaceutical carriers (e.g., sterile liquids (e.g., water and oil (oils of petroleum, animal, plant, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)))). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and aqueous glycerol solutions, may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition may also optionally contain trace amounts of wetting or emulsifying agents or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Their formulations correspond to the mode of administration.

[0166] Various delivery systems are known and can be used to administer the anti-CD37 antibody-drug conjugate of the present invention. Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration may be, for example, by infusion or bolus injection. In certain preferred embodiments, the antibody-drug conjugate is administered by infusion. Parenteral administration is a preferred route of administration.

[0167] In typical embodiments, the pharmaceutical composition is formulated according to a standard procedure as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the pharmacopoeia may also contain a solubilizer and a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the components are supplied either separately (e.g., as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of the activator) or mixed together in a unit dosage form. If the pharmacopoeia is to be administered by infusion, it may be administered in an infusion bottle containing, for example, sterile pharmaceutical-grade water or saline. If the pharmacopoeia is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the components can be mixed before administration. The saline may be, for example, physiological saline.

[0168] Such pharmaceutical compositions may be formulated as lyophilized or liquid formulations having the selected composition and required purity. When formulated as a lyophilized formulation, it may contain appropriate pharmaceutical additives used in this field. Similarly, liquid formulations can also be formulated as liquid formulations containing various pharmaceutical additives used in this field.

[0169] The composition and concentration of the pharmaceutical composition vary depending on the administration method, but the anti-CD37 antibody-drug conjugate contained in the pharmaceutical composition of the present invention exhibits therapeutic effects even at small doses if its affinity for the antigen is high (low Kd value), i.e., the dissociation constant (Kd value) for the antigen is high. Therefore, when determining the dosage of the antibody-drug conjugate, the dosage can also be set based on the affinity between the antibody-drug conjugate and the antigen. When administering the antibody-drug conjugate of the present invention to humans, for example, approximately 0.001 to 100 mg / kg may be administered once or multiple times at intervals of 1 to 180 days. Preferably, 0.1 to 50 mg / kg, and more preferably, 0.1 to 30 mg / kg may be administered multiple times at intervals of 1 to 4 weeks, preferably once every 2 to 3 weeks. [Examples]

[0170] [Example 1: Preparation of humanized anti-CD37 antibody] 1)-1 Design of humanized anti-CD37 antibodies 1)-1-1 Molecular modeling of the variable region of anti-CD37 antibodies A known homology modeling method (Methods in Enzymology, 203, 121-153 (1991)) was used. Using the commercially available protein structure analysis program DiscoveryStudio (Dassault Systèmes), structures registered in the Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) with high sequence homology to the variable region were searched. Three-dimensional model structures were created using the hit heavy chain, light chain, and heavy-chain-light chain interface structures as templates.

[0171] 1)-1-2 Design Method for Humanization Humanized antibodies of the anti-CD37 mouse monoclonal antibody HH1 (Smeland E, et al., Scand J Immunol, 21(3), 205-214 (1985)) were constructed using the commonly known method of CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The framework regions of the anti-CD37 human chimeric antibody exhibit high homology to the consensus sequences of human κ-chain subgroup 1 and human γ-chain subgroup 1 as defined by KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)). Therefore, these were selected as the light chain and heavy chain acceptors, respectively, of the anti-CD37 human chimeric antibody. Furthermore, IGHV1-2*02 and IGHJ6*01 of the human γ chain, as defined in IMGT (registered trademark) (THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM), were selected as heavy chain acceptors to improve the physical properties of the anti-CD37 humanized antibody-drug conjugate. The donor residues to be transferred onto the acceptors were uniquely designed for each sequence by analyzing a three-dimensional model, referencing criteria such as those provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)).

[0172] 1)-1-3 Humanization of the light chain of an anti-CD37 human chimeric antibody A humanized antibody light chain was designed by ligating the constant region of the κ chain of human IgG1 to the variable region of the designed anti-CD37 humanized antibody light chain, and named hmAb-L11. The full amino acid sequence of hmAb-L11 is described in SEQ ID NO: 2. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 is described in SEQ ID NO: 1.

[0173] 1)-1-4 Humanized heavy chain of anti-CD37 human chimeric antibody hmAb-H11 A humanized antibody heavy chain was designed by grafting the human γ-chain constant region of human IgG1 onto the consensus sequence of human γ-chain subgroup 1, which has the highest homology to the anti-CD37 human chimeric antibody, and affixing it to the variable region of the designed anti-CD37 humanized antibody heavy chain. This humanized antibody heavy chain was named hmAb-H11. The full-length amino acid sequence of hmAb-H11 is described in SEQ ID NO: 4. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 is described in SEQ ID NO: 3.

[0174] 1)-1-5 Humanization of anti-CD37 human chimeric antibody heavy chains: hmAb-H541, hmAb-H551, hmAb-H11a To improve the physical properties of anti-CD37 humanized antibody-drug conjugates, humanized antibody heavy chains were designed by ligating the constant region of the gamma chain of human IgG1 to the variable region of the anti-CD37 humanized antibody heavy chain. These humanized antibody heavy chains were named hmAb-H541, hmAb-H551, and hmAb-H11a, respectively. The full-length amino acid sequence of hmAb-H541 is described in SEQ ID NO: 6. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 is described in SEQ ID NO: 5. The full-length amino acid sequence of hmAb-H551 is described in SEQ ID NO: 8. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8 is described in SEQ ID NO: 7. The full-length amino acid sequence of hmAb-H11a is described in SEQ ID NO: 10. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10 is described in SEQ ID NO: 9.

[0175] 1)-2 Construction of an expression vector for humanized anti-CD37 antibodies and preparation of the antibodies. 1)-2-1 Construction of the light chain expression vector pCMA-LK pcDNA3.3 / LK was synthesized by ligating a fragment of approximately 5.4 kb obtained by digesting plasmid pcDNA3.3-TOPO / LacZ (Thermo Fisher Scientific) with restriction enzymes XbaI and PmeI, and a DNA fragment containing a nucleotide sequence encoding the human light chain signal sequence and human κ chain constant region shown in SEQ ID NO: 11, using an In-Fusion HD PCR cloning kit (Takra Bio USA).

