Antibody cleavage site-binding molecule

Abstract

The present disclosure provides pharmaceutical compositions comprising an antibody having ADCC activity, a T cell redirected antibody or a cell expressing a chimeric receptor for use in combination with administration of an antigen binding molecule capable of binding to a target antigen wherein the primary molecule comprises a protease cleavable linker and the secondary molecule comprises a protease cleavable linker. The antigen binding molecule with the cleaved linker has binding capacity to a target antigen; a variable region of an antibody having ADCC activity or a T-cell redirection antibody and an extracellular binding domain of a chimeric receptor bind to a cell expressing a target antigen by binding to a linker-cleaved antigen-binding molecule generated after cleaving a cleavable linker.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511982312.1 (22) Application Date 2020.06.05 (30) Priority Data 2019-105761 2019.06.05 JP (62) Divisional Application Data 202080040523.2 2020.06.05 (71) Applicant Chugai Pharmaceutical Co., Ltd. Address Japan Applicant National University Corporation Yamaguchi University (72) Inventors Sakurai Minoru, Igawa Tomoyuki, Komori Yasunori, Tamada Koji, Sasa, Furuta Yukimi (74) Patent Agency Zhongke Patent & Trademark Agency Co., Ltd. 11021 Patent Attorney Zhang Guoliang (51) Int.Cl. A61K 40 / 11 (2025.01) C07K 19 / 00(2006.01) A61K 40 / 31(2025.01) A61K 40 / 42(2025.01) A61P 35 / 00(2006.01) A61P 35 / 02(2006.01) A61P 35 / 04(2006.01) (54) Invention Title Antibody Cleavage Site Binding Molecule (57) Abstract This disclosure provides a pharmaceutical composition comprising an antibody having ADCC activity, a T-cell redirection antibody, or a cell expressing a chimeric receptor, for use in combination with the administration of an antigen-binding molecule capable of binding a target antigen, wherein the primary molecule comprises a protease-cleavable linker, the linker-cleaved antigen-binding molecule having the ability to bind to the target antigen, and the variable region of the antibody having ADCC activity or the T-cell redirection antibody and the extracellular binding domain of the chimeric receptor bind to the cell expressing the target antigen by binding to the linker-cleaved antigen-binding molecule resulting from the cleavage of the linker. Claims (2 pages), Description (139 pages), Sequence List (electronic publication), Drawings (13 pages), CN 121818917 A, 2026.04.10, CN 1 21 81 89 17 A. 1. A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with an antigen-binding molecule, wherein the antigen-binding molecule comprises a protease-cleavable linker that, upon linker cleavage, has binding capacity for a target antigen, and the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain, the extracellular binding domain having binding capacity for the linker-cleaved antigen-binding molecule, and binding to cells expressing the target antigen via binding to the linker-cleaved antigen-binding molecule. 2. A pharmaceutical composition comprising an antigen-binding molecule for use in combination with cells expressing a chimeric receptor, wherein...The antigen-binding molecule comprises a protease-cleavable linker, which, after cleavage, has the ability to bind to the target antigen. The chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain has the ability to bind to the antigen-binding molecule after linker cleavage. Through binding to the linker-cleaved antigen-binding molecule, the antigen-binding molecule can bind to cells expressing the target antigen. 3. A pharmaceutical composition comprising a bispecific antibody for use in combination with the administration of an antigen-binding molecule, wherein the antigen-binding molecule comprises a protease-cleavable linker, which, after linker cleavage, has the ability to bind to the target antigen; the bispecific antibody comprises an antibody variable region active for binding to the protease-cleaved antigen-binding molecule and an antibody variable region active for binding to molecules expressed on the surface of T cells; the bispecific antibody, through binding to the linker-cleaved antigen-binding molecule, can bind to cells expressing the target antigen. 4. A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of a bispecific antibody, wherein the antigen-binding molecule comprises a protease-cleavable linker, which, upon cleavage by the linker, has the ability to bind to a target antigen; the bispecific antibody comprises an antibody variable region active for binding to the protease-cleaved antigen-binding molecule and an antibody variable region active for binding to molecules expressed on the surface of T cells; the bispecific antibody is capable of binding to cells expressing the target antigen by binding to the linker-cleaved antigen-binding molecule. 5. A pharmaceutical composition comprising an IgG antibody characterized by enhanced antibody-dependent cytotoxicity for use in combination with the administration of an antigen-binding molecule, wherein the antigen-binding molecule comprises a protease-cleavable linker, which, upon cleavage by the protease, has the ability to bind to an antigen expressed on the surface of a target cell; the IgG antibody comprises an antibody variable region active for binding to the protease-cleaved antigen-binding molecule; the IgG antibody is capable of binding to target cells by binding to the linker-cleaved antigen-binding molecule. 6. A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of an IgG antibody characterized by enhanced antibody-dependent cytotoxicity, wherein the antigen-binding molecule comprises a protease-cleavable linker, which, upon protease cleavage of the linker, is active for binding to an antigen expressed on the surface of a target cell, and the IgG comprises an antibody variable region active for binding to the antigen-binding molecule after protease cleavage of the linker, and the IgG antibody is capable of binding to target cells by binding to the linker-cleaved antigen-binding molecule. Claims 1 / 2 page 2 CN 121818917 A 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the linker-cleaved antigen-binding molecule is active for binding to an antigen expressed on the surface of a target cell, and the IgG comprises an antibody variable region active for binding to the linker-cleaved antigen-binding molecule.The KD value of the antigen, relative to the ratio of the KD value of the antigen-binding molecule to the antigen before cleavage (KD(after cleavage) / KD(before cleavage)), is 0.1 or less. 8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a single-domain antibody. 9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a protease-cleavable linker, wherein the antibody is selected from IgG antibodies, IgG antibody-like molecules, or heavy chain antibodies, and the protease-cleaved antigen-binding molecule comprises an antigen-binding domain and a portion of the cleaved linker. 10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the protease-cleavable linker of the antigen-binding molecule is located near the boundary between the variable region and the constant region or near the boundary between CH1 and CH2 within the constant region. 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the antigen-binding molecule is an antibody or IgG antibody-like molecule comprising a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is a VL, VH, VHH, or antigen-binding fragment thereof of the antibody. 12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the antigen-binding molecule is a single-domain antibody comprising a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is a portion of the antigen-binding domain and linker of the single-domain antibody. 13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the protease-cleavable linker comprises a protease-cleaving sequence. 14. The pharmaceutical composition according to any one of claims 1 to 12, wherein the protease-cleavable linker comprises a peptide having a protease-cleaving sequence having any sequence number from 1 to 725. 15. The pharmaceutical composition according to any one of claims 1 to 14, for the treatment or prevention of cancer. Claims 2 / 2 Page 3 CN 121818917 A Antibody Cleavage Site Binding Molecule