[0176] pCMA-LK was constructed by removing the neomycin resistance gene from pcDNA3.3 / LK.

[0177] 1)-2-1-1 Construction of hmAb-L11 expression vector A DNA fragment of the nucleotide sequence of the hmAb-L11 variable region shown in SEQ ID NO: 12 was synthesized (manufactured by Thermo Fisher Scientific). Using the In-Fusion HD PCR Cloning Kit, a hmAb-L11 expression vector was constructed by inserting the synthesized DNA fragment into the site of pCMA-LK constructed in Example 1)-2-1 that had been cleaved with the restriction enzyme BsiWI.

[0178] 1)-2-2 Construction of heavy chain expression vector pCMA-G1 A DNA fragment containing the nucleotide sequence encoding the heavy chain signal sequence shown in SEQ ID NO: 13 and the human heavy chain G1 constant region was synthesized (manufactured by Eurofins Genomics). After cleaving this DNA fragment with the restriction enzymes XbaI and PmeI, a 1.1 kb DNA fragment was excised by agarose gel electrophoresis and purified using the Wizard SV Gel and PCR Clean-Up System (manufactured by Promega). The approximately 3.4 kb fragment obtained by digesting pCMA-LK constructed in Example 1)-2-1 with the restriction enzymes XbaI and PmeI and the 1.1 kb DNA fragment were ligated using Ligation High (manufactured by Toyobo) to construct pCMA-G1.

[0179] 1)-2-2-1 Construction of hmAb-H11 expression vector A DNA fragment of the nucleotide sequence of hmAb-H11 shown in SEQ ID NO: 14 was synthesized. Using the In-Fusion HD PCR Cloning Kit, a hmAb-H11 expression vector was constructed by inserting the synthesized DNA fragment into the site of pCMA-G1 constructed in Example 1)-2-2 that had been cleaved with the restriction enzyme BlpI.

[0180] 1)-2-2-2 Construction of hmAb-H541 expression vector A DNA fragment of the nucleotide sequence of hmAb-H541 shown in SEQ ID NO: 15 was synthesized. An hmAb-H541 expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G1 constructed in Example 1)-2-2 was cleaved with the restriction enzyme BlpI using an In-Fusion HD PCR cloning kit.

[0181] 1)-2-2-3 Construction of the hmAb-H551 expression vector A DNA fragment of the nucleotide sequence of hmAb-H551 shown in SEQ ID NO: 16 was synthesized. An hmAb-H551 expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G1 constructed in Example 1)-2-2 was cleaved with the restriction enzyme BlpI using an In-Fusion HD PCR cloning kit.

[0182] 1-2-2-4 Construction of an hmAb-H11a expression vector A DNA fragment of the nucleotide sequence of hmAb-H11a shown in Sequence ID No. 17 was synthesized. An hmAb-H11a expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G1, constructed in Example 1)-2-2, was cleaved with the restriction enzyme BlpI, using an In-Fusion HD PCR cloning kit.

[0183] 1)-2-2-5 Combination of heavy chain expression vector and light chain expression vector for anti-CD37 humanized antibody The anti-CD37 humanized antibody with hmAb-H11 as the heavy chain and hmAb-L11 as the light chain was named hmAb-H11L11. The anti-CD37 humanized antibody with hmAb-H541 as the heavy chain and hmAb-L11 as the light chain was named hmAb-H541L11. The anti-CD37 humanized antibody with hmAb-H551 as the heavy chain and hmAb-L11 as the light chain was named hmAb-H551L11. The anti-CD37 humanized antibody with hmAb-H11a as the heavy chain and hmAb-L11 as the light chain was named hmAb-H11aL11.

[0184] 1)-2-3 Production of humanized anti-CD37 antibodies FreeStyle 293F cells (Thermo Fisher Scientific) were subcultured and cultured according to the manual. Logarithmic growth phase FreeStyle 293F cells were diluted with FreeStyle293 expression medium (Thermo Fisher Scientific) and cultured to 2.0 × 10⁶ 6 The cells were adjusted to the required concentration per mL and seeded in 600 mL of 3 L Fernbach Erlenmeyer Flask (CORNING). 1.8 mg of Polyethyleneimine (Polysciences) was added to 20 mL of Opti-Pro SFM medium (Thermo Fisher Scientific). Next, 300 μg of heavy chain expression vector and 300 μg of light chain expression vector were added to 20 mL of Opti-Pro SFM medium. The expression vector / Opti-Pro SFM mixture was added to the Polyethyleneimine / Opti-Pro SFM mixture, gently mixed, and allowed to stand for 5 minutes before being added to FreeStyle 293F cells. After incubation at 37°C in an 8% CO2 incubator for 4 hours with shaking at 95 rpm, 600 mL of EX-CELL VPRO medium (SAFC Biosciences) and 30 mL of 43.4 g / L BD Recharge CD (BD Biosciences) were added, and the culture supernatant obtained after incubation at 37°C in an 8% CO2 incubator for 6 days with shaking at 95 rpm was filtered through a bottle-top filter with a pore size of 0.2 μm (Thermo Fisher Scientific).

[0185] 1)-2-4 Purification of humanized anti-CD37 antibody Antibodies were purified from the culture supernatant obtained in Example 1)-2-3 using a two-step process of rProtein A affinity chromatography and ceramic hydroxyapatite. The culture supernatant was equilibrated with PBS and applied to a column packed with MabSelectSuRe (Cytiva), after which the column was washed with more than twice the column volume of PBS. Next, the antibodies were eluted with 2M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was buffer-replaced with PBS by dialysis using a Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific), diluted 5-fold with a 5mM sodium phosphate / 50mM MES / pH 7.0 buffer, and then applied to a ceramic hydroxyapatite column (Bio-Rad Laboratories) equilibrated with a 5mM NaPi / 50mM MES / 30mM NaCl / pH 7.0 buffer. Linear concentration gradient elution with sodium chloride was performed, and the antibody-containing fraction was collected. This fraction was buffer-replaced with HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis using a Dialysis Cassette. The antibody was concentrated using VIVASPIN 20 (molecular weight cutoff UF10K, Sartorius Stedim Biotech) to adjust the IgG concentration to 20-25 mg / mL. Finally, the sample was filtered using Minisart Plus (Sartorius Stedim Biotech) to obtain a purified sample.