[0001] This application is a divisional application of international application number PCT / JP2020 / 022218, international application date June 5, 2020, Chinese application number 202080040523.2, entitled "Antibody Cleavage Site Binding Molecule". Technical Field

[0002] This disclosure relates to antibodies with ADCC activity, T cell redirection antibodies, chimeric receptors, cells expressing chimeric receptors, and disease treatment methods using the cells or antibodies, particularly to therapies utilizing the ADCC activity of antibodies, CAR-T therapy utilizing the cells, and T cell redirection antibody therapy. Background Art

[0003] Antibody drugs use immunoglobulins or analogues thereof, which are part of the immune system present in living organisms, as the main component.Drugs containing these components (Non-Patent Literature 1 and Non-Patent Literature 2). Compared with existing small molecule compounds, antibody drugs have large molecular weights and can recognize complex molecules, thus exhibiting high specificity for target recognition and fewer unexpected side effects. Furthermore, foreign substances in the blood are generally taken up by cells and broken down via endocytosis; however, because antibodies possess a mechanism for antibody recovery through specific receptors FcRn and the antibody Fc region, they have long retention in the blood, allowing for prolonged efficacy with a single administration. Further, since antibody drugs are prepared as recombinant proteins, their function can be altered using genetic engineering.

[0004] For example, antibody constant regions induce antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to FcγR on NK cells or macrophages. When using antibodies with such constant regions, the addition of alterations to enhance binding to FcγR is known to induce stronger cytotoxicity (Non-Patent Literature 3).

[0005] In addition, conventional antibodies only recognize and bind to one epitope of an antigen. However, by modifying natural IgG antibodies, antibodies that bind to two or more antigens with a single molecule (called bispecific antibodies) (Non-Patent Document 11) can be developed, which can bind to proteins expressed by T cells (CD3ε or TCR) and proteins expressed by cancer cells (cancer antigens). As a type of bispecific antibody, T cell-redirecting antibodies have been known since the 1980s. These antibodies use T cells as effector cells and mobilize them to achieve their anti-tumor effect through cytotoxicity (Non-Patent Documents 12, 13, 14). Unlike antibodies that use NK cells or macrophages as effector cells and mobilize ADCC as their anti-tumor mechanism, T-cell redirection antibodies have a binding domain targeting any of the constituent subunits of the T-cell receptor (TCR) complex, particularly the domain that binds to the CD3ε chain, and bind to antigens on cancer cells as targets, forming an intercellular bridge between T cells and cancer antigen-expressing cells. Using T cells as effector cells, they induce strong cytotoxicity (T-cell dependent cellular cytotoxicity; TDCC) on cancer antigen-expressing cells (Non-Patent Literature 4, 12, 13, 14).

[0006] Furthermore, in recent years, anti-tumor therapies that utilize the high specificity of antibodies for antigen recognition by effector cells have been developed, showing significant efficacy in some cancers. This is known as chimeric antigen receptor (CAR) adoptive immunotherapy.In immunotherapy, an extracellular domain with antigen-binding ability, primarily scFv derived from antibodies, is artificially fused with an intracellular signal transduction domain to form a CAR, which is expressed in effector cells such as T cells. When this CAR-expressing T cell (hereinafter also referred to as "CAR-T cell") is transferred into a cancer patient, the intracellular domain is activated when the tumor antigen is recognized by the CAR, inducing cytotoxicity in effector cells and exerting a therapeutic effect by killing tumor cells (Non-Patent Literature 5).

[0007] Clinical trials of cancer immunotherapy using CAR-T cells (Non-Patent Literature 10) have shown effectiveness in cancer immunotherapy using CAR-T cells in hematopoietic organ malignancies such as leukemia or lymphoma. In 2017, CAR-T inhibitors Kymriah (registered trademark) (Novartis, tisagenlecleucel, CTL-019, CD3ζ-CD137) and Yescarta (registered trademark) (KiTE, axicabtagene ciloleucel, CD3ζ-CD28), using CD19 as the antigen, were approved as drugs in the United States, and in 2019 in Japan.

[0008] In addition, methods have been proposed that utilize single-domain antibodies from camelids to target a variety of antigens, thereby simplifying antibody preparation. In summary, antibody drugs have many advantages and are therefore applicable to a wide range of diseases such as tumors, autoimmune diseases, and infectious diseases (Non-Patent Literature 6).

[0009] On the other hand, limitations of antibody drugs have also been pointed out. One of these is the problem of antigen lesion site specificity. Surface antigens that become antibody targets may also be expressed in normal tissues outside the lesion site. Although the expression level of the antigen is lower than that in the lesion site, the antibody action produces side effects for the normal tissue expressing the antigen.

[0010] Regarding this problem, a possible solution is to identify lesions targeting protease activity specific to the lesion site. For example, by applying a protease substrate containing a cleavage site of a synthetic peptide chain with a fluorescent dye, and measuring the fluorescence change accompanying the cleavage, it was shown that the lesion site can be identified by protease activity (Non-Patent Document 7).