[0186] [Example 2: Preparation of anti-CD37 antibody-drug conjugate 1] 2)-1 Preparation of antibody-drug conjugates (1) hmAb-H11L11-DXd hmAb-H11L11-DXd was synthesized by the following procedure.

[0187] [ka]

[0188] Antibody reduction: The hmAb-H11L11 prepared in Example 1)-2 was reduced to 1.50 mL mg of the common operation B described in Manufacturing Method 1 (as an extinction coefficient of 280 nm). -1 cm -1 Using (and C), the antibody was prepared to a concentration of 10.67 mg / mL in PBS 6.0 / EDTA. To this solution (0.5 mL), 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0075 mL) and 10 mM TCEP (Tokyo Chemical Industries, Ltd.) aqueous solution (0.022 mL; 6.0 equivalents per antibody molecule) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the disulfide bonds in the interchain region of the antibody.

[0189] Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Then, a 10 mM dimethyl sulfoxide solution (0.0367 mL; 10.0 equivalents per molecule of antibody) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added and incubated at 15°C for 1 hour to conjugate the drug linker to the antibody. Next, 0.0037 mL of 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution was added and stirred. The mixture was then allowed to stand at room temperature for 20 minutes to stop the reaction of the drug linker.

[0190] Purification: The above solution was purified using common procedure D described in Manufacturing Method 1 to obtain 3.5 mL of a solution containing the antibody-drug conjugate "hmAb-H11L11-ADC".

[0191] Characterization: Common operations E and F(ε) described in Manufacturing Method 1 D,280=5440, ε D,370 Using (=21800), the following characteristic values ​​were obtained. Antibody concentration: 1.30 mg / mL, antibody yield: 4.57 mg (86%), average number of drug-bound antibodies per molecule measured by common procedure E (n): 5.6; average number of drug-bound antibodies per molecule measured by common procedure F (n): 7.5.

[0192] 2)-2 Preparation of antibody-drug conjugates (2) hmAb-H11L11-DXd hmAb-H11L11-DXd was synthesized by the following procedure.

[0193] [ka]

[0194] Antibody reduction: The hmAb-H11L11 prepared in Example 1)-2 was reduced to 1.50 mL mg of the common operation B described in Manufacturing Method 1 (as an extinction coefficient of 280 nm). -1 cm -1 Using (and C), the antibody was prepared to a concentration of 10.67 mg / mL in PBS 6.0 / EDTA. To this solution (0.5 mL), 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0075 mL) and 10 mM TCEP (Tokyo Chemical Industries, Ltd.) aqueous solution (0.0294 mL; 8.0 equivalents per antibody molecule) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the disulfide bonds in the interchain region of the antibody.

[0195] Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Then, a 10 mM dimethyl sulfoxide solution (0.0441 mL; 12.0 equivalents per molecule of antibody) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added and incubated at 15°C for 1 hour to conjugate the drug linker to the antibody. Next, 0.0044 mL of 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution was added and stirred. The mixture was then left to stand at room temperature for 20 minutes to stop the reaction of the drug linker.

[0196] Purification: The above solution was purified using common procedure D described in Manufacturing Method 1 to obtain 3.5 mL of a solution containing the antibody-drug conjugate "hmAb-H11L11-ADC".

[0197] Characterization: Common operations E and F(ε) described in Manufacturing Method 1 D,280 =5440, ε D,370 Using (=21800), the following characteristic values ​​were obtained. Antibody concentration: 1.34 mg / mL, antibody yield: 4.69 mg (88%), average number of drug-bound antibodies per molecule measured by common procedure E (n): 5.9; average number of drug-bound antibodies per molecule measured by common procedure F (n): 7.7.

[0198] 2)-3 Preparation of antibody-drug conjugates (3) hmAb-H11L11-DXd hmAb-H11L11-DXd was synthesized by the following procedure.

[0199] [ka]

[0200] Antibody reduction: The hmAb-H11L11 prepared in Example 1)-2 was reduced to 1.50 mL mg of the common operation B described in Manufacturing Method 1 (as an extinction coefficient of 280 nm). -1 cm -1 Using (and C), the antibody was prepared to a concentration of 10.67 mg / mL in PBS 6.0 / EDTA. To this solution (8.3 mL), 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.124 mL) and 10 mM TCEP (Tokyo Chemical Industries, Ltd.) aqueous solution (0.486 mL; 8.0 equivalents per antibody molecule) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the disulfide bonds in the interchain region of the antibody.

[0201] Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Then, a 10 mM dimethyl sulfoxide solution (0.728 mL; 12.0 equivalents per molecule of antibody) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added and incubated at 15°C for 1 hour to conjugate the drug linker to the antibody. Next, 0.073 mL of 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution was added and stirred. The mixture was then allowed to stand at room temperature for 20 minutes to stop the reaction of the drug linker.

[0202] Purification: The above solution was purified using common operation D described in Manufacturing Method 1 to obtain 31.5 mL of a solution containing the antibody-drug conjugate "hmAb-H11L11-ADC".

[0203] Characterization: Common operations E and F(ε) described in Manufacturing Method 1 D,280 =5440, ε D,370 Using (=21800), the following characteristic values ​​were obtained. Antibody concentration: 2.23 mg / mL, antibody yield: 70.29 mg (80%), average number of drug-bound antibodies per molecule measured by common procedure E (n): 5.6; average number of drug-bound antibodies per molecule measured by common procedure F (n): 7.4.

[0204] 2)-4 Preparation of antibody-drug conjugates (4) hmAb-H541L11-DXd hmAb-H541L11-DXd was synthesized by the following process.

[0205] [ka]

[0206] Antibody reduction: The hmAb-H541L11 prepared in Example 1)-2 was reduced to 1.50 mL mg of the common operation B described in Manufacturing Method 1 (as an extinction coefficient of 280 nm). -1 cm -1 Using (and C), the antibody was prepared to a concentration of 10.63 mg / mL in PBS 6.0 / EDTA. To this solution (8.9 mL), 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.133 mL) and 10 mM TCEP (Tokyo Chemical Industries, Ltd.) aqueous solution (0.389 mL; 6.0 equivalents per antibody molecule) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the disulfide bonds in the interchain region of the antibody.

[0207] Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Then, a 10 mM dimethyl sulfoxide solution (0.649 mL; 10.0 equivalents per molecule of antibody) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added and incubated at 15°C for 1 hour to conjugate the drug linker to the antibody. Next, 0.065 mL of 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution was added and stirred. The mixture was then allowed to stand at room temperature for 20 minutes to stop the reaction of the drug linker.

[0208] Purification: The above solution was purified using common operation D described in Manufacturing Method 1 to obtain 31.5 mL of a solution containing the antibody-drug conjugate "hmAb-H541L11-ADC".

[0209] Characterization: Common operations E and F(ε) described in Manufacturing Method 1 D,280 =5440, ε D,370 Using (=21800), the following characteristic values ​​were obtained. Antibody concentration: 2.68 mg / mL, antibody yield: 84.26 mg (89%), average number of drug-bound antibodies per molecule measured by common procedure E (n): 5.9; average number of drug-bound antibodies per molecule measured by common procedure F (n): 7.8.

[0210] 2)-5 Preparation of antibody-drug conjugates (5) hmAb-H551L11-DXd hmAb-H551L11-DXd was synthesized by the following process.

[0211] [ka]

[0212] Antibody reduction: The hmAb-H551L11 prepared in Example 1)-2 was reduced to 1.50 mL mg of the common operation B described in Manufacturing Method 1 (as an extinction coefficient of 280 nm). -1 cm -1 Using (and C), the antibody was prepared to a concentration of 10.62 mg / mL in PBS 6.0 / EDTA. To this solution (9.4 mL), 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.141 mL) and 10 mM TCEP (Tokyo Chemical Industries, Ltd.) aqueous solution (0.411 mL; 6.0 equivalents per antibody molecule) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the disulfide bonds in the interchain region of the antibody.

[0213] Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Then, a 10 mM dimethyl sulfoxide solution (0.686 mL; 10.0 equivalents per molecule of antibody) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added and incubated at 15°C for 1 hour to conjugate the drug linker to the antibody. Next, 0.069 mL of 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution was added and stirred. The mixture was then allowed to stand at room temperature for 20 minutes to stop the reaction of the drug linker.

[0214] Purification: The above solution was purified using common operation D described in Manufacturing Method 1 to obtain 35.0 mL of a solution containing the antibody-drug conjugate "hmAb-H551L11-ADC".

[0215] Characterization: Common operations E and F(ε) described in Manufacturing Method 1 D,280 =5440, ε D,370 Using (=21800), the following characteristic values ​​were obtained. Antibody concentration: 2.43 mg / mL, antibody yield: 85.08 mg (85%), average number of drug-bound antibodies per molecule measured by common procedure E (n): 5.7; average number of drug-bound antibodies per molecule measured by common procedure F (n): 7.8.

[0216] 2)-6 Preparation of antibody-drug conjugates (6) hmAb-H11aL11-DXd hmAb-H11aL11-DXd was synthesized by the following procedure.

[0217] [ka]

[0218] Antibody reduction: The hmAb-H11aL11 prepared in Example 1)-2 was reduced to 1.50 mL mg of the common operation B described in Manufacturing Method 1 (as an extinction coefficient of 280 nm). -1 cm -1 Using (and C), the antibody was prepared to a concentration of 10.59 mg / mL in PBS 6.0 / EDTA. To this solution (10.0 mL), 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.150 mL) and 10 mM TCEP (Tokyo Chemical Industries, Ltd.) aqueous solution (0.510 mL; 7.0 equivalents per antibody molecule) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the disulfide bonds in the interchain region of the antibody.

[0219] Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Then, a 10 mM dimethyl sulfoxide solution (0.801 mL; 11.0 equivalents per molecule of antibody) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added and incubated at 15°C for 1 hour to conjugate the drug linker to the antibody. Next, 0.080 mL of 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution was added and stirred. The mixture was then left to stand at room temperature for 20 minutes to stop the reaction of the drug linker.

[0220] Purification: The above solution was purified using common operation D described in Manufacturing Method 1 to obtain 35.0 mL of a solution containing the antibody-drug conjugate "hmAb-H11aL11-ADC".

[0221] Characterization: Common operations E and F(ε) described in Manufacturing Method 1 D,280 =5440, ε D,370 Using (=21800), the following characteristic values ​​were obtained. Antibody concentration: 2.62 mg / mL, antibody yield: 91.57 mg (86%), average number of drug-bound antibodies per molecule measured by common procedure E (n): 5.7; average number of drug-bound antibodies per molecule measured by common procedure F (n): 7.6.

[0222] In the preparation of hmAb-H11L11-DXd, using 6.0 equivalents of 10mM TCEP aqueous solution per antibody molecule at a 5mg scale, the average drug binding count was 7.5. When the amount of TCEP aqueous solution was increased to 8.0 equivalents per antibody molecule, the average drug binding count improved to 7.7. However, in the preparation at a 100mg scale, even using 8.0 equivalents of 10mM TCEP aqueous solution per antibody molecule, the average drug binding count was only 7.4. It is presumed that a larger amount of 10mM TCEP aqueous solution is needed to increase the average drug binding count of hmAb-H11L11-DXd when preparing at a 100mg scale. In contrast, when other hmAb-H541L11-DXd, hmAb-H551L11-DXd, and hmAb-H11aL11-DXd were prepared on a 100 mg scale, the average number of drug-bound molecules reached 7.6-7.8 at 6.0-7.0 equivalents per antibody molecule in a 10 mM TCEP aqueous solution.