[0011] A study on detecting lesions by recognizing protease cleavage products generated at the lesion site with antibodies has been reported (Non-Patent Document 8). In this study, it was reported that the cleavage product of the IdeS protease expressed by actinomycetes could be specifically recognized in vivo using an antibody that specifically binds to the cleavage product (Non-Patent Document 8).

[0012] In the field of chronic diseases, there are also antibodies developed to identify type II collagen cleaved by activated MMP (matrix metalloproteinase) in the lesion site of osteoarthritis of the knee, with the aim of developing reagents for detecting lesions of osteoarthritis of the knee (Non-Patent Document 9).

[0013] As an example of applying existing protease-activated antibody technology to treatment, the "Probody (registered trademark) technology" (Figure 1) can be cited, which expands tissue specificity and therapeutic window by conferring sensitivity of antibodies to proteases expressed or activated to an increased degree in lesion sites such as cancerous or inflamed tissues.

[0014] "Probody (registered trademark)" is a molecule formed by linking the antigen-binding site of an antibody and a masking peptide of a masking antibody with a cleaving peptide sequence of a protease expressed at the lesion site (Non-Patent Document 15). The antigen-binding site of the antibody is masked by the masking peptide in the uncleaved state of the peptide sequence, and therefore cannot bind to the antigen. Probody (registered trademark) cleaves its peptide sequence by a protease expressed at the target lesion site, thereby masking peptide dissociation and generating an antibody molecule with antigen-binding activity, enabling binding to specific antigens of the target lesion tissue. In non-lesion sites where the protease is absent, Probody (registered trademark) allows for the administration of larger doses than conventional antibodies due to the inhibition of antigen-antibody binding, potentially expanding the therapeutic window.

[0015] Prior Art Documents

[0016] Patent Documents

[0017] Patent Document 1: WO2009 / 025846

[0018] Patent Document 2: WO2017 / 143094

[0019] Patent Document 3: WO2018 / 097307

[0020] Non-Patent Document Specification 2 / 139 pages 5 CN 121818917 A

[0021] Non-Patent Document 1: Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic., Nat. Biotechnol. (2005)23, 1073-1078

[0022] Non-Patent Document 2: Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. (2005) 59(3), 389-396

[0023] Non-Patent Literature 3: The impactof Fc engineering on an anti-CD19 antibody: increased Fcgamma receptor affinity enhances B-cell clearing in nonhuman primates. Zalevsky J, Leung IW, Karki S, Chu SY, Zhukovsky EA, Desjarlais JR, Carmichael DF, Lawrence CE. Blood. 2009 Apr 16;113(16):3735-43.

[0024] Non-patent document 4: Advances in bispecific biotherapeutics for the treatment of cancer Biochem Pharmacol. 2012 Nov 1; 84(9): 1105-12

[0025] Non-patent document 5: Chimeric Antigen Receptor Therapy N Engl J Med 2018; 379: 64-73

[0026] Non-patent document 6: Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol. 2016; 38(1): 21-8

[0027] Non-Patent Literature 7: Shedding light onto live molecular targets Nat Med. 2003 Jan; 9(1): 123-8

[0028] Non-Patent Literature 8: Structure and specificity of an antibody targeting a proteolytically cleaved IgG hinge Malia TJ1, Teplyakov A, Brezski RJ, Luo J, Kinder M, Sweet RW, Almagro JC, Jordan RE, Gilliland GL. Proteins. 2014 Aug; 82(8): 1656-67

[0029] Non-Patent Literature 9: Development of a novel immunoassay for the measurement of type II collagen neoepitopeGenerated by collagenase cleavage Clin Chim Acta. 2012 Oct 9; 413(19-20): 1591-9.

[0030] Non-patent literature 10: Grupp et al., 2013 N Engl J Med 368(16): 1509-18.

[0031] Non-patent literature 11: Kontermann, mAbs 2012; 4: 182-197.

[0032] Non-patent literature 12: Mezzanzanica et al., International journal of cancer 1988; 41: 609-615.

[0033] Non-patent literature 13: Staerz and Bevan, Proceedings of the National Academy of Sciences of the United States of America 1986; 83: 1453-1457.

[0034] Non-patent literature 14: Staerz et al., Nature 1985; 314: 628-631.

[0035] Non-Patent Literature 15: Desnoyers LR et al., Sci Transl Med. 2013 Oct 16; 5(207): 207ra144. Summary of the Invention

[0036] Problems to be Solved by the Invention

[0037] Probody (registered trademark) exerts cytotoxicity in normal tissues due to the high blood retention of activated Probody (registered trademark) and its antigen-binding activity even in its inactive state without protease cleavage. In addition, it is known that high cytotoxicity is obtained even in T-cell redirection antibody therapy and CAR-T therapy; on the other hand, its activity can also occur in normal cells, causing serious side effects, and there is a need to improve the safety of these therapies.

[0038] Furthermore, single tumor antigens are not universally expressed in all cancers, therefore these therapies require the construction of antigen recognition sites for each tumor antigen as a target, which presents significant challenges due to the economic cost and labor involved. Moreover, tumor antigens as targets may exhibit reduced expression or sudden mutations in response to treatment, leading to immune escape and reduced or absent therapeutic efficacy.

[0039] For tumor antigens that change recognition in response to treatment, potential CAR-T cells and T-cell redirection antibodies, and therapeutic methods utilizing them, are being investigated, with the expectation of providing sufficient therapeutic efficacy and high safety when administered to patients.The development of treatment methods and widely available technologies for sexual treatments leads to safer and cheaper treatments.