[0223] [Example 3: Evaluation of the recovery rate of anti-CD37 humanized antibody-drug conjugate in physiological saline] Anti-CD37 humanized antibody-drug conjugates dissolved in ABSor (Nacalai Tesque) at 20 mg / mL were diluted to 2 mg / mL with Otsuka physiological saline (Otsuka Pharmaceutical Factory Co., Ltd.) and allowed to stand at room temperature or 4°C for 5 hours. 10 μL of the supernatant was injected into a YMC-Pack Diol-300 SEC, 30 nm, S-2 μm, 300 × 4.6 mm (YMC Corporation) using Prominence (Shimadzu Corporation), and analyzed by size exclusion chromatography using 3 × PBS and 8% isopropanol (a solution prepared by dissolving 3 PBS tablets (Takara Bio Inc.) in 920 mL of ultrapure water and adding 80 mL of isopropanol) as the mobile phase. The recovery rate of the anti-CD37 humanized antibody-drug conjugate was calculated using the following formula. Recovery rate (%) = (Recovery rate of samples left standing at 4°C / Recovery rate of samples left standing at room temperature) × 100 As shown in Table 1, the antibody-drug conjugate containing the antibody whose amino acid sequence was designed in Example 1)-1-5 for the purpose of improving the physical properties of the anti-CD37 humanized antibody-drug conjugate showed improved recovery rate at 4°C in physiological saline compared to the antibody-drug conjugate containing the antibody whose amino acid sequence was designed in Example 1)-1-4. The improved recovery rate at 4°C in physiological saline indicates that the anti-CD37 humanized antibody-drug conjugate can be handled in physiological saline. On the other hand, hmAb-H11L11-DXd was shown to be difficult to handle in physiological saline. In this case, the use of glucose solution may be considered, but this solution is known to cause glycation of antibodies (MAbs, v.9(4), 586-594, (2017)), which may lead to a decrease in the efficacy of the antibody-drug conjugate. In addition, protein aggregation has been reported when mixed with glucose solution for some antibody drugs, so it is desirable to have a choice of diluents. Furthermore, when using glucose solution, careful administration is necessary in patients with diabetes, diabetes insipidus, or renal failure due to the risk of electrolyte loss. hmAb-H11L11 was selected as a humanized antibody because it was humanized using the human consensus sequence with the highest homology to the anti-CD37 mouse monoclonal antibody HH1 as the acceptor, and maintained antigen-binding activity. However, for the reasons mentioned above, modification was deemed necessary, and the amino acid sequence shown in Example 1)-1-5 was newly designed.

[0224] [Table 1]

[0225] [Example 4: In vitro activity evaluation of antibody-drug conjugates] 4)-1 Evaluation of binding affinity of humanized anti-CD37 antibody-drug conjugates The binding affinity of the four antibody-drug conjugates (clone names: hmAb-H11L11-DXd, hmAb-H541L11-DXd, hmAb-H551L11-DXd, and hmAb-H11aL11-DXd) prepared in Example 2 was evaluated by flow cytometry. CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7 (DSMZ), cultured in IMDM medium containing 20% ​​FBS at 37°C and 5% CO2, was collected and centrifuged. After removing the supernatant, the four antibody-drug conjugates at each concentration, or an antibody-drug conjugate prepared using human IgG as a negative control (hmAb-IgG1-DXd), was added and the mixture was suspended and allowed to stand at 4°C for 1 hour. After washing twice with PBS containing 5% FBS, the cells were suspended in 100-fold diluted Fluorescein (FITC)-AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson Immuno Research) in PBS containing 5% FBS and allowed to stand at 4°C for 30 minutes. After washing twice with PBS containing 5% FBS, detection was performed using a flow cytometer (BD LSRFortessa™ X-20, BD Bioscience). Data analysis was performed using FlowJo (TreeStar). The results are shown in Figure 6. In Figure 6, the horizontal axis represents antibody concentration (μg / ml), and the vertical axis represents the amount of antibody bound by MFI (mean fluorescence intensity). As shown in Figure 6, humanized anti-CD37 antibody and antibody-drug conjugate showed a concentration-dependent increase in binding in the CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7.

[0226] 4)-2 Evaluation of the cell proliferation inhibitory activity of humanized anti-CD37 antibody-drug conjugates CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7 (DSMZ) was placed in IMDM medium containing 20% ​​FBS in 5x10 cells. 2Cells were seeded into 96-well plates at a concentration of 100 μL / well. Four antibody-drug conjugates prepared in Example 2 (clone names: hmAb-H11L11-DXd, hmAb-H541L11-DXd, hmAb-H551L11-DXd, and hmAb-H11aL11-DXd) were added to a final concentration ranging from 0.0064 nM to 20 nM. After culturing for 6 days at 37°C and 5% CO2, the number of viable cells was measured using CellTiter-Glo. TM Cell viability was quantified by measuring ATP using the Luminescent Cell Viability Assay (Promega), with the vehicle group set to 100%. Figure 7 shows the concentration-dependent cell proliferation inhibitory activity when each antibody-drug conjugate was added. The hmAb-IgG-DXd used in the experiment was an antibody-drug conjugate prepared from human IgG1 that recognizes an antigen unrelated to CD37, and was used as a negative control.

[0227] [Example 5: In vivo antitumor effect of antibody-drug conjugate 1] The antitumor effects of antibody-drug conjugates were evaluated using an animal model in which cells from a CD37-positive human tumor cell line were transplanted into immunodeficient mice. Five-week-old SCID mice (CB17 / Icr-Prkdc[scid] / CrlCrlj, Charles River, Japan) were acclimatized for at least three days under SPF conditions before use in the experiment. The mice were fed sterile solid feed (FR-2, Funabashi Farms Co., Ltd.) and sterile tap water (prepared by adding 5-15 ppm sodium hypochlorite solution). The long and short diameters of the transplanted tumors were measured twice a week using an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and the tumor volume was calculated using the formula shown below. Tumor volume (mm 3 ) = 1 / 2 × major axis (mm) × minor axis (mm) 2 All antibody-drug conjugates were diluted with ABS buffer (10 mM Acetate Buffer, 5% Sorbitol, pH 5.5) (NACALAI) and administered intravenously at the doses shown in each example. ABS buffer was also administered to the control group (Vehicle group) in the same manner. Six mice were used in each group for the experiment.