[0040] Methods for Solving the Problems

[0041] In order to solve these problems, the inventors have repeatedly studied and found that antibodies, T-cell redirection antibodies, or CAR-T cells with ADCC activity that bind to newly generated antigen-binding domains through protease cleavage have selective action on therapeutic target cells and are effective in treatment, thus completing the present invention. In one aspect of this disclosure, a group of therapeutic molecules with general applicability is disclosed, which are molecules containing antigen-binding domains generated by protease action that have a short half-life in the blood and do not exert pharmacological effects without protease cleavage.

[0042] This disclosure provides, for example, a pharmaceutical composition comprising a molecule having antigen-binding capacity and a pharmaceutical composition comprising a molecule having effector activation capacity, wherein the pharmaceutical composition is used to activate effector cells by cleavage by a protease specifically expressed in the target tissue, thereby bridging target cells expressing antigens and effector cells, a pharmaceutical composition for treating diseases originating from the target tissue, and molecules having antigen-binding capacity and molecules having effector activation capacity used as active ingredients of the pharmaceutical composition. Further, a method for manufacturing the pharmaceutical composition and the molecules having antigen-binding capacity and molecules having effector activation capacity used as active ingredients is provided.

[0043] Further, according to this disclosure, by using a molecule that commonly binds to tumor antigens, it is possible to select the most suitable therapy from a variety of therapies, including CAR-T therapy, bispecific antibody therapy, and antibody therapy with ADCC activity, based on the patient's treatment suitability, or to change or add therapy depending on the treatment status.

[0044] Further, according to this disclosure, by using multiple common molecules that bind to tumor antigens, the most suitable therapy can be selected from a variety of therapies, including CAR-T therapy, bispecific antibody therapy, and antibody therapy with ADCC activity, based on the patient's treatment suitability, or the therapy can be changed or added depending on the treatment status.

[0045] One aspect of this disclosure is an antibody, bispecific antibody, or CAR-T cell with ADCC activity that binds to a target cell expressing a target antigen, said CAR-T cell, bispecific antibody, or ADCC-active antibody by binding to an antigen-binding molecule. The antigen-binding molecule contains a protease-cleavable linker, which, after being cleaved by a protease, has the ability to bind to the target antigen. This disclosure relates to the aforementioned CAR-T cells, bispecific antibodies, or ADCC-active antibodies.

[0046] Another aspect of this disclosure relates to isolated nucleic acid molecules, said isolated nucleic acid molecules being encoding nucleic acid molecules of ADCC-active antibodies, bispecific antibodies, and / or CARs that can be used to target cells expressing a target antigen.

[0047] Another aspect of this disclosure relates to a vector comprising an isolated nucleic acid molecule, said vector being a vector encoding a nucleic acid molecule of an antibody, bispecific antibody, and / or CAR that can be used to target cells expressing a target antigen.

[0048] Another aspect of this disclosure relates to cells expressing the CAR of this disclosure, or cells transfected or transduced with the nucleic acid molecule or vector of this disclosure.

[0049] More specifically, one aspect of this disclosure provides the following invention. Instructions for Use 4 / 139 pages 7 CN 121818917 A

[0050] [1] A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with an antigen-binding molecule, wherein

[0051] the antigen-binding molecule comprises a protease-cleavable linker that has the ability to bind to a target antigen after linker cleavage,

[0052] the chimeric receptor comprises an extracellular binding domain, a transmembrane domain and an intracellular signal transduction domain, the extracellular binding domain having the ability to bind to the antigen-binding molecule after linker cleavage, and is able to bind to cells expressing the target antigen by binding to the antigen-binding molecule after linker cleavage.

[0053] [2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with an administration of a cell expressing a chimeric receptor, wherein

[0054] the antigen-binding molecule comprises a protease-cleavable linker that has the ability to bind to a target antigen after linker cleavage,

[0055] the chimeric receptor comprises an extracellular binding domain, a transmembrane domain and an intracellular signal transduction domain, the extracellular binding domain having the ability to bind to the linker-cleaved antigen-binding molecule, thereby enabling binding to a cell expressing the target antigen.

[0056] [3] A pharmaceutical composition comprising a bispecific antibody for use in combination with the administration of an antigen-binding molecule, wherein

[0057] the antigen-binding molecule comprises a protease-cleavable linker and has binding ability to a target antigen after linker cleavage,

[0058] the bispecific antibody comprises an antibody variable region having binding activity to the antigen-binding molecule after protease cleavage and an antibody variable region having binding activity to a molecule expressed on the surface of T cells,

[0059] the bispecific antibody is able to bind to cells expressing the target antigen by binding to the antigen-binding molecule after linker cleavage.

[0060] [4] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of a bispecific antibody, wherein

[0061] the antigen-binding molecule comprises a protease-cleavable linker and has binding ability to a target antigen after linker cleavage,

[0062] the bispecific antibody comprises an antibody having binding activity to the antigen-binding molecule after protease cleavage.The variable region and the antibody variable region having binding activity to molecules expressed on the surface of T cells,

[0063] The bispecific antibody is able to bind to cells expressing target antigens by binding to antigen-binding molecules cleaved by the linker.

[0064] [5] A pharmaceutical composition comprising an IgG antibody characterized by enhanced antibody-dependent cytotoxicity for use in combination with the administration of an antigen-binding molecule, wherein

[0065] the antigen-binding molecule comprises a protease-cleavable linker and, after protease cleavage of the linker, has binding activity to antigens expressed on the surface of target cells,

[0066] the IgG antibody comprises an antibody variable region having binding activity to antigen-binding molecules cleaved by the protease,

[0067] the IgG antibody is able to bind to target cells by binding to antigen-binding molecules cleaved by the linker.

[0068] [6] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of an IgG antibody characterized by enhanced antibody-dependent cytotoxicity, wherein

[0069] the antigen-binding molecule comprises a protease-cleavable linker, which, after being cleaved by a protease, has binding activity to an antigen expressed on the surface of a target cell,

[0070] the IgG comprises an antibody variable region that has binding activity to the antigen-binding molecule after being cleaved by a protease,

[0071] the IgG antibody is able to bind to the target cell by binding to the antigen-binding molecule after being cleaved by the linker. Specification 5 / 139 pages 8 CN 121818917 A

[0072] [7] The pharmaceutical composition according to any one of [1] to [6], wherein the ratio of the KD value of the antigen-binding molecule after being cleaved to the antigen to the KD value of the antigen-binding molecule before being cleaved (KD(after cleavage) / KD(before cleavage)) is 0.1 or less.