[0228] 5)-1 Antitumor effect (1) CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7 (DSMZ) was suspended in 50% Matrigel (Corning, diluted with physiological saline) and 1 × 10⁶ cells were prepared. 7 Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and random group assignment was performed on Day 9. On the day of group assignment, four antibody-drug conjugates prepared in Example 2 (clone names: hmAb-H11L11-DXd, hmAb-H541L11-DXd, hmAb-H551L11-DXd, and hmAb-H11aL11-DXd) were administered intravenously at doses of 1 mg / kg and 3 mg / kg. As a negative control, an antibody-drug conjugate prepared using human IgG (hmAb-IgG1-DXd) was administered similarly at a dose of 3 mg / kg. The results are shown in Figure 8. The horizontal axis represents days, the vertical axis represents tumor volume, and the error range is the SE value.

[0229] All four antibody-drug conjugates prepared in Example 2 showed a significant dose-dependent reduction in tumor volume, and tumor growth was completely suppressed at a dose of 3 mg / kg.

[0230] 5)-2 Antitumor effect (2) Similar to Example 5)-1, CD37-positive human diffuse large B-cell lymphoma cell line WSU-DLCL2(DSMZ) was subjected to 1 × 10⁻¹⁴ treatment. 7 Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and random group assignment was performed on Day 11. On the day of group assignment, four antibody-drug conjugates prepared in Example 2 and hmAb-IgG1-DXd (negative control) were administered intravenously at doses of 1 mg / kg and 3 mg / kg. The results are shown in Figure 9. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the error range is the SE value.

[0231] All four antibody-drug conjugates prepared in Example 2 showed a significant dose-dependent reduction in tumor volume, demonstrating tumor regression at a dose of 3 mg / kg.

[0232] 5)-3 Antitumor effect (3) Similar to Example 5)-1, CD37-positive human diffuse large B-cell lymphoma cell line SU-DHL-8 (ATCC) was divided into 5 × 10⁻¹⁴ cells. 6 Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and random group assignment was performed on Day 7. On the day of group assignment, four antibody-drug conjugates prepared in Example 2 and hmAb-IgG1-DXd (negative control) were administered intravenously at doses of 1 mg / kg and 3 mg / kg. The results are shown in Figure 10. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the error range is the SE value.

[0233] All four antibody-drug conjugates prepared in Example 2 showed a significant dose-dependent reduction in tumor volume, and tumor growth was completely suppressed at a dose of 3 mg / kg.

[0234] [Example 6: Preparation of anti-CD37 antibody-drug conjugate 2] IMGN529 was synthesized by the following process.

[0235] [ka]

[0236] Antibody preparation: Prepare Naratuximab (anti-CD37 antibody, IMGT / 2D structure-DB card for INN 10239) using common procedure B described in Manufacturing Method 1 (1.531 mL mg as the 280 nm extinction coefficient). -1 cm -1 Using (and C), the solution was prepared to 12.12 mg / mL in PBS6.0 / EDTA.

[0237] Conjugation of antibody and drug linker: To 0.4 mL of the above antibody solution, a 10 mM N,N-dimethylacetamide solution of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (0.0338 mL; 10.0 equivalents per antibody molecule) and a 10 mM N,N-dimethylacetamide solution of maytansine derivative (DM1) (0.0507 mL; 15.0 equivalents per antibody molecule) were added, mixed, and rotated at room temperature for 16 hours.

[0238] Purification: The above solution was purified using common procedure D described in Manufacturing Method 1 to obtain 2.5 mL of a solution containing the antibody-drug conjugate "IMGN529". This 0.4 mL scale reaction was repeated six times, and the results were obtained by combining the findings.

[0239] Characterization: Common operation E(ε) described in Manufacturing Method 1 D,280 =5700, ε D,252 Using (=26790), the following characteristic values ​​were obtained. Antibody concentration: 1.29 mg / mL, antibody yield: 15.61 mg (54%), average number of drug-bound antibodies per molecule (n) measured by common procedure E: 3.6.

[0240] [Example 7: In vivo antitumor effect of antibody-drug conjugates 2] The antitumor effects of antibody-drug conjugates were evaluated using an animal model in which cells from CD37-positive human tumor cell lines were transplanted into immunodeficient mice. 4-6 week old SCID mice (CB17 / Icr-Prkdc[scid] / CrlCrlj: Charles River Japan, CB17 / IcrJcl-Prkdc[scid]: CREA Japan) were acclimatized for at least 3 days under SPF conditions before use in the experiment. Mice were fed sterile solid feed (FR-2, Funabashi Farms Co., Ltd.) and sterile tap water (prepared by adding 5-15 ppm sodium hypochlorite solution). The long and short diameters of the transplanted tumors were measured twice a week using an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and the tumor volume was calculated using the formula shown below. Tumor volume (mm 3 ) = 1 / 2 × major axis (mm) × [minor axis (mm)] 2 All antibody-drug conjugates were diluted with ABS buffer (10 mM-Acetate Buffer, 5% Sorbitol, pH 5.5) (NACALAI) and administered intravenously into the tail vein at the doses shown in each example. In addition, ABS buffer was similarly administered as a control group (Vehicle group). As a control group, POLIVY (manufactured by Genentech), IMGN529, and RITUXAN (manufactured by Zenyaku Kogyo Co., Ltd.) were administered intravenously into the tail vein, Ibrutinib (synthesized by a method well known to those skilled in the art) and Venetoclax (synthesized by a method well known to those skilled in the art) were orally administered once a day, and TREAKISYM (manufactured by Simcere Pharmaceutical Group) was administered intraperitoneally once a day for 2 days. 5-6 mice were used in each experiment.

[0241] 7)-1 Antitumor effect (1) The CD37-positive human diffuse large B-cell lymphoma cell line OCI-LY7 (DSMZ) was suspended in 50% Matrigel (Corning, diluted with physiological saline), and 1×10 7 cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and random grouping was performed on Day 10. On the day of grouping, each antibody-drug conjugate was administered intravenously into the tail vein. The results are shown in Fig. 11. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the error range represents the SE value.

[0242] In the POLIVY, IMGN529, and hmAb-IgG1-DXd administration groups, which are negative controls, no tumor regression was observed. In contrast, in the hmAb-H541L11-DXd administration groups prepared in Examples 2)-4, tumor growth was significantly suppressed at a dose of 1 mg / kg, and the tumors completely regressed at a dose of 3 mg / kg.

[0243] 7)-2 Antitumor effect (2) Similar to Example 7)-1, the CD37-positive human diffuse large B-cell lymphoma cell line SU-DHL-8 (ATCC) was used at 1×10 7Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and randomization was performed on Day 8. On the day of randomization, each antibody-drug conjugate was administered intravenously via the tail vein. The results are shown in Figure 12. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the margin of error is the SE value.

[0244] While no tumor regression was observed in the POLIVY, IMGN529, or the negative control group treated with hmAb-IgG1-DXd, the hmAb-H541L11-DXd group prepared in Example 2)-4 showed complete tumor regression with a dose of 3 mg / kg.

[0245] 7)-3 Antitumor effect (3) Similar to Example 7)-1, CD37-positive human diffuse large B-cell lymphoma cell line NU-DUL-1 (DSMZ) was subjected to 1 × 10⁻¹⁴ treatment. 7 Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and random group assignment was performed on Day 14. On the day of group assignment, each antibody-drug conjugate was administered intraveinally to the tail vein. The results are shown in Figure 13. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the margin of error is the SE value.

[0246] In the POLIVY, IMGN529, and negative control groups treated with hmAb-IgG1-DXd, tumor regression was not observed, or tumors proliferated again after regression. In contrast, in the hmAb-H541L11-DXd group prepared in Example 2)-4, a dose of 1 mg / kg significantly suppressed tumor growth, and a dose of 3 mg / kg resulted in complete tumor regression.

[0247] 7)-4 Antitumor effect (4) Similar to Example 7)-1, CD37-positive human diffuse large B-cell lymphoma cell line SU-DHL-4 (DSMZ) was subjected to 1 × 10⁻¹⁴ treatment. 7 Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and randomization was performed on Day 16. On the day of randomization, each antibody-drug conjugate was administered intraveinally to the tail vein. The results are shown in Figure 14. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the margin of error is the SE value.

[0248] The group administered 3 mg / kg of hmAb-H541L11-DXd prepared in Example 2)-4 showed antitumor effects equivalent to or better than those of the group administered 10 mg / kg of IMGN529.

[0249] 7)-5 Antitumor effect (5) Similar to Example 7)-1, CD37-positive human chronic lymphocytic leukemia cell line JVM-3 (DSMZ) was used in 3 × 10⁻¹⁴ samples. 6 Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and random group assignment was performed on Day 13. On the day of group assignment, each antibody-drug conjugate was administered intravenously via the tail vein. In addition, RITUXAN was administered intravenously via the tail vein, Ibrutinib and Venetoclax were administered orally once daily, and TREAKISYM was administered intraperitoneally once daily for two days. The results are shown in Figure 15. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the margin of error is the SE value.

[0250] While no tumor regression was observed in the control drug administration group, in the hmAb-H541L11-DXd administration group prepared in Example 2)-4, a dose of 1 mg / kg significantly suppressed tumor growth, and a dose of 3 mg / kg resulted in complete tumor regression.

[0251] 7)-6 Antitumor effect (6) Similar to Example 7)-1, CD37-positive human follicular lymphoma cell line DOHH-2 (DSMZ) was subjected to 1 × 10⁻¹⁴ treatment. 6 Cells were subcutaneously transplanted into the right flank of female SCID mice (Day 0), and random group assignment was performed on Day 21. On the day of group assignment, hmAb-H541L11-DXd was administered intraveinally to the tail vein. The results are shown in Figure 16. The horizontal axis represents days, the vertical axis represents tumor volume, and the margin of error is the SE value.

[0252] In IMGN529, no tumor regression was observed in the negative control group treated with hmAb-IgG1-DXd. In contrast, in the group treated with hmAb-H541L11-DXd prepared in Example 2)-4, tumor regression occurred at 1 mg / kg, and at 3 mg / kg, the tumor completely regressed, similar to the POLIVY treatment group. [Industrial applicability]

[0253] The present invention provides an anti-CD37 antibody having internalization activity and an antibody-drug conjugate containing the antibody. The antibody-drug conjugate can be used as a therapeutic agent for B-cell malignant lymphoma and the like. [Sequence Listing Free Text]

[0254] Sequence ID 1: Nucleotide sequence encoding the hmAb-L11 light chain Sequence ID 2: Amino acid sequence of the hmAb-L11 light chain Sequence ID 3: Nucleotide sequence encoding the hmAb-H11 heavy chain SEQ ID NO: 4: Amino acid sequence of the hmAb-H11 heavy chain Sequence ID 5: Nucleotide sequence encoding the hmAb-H541 heavy chain SEQ ID NO: hmAb-H541 heavy chain amino acid sequence Sequence ID 7: Nucleotide sequence encoding the hmAb-H551 heavy chain Sequence ID 8: Amino acid sequence of the hmAb-H551 heavy chain Sequence ID 9: Nucleotide sequence encoding the hmAb-H11a heavy chain SEQ ID NO: 10: Amino acid sequence of the hmAb-H11a heavy chain Sequence ID 11: Nucleotide fragment containing a nucleotide sequence encoding a light chain signal sequence and a human κ light chain constant region. Sequence ID 12: Nucleotide sequence encoding the variable region of the hmAb-L11 light chain Sequence ID 13: Nucleotide fragment containing a nucleotide sequence encoding a heavy chain signal sequence and a human G1 heavy chain constant region. Sequence ID 14: Nucleotide sequence encoding the variable region of the hmAb-H11 heavy chain Sequence ID 15: Nucleotide sequence encoding the variable region of the hmAb-H541 heavy chain Sequence ID 16: Nucleotide sequence encoding the variable region of the hmAb-H551 heavy chain Sequence ID 17: Nucleotide sequence encoding the variable region of the hmAb-H11a heavy chain Sequence ID 18: Amino acid sequence of human CD37 Sequence ID 19: CDRL1 sequence of humanized anti-CD37 antibody Sequence ID 20: CDRL2 sequence of humanized anti-CD37 antibody Sequence ID 21: CDRL3 sequence of humanized anti-CD37 antibody Sequence ID 22: CDRH1 sequence of humanized anti-CD37 antibody Sequence ID 23: CDRH2 sequence of humanized anti-CD37 antibody Sequence ID 24: CDRH3 sequence of humanized anti-CD37 antibody

Claims

1. The anti-CD37 antibody has the following formula (wherein A indicates the binding site with the anti-CD37 antibody): This is an antibody-drug conjugate bound to a drug-linker structure shown as follows: Anti-CD37 antibodies bind to the drug-linker structure via sulfhydryl groups derived from the antibody. Antibody-drug conjugates in which the anti-CD37 antibody contains both a heavy chain variable region and a light chain variable region in any one combination selected from the group consisting of the following combinations (k) to (n): (k) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 4; (l) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2, and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 6; (m) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (n) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

10.