[0073] [8] The pharmaceutical composition according to any one of [1] to [7], wherein the antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a single-domain antibody.

[0074] [9] The pharmaceutical composition according to any one of [1] to [8], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a protease-cleavable linker, wherein the antibody is selected from IgG antibodies, IgG antibody-like molecules, or heavy chain antibodies, and the protease-cleaved linker comprises an antigen-binding domain and a portion of the cleaved linker.

[0075]

[10] The pharmaceutical composition according to any one of [1] to [9], wherein the protease-cleavable linker of the antigen-binding molecule is located near the boundary between the variable region and the constant region or near the boundary between CH1 and CH2 within the constant region.

[0076]

[11] The pharmaceutical composition according to any one of [1] to

[10] , wherein the antigen-binding molecule is an antibody or IgG antibody-like molecule containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is the VL, VH, VHH or antigen-binding fragment of the antibody.

[0077]

[12] The pharmaceutical composition according to any one of [1] to

[11] , wherein the antigen-binding molecule is a single-domain antibody containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is part of the antigen-binding domain and linker of the single-domain antibody.

[0078]

[13] The pharmaceutical composition according to any one of [1] to

[12] , wherein the protease-cleavable linker contains a protease-cleaving sequence.

[0079]

[14] The pharmaceutical composition according to any one of [1] to

[12] , wherein the protease-cleavable linker contains a peptide having a protease-cleaving sequence having any sequence number 1 to 725.

[0080]

[15] The pharmaceutical composition according to any one of [1] to

[14] is used for the treatment or prevention of cancer.

[0081] [A1-1] A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with an antigen-binding molecule,

[0082] wherein the antigen-binding molecule comprises a protease-cleavable linker that has the ability to bind to a target antigen after linker cleavage,

[0083] wherein the chimeric receptor comprises an extracellular binding domain, a transmembrane domain and an intracellular signal transduction domain, the extracellular binding domain having the ability to bind to the linker-cleaved antigen-binding molecule, thereby enabling binding to cells expressing the target antigen.

[0084] [A1-2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with administration to cells expressing a chimeric receptor,

[0085] the antigen-binding molecule comprising a protease-cleavable linker, which, after linker cleavage, has the ability to bind to a target antigen,

[0086] the chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain, the extracellular binding domain having the ability to bind to the linker-cleaved antigen-binding molecule, thereby enabling binding to cells expressing the target antigen.

[0087] [A1-3] The pharmaceutical composition according to [A1-1] or [A1-2] wherein the ratio of the KD value of the linker-cleaved antigen-binding molecule to the antigen (KD(after cleavage) / KD(before cleavage)) to the KD value of the linker-uncleaved antigen-binding molecule to the antigen is 0.1 or less or 0.01 or less.

[0088] [A1-4] The pharmaceutical composition according to any one of [A1-1] to [A1-3], wherein the above-mentioned antigen-binding component is... (See page 6 / 139 of the specification, 9 CN)121818917 A is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a single-domain antibody.

[0089] [A1-5] The pharmaceutical composition according to any one of [A1-1] to [A1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a protease-cleavable linker, wherein the antibody is selected from IgG antibodies, IgG antibody-like molecules, or heavy chain antibodies, and the protease-cleaved antigen-binding molecule comprises an antigen-binding domain and a portion of the cleaved linker.

[0090] [A1-6] The pharmaceutical composition according to any one of [A1-1] to [A1-4], wherein the antigen-binding molecule comprises a VHH of a single-domain antibody and a protease-cleavable linker, and the protease-cleaved antigen-binding molecule comprises an antigen-binding domain and a portion of the cleaved linker.

[0091] [A1-7] The pharmaceutical composition according to any one of [A1-1] to [A1-6], wherein the protease-cleavable linker of the antigen-binding molecule is located near the boundary between the variable region and the constant region or near the boundary between CH1 and CH2 within the constant region.

[0092] [A1-8] The pharmaceutical composition according to any one of [A1-1] to [A1-7], wherein the protease-cleavable linker of the antigen-binding molecule is located near the hinge region.

[0093] [A1-9] The pharmaceutical composition according to any one of [A1-1] to [A1-8], wherein the antigen-binding molecule is an antibody or IgG antibody-like molecule containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is VL, VH, VHH of the antibody or an antigen-binding fragment thereof.

[0094] [A1-10] The pharmaceutical composition according to any one of [A1-1] to [A1-9], wherein the antigen-binding molecule is a heavy-chain antibody or a single-domain antibody containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is a VHH or a portion thereof containing the antigen-binding domain of the antigen-binding molecule.

[0095] [A1-11] The pharmaceutical composition according to any one of [A1-1] to [A1-10], wherein the antigen-binding molecule after linker cleavage is scFv, Fv, Fab, Fab', F(ab')2, VH, or VHH.

[0096] [A1-12] The pharmaceutical composition according to any one of [A1-1] to [A1-11], wherein the extracellular binding domain of the chimeric receptor recognizes the cleaved linker, a portion thereof, or a portion thereof containing the linker.

[0097] [A1-13] The pharmaceutical composition according to any one of [A1-1] to [A1-12], wherein the KD value of the extracellular binding domain of the chimeric receptor to the antigen-binding molecule after cleavage of the linker is relative to the antigen-binding molecule before cleavage of the linker.The ratio of KD values ​​(KD(after cleavage) / KD(before cleavage)) is less than 0.1 or less than 0.01.

[0098] [A1-14] The pharmaceutical composition according to any one of [A1-1] to [A1-13], wherein the protease-cleavable linker comprises a protease-cleaving sequence.

[0099] [A1-15] The pharmaceutical composition according to any one of [A1-1] to [A1-14], wherein the protease-cleavable linker comprises a peptide having a protease-cleaving sequence having any sequence number 1 to 725.

[0100] [A1-16] The pharmaceutical composition according to any one of [A1-1] to [A1-15], wherein the protease-cleavable linker further comprises a flexible linker.

[0101] [A1-17] The pharmaceutical composition according to any one of [A1-1] to [A1-16], wherein the protease is a protease specifically expressed in the target tissue.

[0102] [A1-18] The pharmaceutical composition according to any one of [A1-1] to [A1-17], wherein the target cell is a tumor cell and the protease is a tumor protease.

[0103] [A1-19] The pharmaceutical composition according to any one of [A1-1] to [A1-18] is used for the treatment or prevention of cancer.

[0104] [A1-20] The pharmaceutical composition according to [A1-19], wherein the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, embryonal cell carcinoma, and leukemia.

[0105] [A1-21] The pharmaceutical composition according to [A1-19], wherein the cancer is selected from the group consisting of B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma.

[0106] [A1-22] The pharmaceutical composition according to any one of [A1-1] to [A1-21] is used in CAR-T therapy.

[0107] [A2-1] A chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein, in an antigen-binding molecule comprising a protease-cleavable adaptor, the extracellular binding domain binds to an antigen-binding molecule whose adaptor has been cleaved by a protease, and the extracellular binding domain binds to a cell expressing an antigen by binding to the antigen-binding molecule cleaved by the adaptor.

[0108] [A2-2] The chimeric receptor according to [A2-1], wherein the extracellular binding domain recognizes a protease-cleaved adaptor, a portion of the adaptor, or a portion comprising the adaptor.

[0109] [A2-3]The chimeric receptor according to [A2-1] or [A2-2], wherein the transmembrane domain comprises CD28.

[0110] [A2-4] The chimeric receptor according to any one of [A2-1] to [A2-3] further comprises one or more co-stimulatory molecules located between the transmembrane domain and the intracellular signal transduction domain.

[0111] [A2-5] The chimeric receptor according to [A2-4], wherein the co-stimulatory molecule is CD3ζ, CD28, 4-1BB, 4-1BBL, ICOS, or OX40.

[0112] [A2-6] The chimeric receptor according to any one of [A2-1] to [A2-5], wherein the intracellular signal transduction domain comprises CD3ζ.

[0113] [A2-7] The chimeric receptor according to any one of [A2-1] to [A2-6], wherein the protease-cleavable linker comprises a peptide having a protease-cleaving sequence having any one of sequence numbers 1 to 725.

[0114] [A2-8] Nucleic acid encoding the chimeric receptor described in any one of [A2-1] to [A2-7].

[0115] [A2-9] Vector comprising the nucleic acid described in [A2-8].

[0116] [A2-10] Cell comprising the vector described in [A2-8].

[0117] [A2-11] Cell according to [A2-9], wherein the cell is a T cell.

[0118] [A2-12] Cell according to [A2-11], wherein the T cell is a CD4+ or CD8+ T cell.

[0119] [A2-13] Cell according to [A2-11], wherein the T cell is a regulatory T cell (Treg) or a follicular regulatory T cell (TFR).

[0120] [A3-1] An antigen-binding molecule comprising a protease-cleavable linker,

[0121] the linker-cleaved antigen-binding molecule is capable of binding to an antigen and can bind to target cells expressing an antigen by binding the extracellular binding domain of a chimeric receptor to the linker-cleaved antigen-binding molecule.

[0122] [A3-2] The antigen-binding molecule according to [A3-1], wherein the protease-cleavable linker comprises a peptide having a protease-cleaving sequence having any of sequence numbers 1 to 725.

[0123] [B1-1] A pharmaceutical composition comprising a bispecific antibody for use in combination with the administration of an antigen-binding molecule,

[0124] the antigen-binding molecule comprising a protease-cleavable linker and being capable of binding to a target antigen after linker cleavage,

[0125] the bispecific antibody comprising an antibody variable region having binding activity against the protease-cleaved antigen-binding molecule and an antibody variable region having binding activity against molecules expressed on the surface of T cells, (Specification 8 / 139 pages 11 CN)121818917 A

[0126] Bispecific antibodies can bind to cells expressing target antigens by binding to antigen-binding molecules cleaved by a linker.

[0127] [B1-2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of a bispecific antibody,

[0128] the antigen-binding molecule comprising a protease-cleavable linker that has the ability to bind to a target antigen after linker cleavage,

[0129] the bispecific antibody comprising an antibody variable region having binding activity to antigen-binding molecules cleaved by a protease and an antibody variable region having binding activity to molecules expressed on the surface of T cells,

[0130] the bispecific antibody can bind to cells expressing target antigens by binding to antigen-binding molecules cleaved by a linker.

[0131] [B1-3] The pharmaceutical composition according to [B1-1] or [B1-2], wherein the KD value of the antigen-binding molecule after linker cleavage to the antigen is less than or equal to the KD value of the antigen-binding molecule before linker cleavage (KD(after cleavage) / KD(before cleavage)) of the antigen-binding molecule is less than or equal to 0.1 or less.

[0132] [B1-4] The pharmaceutical composition according to any one of [B1-1] to [B1-3], wherein the antigen-binding molecule is an IgG antibody or a heavy chain antibody.

[0133] [B1-5] The pharmaceutical composition according to any one of [B1-1] to [B1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a protease-cleavable linker, and the antigen-binding molecule whose linker has been cleaved by a protease comprises the variable region or an antigen-binding fragment thereof.

[0134] [B1-6] The pharmaceutical composition according to any one of [B1-1] to [B1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a protease-cleavable linker located near the boundary between the variable region and the constant region or near the boundary between CH1 and CH2 within the constant region.