2. The antibody-drug conjugate according to claim 1, wherein the anti-CD37 antibody is an antibody comprising a heavy chain variable region and a light chain variable region in combination (l): (l) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

6.

3. The antibody-drug conjugate according to claim 2, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

4. The antibody-drug conjugate according to claim 1, wherein the anti-CD37 antibody is an antibody comprising a heavy chain variable region and a light chain variable region in the combination of (m): (m) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

8.

5. The antibody-drug conjugate according to claim 4, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

6. The antibody-drug conjugate according to claim 1, wherein the anti-CD37 antibody is an antibody comprising a heavy chain variable region and a light chain variable region in combination (n): (n) A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acid sequences 20 to 468 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

10.

7. The antibody-drug conjugate according to claim 6, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

8. The anti-CD37 antibody has the following formula (wherein A indicates the binding site with the anti-CD37 antibody): This is an antibody-drug conjugate bound to a drug-linker structure shown as follows: Anti-CD37 antibodies bind to the drug-linker structure via sulfhydryl groups derived from the antibody. Antibody-drug conjugates in which the anti-CD37 antibody contains both a heavy chain variable region and a light chain variable region in any one combination selected from the group consisting of the following combinations (k') to (n'): (k') A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2 and a heavy chain consisting of amino acid sequences 20 to 467 of the full-length heavy chain amino acid sequence shown in Sequence ID No. 4; (l') A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2, and a heavy chain consisting of amino acid sequences 20 to 467 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 6; (m') A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acid sequences 20 to 467 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8; and (n') A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acid sequences 20 to 467 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

10.

9. The antibody-drug conjugate according to claim 8, wherein the anti-CD37 antibody is an antibody comprising a heavy chain variable region and a light chain variable region in the combination of (l'): (l') A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acid sequences 20 to 467 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

6.

10. The antibody-drug conjugate according to claim 9, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

11. The antibody-drug conjugate according to claim 8, wherein the anti-CD37 antibody is an antibody comprising a heavy chain variable region and a light chain variable region in the combination of (m'): (m') A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acid sequences 20 to 467 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

8.

12. The antibody-drug conjugate according to claim 11, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

13. The antibody-drug conjugate according to claim 8, wherein the anti-CD37 antibody is an antibody comprising a heavy chain variable region and a light chain variable region in the combination of (n'): (n') A light chain consisting of amino acid sequences 21 to 234 of the full-length light chain amino acid sequence shown in Sequence ID No. 2, and a heavy chain consisting of amino acid sequences 20 to 467 of the full-length heavy chain amino acid sequence shown in Sequence ID No.

10.

14. The antibody-drug conjugate according to claim 13, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

15. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, which is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 4, where n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

16. The antibody-drug conjugate according to claim 15, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

17. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, the anti-CD37 antibody is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 467 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 4, n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

18. The antibody-drug conjugate according to claim 17, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

19. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, which is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 6, where n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

20. The antibody-drug conjugate according to claim 19, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

21. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, the anti-CD37 antibody is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 467 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 6, n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

22. The antibody-drug conjugate according to claim 21, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

23. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, the anti-CD37 antibody is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8, n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

24. The antibody-drug conjugate according to claim 23, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

25. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, the anti-CD37 antibody is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 467 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 8, n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

26. The antibody-drug conjugate according to claim 25, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

27. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, the anti-CD37 antibody is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 468 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 10, n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

28. The antibody-drug conjugate according to claim 27, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

29. The following formula: An antibody-drug conjugate represented by [this symbol]. (Here, AB represents an anti-CD37 antibody, which is an antibody comprising a light chain consisting of amino acids 21 to 234 of the full-length light chain amino acid sequence shown in SEQ ID NO: 2 and a heavy chain consisting of amino acids 20 to 467 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 10, where n represents the average number of drug-linker structures bound to the anti-CD37 antibody per antibody, and the anti-CD37 antibody and the drug-linker structure are bound via sulfhydryl groups derived from the antibody.)

30. The antibody-drug conjugate according to claim 29, wherein the average number of drug-linker structures bound per antibody is in the range of 7 to 8.

31. A pharmaceutical composition characterized by comprising an antibody-drug conjugate according to any one of claims 1 to 30, a pharmaceutically acceptable salt thereof, or a hydrate thereof.

32. The pharmaceutical composition according to claim 31, characterized in that it is an antitumor drug.

33. The pharmaceutical composition according to claim 32, characterized in that the tumor is a tumor that expresses CD37.

34. The pharmaceutical composition according to claim 32, characterized in that the tumor is one selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal body lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia.

35. The pharmaceutical composition according to claim 33, characterized in that the tumor is one selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal body lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia.

36. The pharmaceutical composition according to claim 32, characterized in that the tumor is one selected from the group consisting of T-cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia.

37. The pharmaceutical composition according to claim 36, characterized in that the T-cell lymphoma is peripheral T-cell lymphoma or cutaneous T-cell lymphoma.

38. The pharmaceutical composition according to claim 33, characterized in that the tumor is one selected from the group consisting of T-cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia.

39. The pharmaceutical composition according to claim 38, characterized in that the T-cell lymphoma is peripheral T-cell lymphoma or cutaneous T-cell lymphoma.

40. A tumor treatment agent comprising an antibody-drug conjugate according to any one of claims 1 to 30, a pharmacochemically acceptable salt thereof, or a hydrate thereof.

41. A physiological saline solution preparation containing 0.001 to 100 mg / kg of an antibody-drug conjugate according to any one of claims 1 to 30, a pharmacokinetically acceptable salt thereof, or a hydrate thereof.

Images