[0135] [B1-7] The pharmaceutical composition according to any one of [B1-1] to [B1-6], wherein the antigen-binding molecule is an antibody comprising a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is VL, VH, or an antigen-binding fragment thereof of the antibody.

[0136] [B1-8] The pharmaceutical composition according to any one of [B1-1] to [B1-6], wherein the antigen-binding molecule is a heavy chain antibody comprising a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is VHH of the heavy chain antibody.

[0137] [B1-9] The pharmaceutical composition according to any one of [B1-1] to [B1-8], wherein the antigen-binding molecule after linker cleavage is scFv, Fv, Fab, Fab', F(ab')2, VH, or VHH.

[0138] [B1-10]The pharmaceutical composition according to any one of [B1-1] to [B1-9], wherein the bispecific antibody recognizes the cleaved linker, a portion of the linker, or a portion comprising the linker.

[0139] [B1-11] The pharmaceutical composition according to any one of [B1-1] to [B1-10], wherein the ratio of the KD value of the bispecific antibody to the antigen-binding molecule after linker cleavage to the KD value of the bispecific antibody to the antigen-binding molecule before linker cleavage (KD(after cleavage) / KD(before cleavage)) is less than 0.1 or less than 0.01.

[0140] [B1-12] The pharmaceutical composition according to any one of [B1-1] to [B1-11], wherein the protease-cleavable linker comprises a protease-cleaving sequence.

[0141] [B1-13] The pharmaceutical composition according to any one of [B1-1] to [B1-12], wherein the protease-cleavable linker comprises a peptide having a protease-cleaving sequence having any one of sequence numbers 1 to 725.

[0142] [B1-14] The pharmaceutical composition according to any one of [B1-1] to [B1-13], wherein the protease cleavable linker further comprises a flexible linker.

[0143] [B1-15] The pharmaceutical composition according to any one of [B1-1] to [B1-14], wherein the protease is a target tissue-specific protease.

[0144] [B1-16] The pharmaceutical composition according to any one of [B1-1] to [B1-15], wherein the target cell is a tumor cell and the protease is a tumor protease.

[0145] [B1-17] The pharmaceutical composition according to any one of [B1-1] to [B1-16], for use in the treatment or prevention of cancer.

[0146] [B1-18] The pharmaceutical composition according to [B1-17], wherein the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, germ cell tumor and leukemia.

[0147] [B1-19] The pharmaceutical composition according to [B1-17], wherein the cancer is selected from the group consisting of B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia and Hodgkin lymphoma.

[0148] [B1-20] The pharmaceutical composition according to any one of [B1-1] to [B1-19], for use in bispecific antibody therapy.

[0149] [B2-1] A bispecific antibody comprising 1) a first...1) An antibody variable region; and 2) a second antibody variable region having binding activity for an antigen-binding molecule containing a protease-cleavable linker;

[0150] The antigen-binding molecule, after being cleaved by a protease linker, has binding activity for an antigen expressed on the surface of a target cell, and the bispecific antibody can bind to the target cell by binding to the antigen-binding molecule after linker cleavage.

[0151] [B2-2] The bispecific antibody according to [B2-1], wherein the molecule expressed on the surface of the T cell is CD3.

[0152] [B2-3] The bispecific antibody according to [B2-1], wherein the antibody variable region having binding activity for a molecule expressed on the surface of the T cell binds to CD3ε.

[0153] [B2-4] The bispecific antibody according to any one of [B2-1] to [B2-3], which recognizes the protease-cleaved linker, a portion of the linker, or a portion containing the linker.

[0154] [B2-5] The bispecific antibody according to any one of [B2-1] to [B2-4], comprising an Fc region with low binding activity to the Fc γ receptor.

[0155] [B2-6] The bispecific antibody according to any one of [B1-1] to [B1-5], wherein the protease-cleavable adapter comprises a peptide having any protease-cleaving sequence.

[0156] [B2-7] The bispecific antibody according to any one of [B1-1] to [B1-5], wherein the protease-cleavable adapter comprises a peptide having any protease-cleaving sequence numbered 1 to 725.

[0157] [B2-8] The bispecific antibody according to any one of [B1-1] to [B1-7], wherein the protease-cleavable adapter further comprises a flexible adapter.

[0158] [B2-9] The bispecific antibody according to any one of [B2-1] to [B2-8] is an IgG antibody.

[0159] [B3-1] Nucleic acid encoding a bispecific antibody according to any one of [B2-1] to [B2-9].

[0160] [B3-2] Vector comprising the nucleic acid according to [B3-1].

[0161] [B3-3] Cell comprising the vector according to [B3-2].

[0162] [B3-4] A method of manufacturing a bispecific antibody, comprising culturing cells according to [B3-3] and recovering the bispecific antibody from the culture supernatant.

[0163] [C1-1] A pharmaceutical composition for use in combination with an antigen-binding molecule, comprising a composition characterized by enhanced antibody-dependent cellular cytotoxicity (ADCC). (See specification 10 / 139 pages 13 CN 121818917 A)The pharmaceutical composition of IgG antibody,

[0164] the antigen-binding molecule contains a protease-cleavable linker, which, after being cleaved by the protease, has binding activity to antigens expressed on the surface of target cells,

[0165] the IgG antibody contains an antibody variable region that has binding activity to the antigen-binding molecule after the linker has been cleaved by the protease,

[0166] the IgG antibody can bind to target cells by binding to the antigen-binding molecule after the linker has been cleaved.

[0167] [C1-2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of an IgG antibody characterized by enhanced antibody-dependent cellular cytotoxicity (ADCC),

[0168] the antigen-binding molecule comprising a protease-cleavable linker, which, upon protease cleavage, has binding activity to an antigen expressed on the surface of a target cell,

[0169] the IgG comprising an antibody variable region having binding activity to the antigen-binding molecule after protease cleavage,

[0170] the IgG antibody is able to bind to the target cell by binding to the linker-cleaved antigen-binding molecule.

[0171] [C1-3] The pharmaceutical composition according to [C1-1] or [C1-2] wherein the ratio of the KD value of the linker-cleaved antigen-binding molecule to the antigen (KD(after cleavage) / KD(before cleavage)) to the KD value of the linker-cleaved antigen-binding molecule to the antigen is 0.1 or less.

[0172] [C1-4] The pharmaceutical composition according to any one of [C1-1] to [C1-3], wherein the antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a single-domain antibody.

[0173] [C1-5] The pharmaceutical composition according to any one of [C1-1] to [C1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a protease-cleavable linker, wherein the antigen-binding molecule with the linker cleaved by a protease comprises the variable region or an antigen-binding fragment thereof.

[0174] [C1-6] The pharmaceutical composition according to any one of [C1-1] to [C1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a protease-cleavable linker located near the boundary between CH1 and CH2 within the constant region.

[0175] [C1-7] The pharmaceutical composition according to any one of [C1-1] to [C1-6], wherein the antigen-binding molecule is an antibody containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is VL, VH, or an antigen-binding fragment of the antibody.

[0176] [C1-8] The pharmaceutical composition according to any one of [C1-1] to [C1-6], wherein the antigen-binding molecule...The component is a heavy chain antibody containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is the VHH of the heavy chain antibody.

[0177] [C1-9] The pharmaceutical composition according to any one of [C1-1] to [C1-8], wherein the antigen-binding molecule after linker cleavage is scFv, Fv, Fab, Fab', F(ab')2, VH or VHH.

[0178] [C1-10] The pharmaceutical composition according to any one of [C1-1] to [C1-9], wherein the IgG antibody recognizes the cleaved linker, a portion of the linker, or a portion containing the linker.

[0179] [C1-11] The pharmaceutical composition according to any one of [C1-1] to [C1-10], wherein the ratio of the KD value of the IgG antibody to the antigen-binding molecule after linker cleavage to the KD value of the IgG antibody to the antigen-binding molecule before linker cleavage (KD(after cleavage) / KD(before cleavage)) is 0.1 or less.

[0180] [C1-12] The pharmaceutical composition according to any one of [C1-1] to [C1-11], wherein the protease-cleavable adapter comprises a protease-cleaving sequence.

[0181] [C1-13] The pharmaceutical composition according to any one of [C1-1] to [C1-12], wherein the protease-cleavable adapter comprises a peptide having a protease-cleaving sequence having any sequence number 1 to 725.

[0182] [C1-14] The pharmaceutical composition according to any one of [C1-1] to [C1-13], wherein the protease-cleavable adapter further comprises a flexible adapter.

[0183] [C1-14] The pharmaceutical composition according to any one of [C1-1] to [C1-13], wherein the protease is a protease specifically expressed in the target tissue.

[0184] [C1-15] The pharmaceutical composition according to any one of [C1-1] to [C1-14], wherein the target cell is a tumor cell and the protease is a tumor protease.

[0185] [C1-16] The pharmaceutical composition according to any one of [C1-1] to [C1-15] is used for treatment or prevention utilizing antibody-dependent cytotoxicity (ADCC) or antibody-dependent phagocytic capacity (ADCP).

[0186] [C1-17] The pharmaceutical composition according to any one of [C1-1] to [C1-16] is used for treatment or prevention of cancer.

[0187] [C1-18] The pharmaceutical composition according to [C1-17], wherein the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, germ cell tumor, and leukemia.

[0188] [C1-19]According to the pharmaceutical composition of [C1-17], wherein the cancer is selected from the group consisting of B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma.

[0189] [C1-20] The pharmaceutical composition according to any one of [C1-1] to [C1-19] is used in IgG antibody therapy.

[0190] [C2-1] An IgG antibody comprising an antibody variable region having binding activity for an antigen-binding molecule containing a protease-cleavable linker,

[0191] the antigen-binding molecule having binding activity for an antigen expressed on the surface of a target cell after being cleaved by a protease linker, and the IgG antibody being able to bind to the target cell by binding to the antigen-binding molecule after being cleaved by the linker.

[0192] [C2-2] The IgG antibody according to [C2-1] has enhanced antibody-dependent cellular cytotoxicity (ADCC).

[0193] [C2-3] The IgG antibody according to [C2-1] or [C2-2] comprises an Fc region with increased binding activity to the Fc γ receptor.

[0194] [C2-4] The pharmaceutical composition according to any one of [C1-1] to [C1-3], wherein the protease-cleavable linker comprises a protease-cleaving sequence.

[0195] [C2-5] The pharmaceutical composition according to any one of [C1-1] to [C1-4], wherein the protease-cleavable linker comprises a peptide having a protease-cleaving sequence having any sequence number 1 to 725.

[0196] [C2-6] The pharmaceutical composition according to any one of [C1-1] to [C1-5], wherein the protease-cleavable linker further comprises a flexible linker.

[0197] [C3-1] A nucleic acid encoding an IgG antibody as described in any one of [C2-1] to [C2-6].

[0198] [C3-2] A vector comprising the nucleic acid according to [C3-1].

[0199] [C3-3] A cell comprising the vector according to [C3-2].

[0200] [C3-4] A method for manufacturing an IgG antibody, comprising culturing cells according to [C3-3] and recovering the IgG antibody from the culture supernatant.

[0201] [D1-1] A pharmaceutical composition comprising a secondary molecule for use in combination with the administration of a primary molecule.

[0202] (Page 12 / 139, CN 121818917 A)The primary molecule contains a protease-cleavable linker and has the ability to bind to the target antigen after linker cleavage.

[0203] The secondary molecule has the ability to bind to the primary molecule after linker cleavage, and can bind to cells expressing the target antigen by binding to the primary molecule after linker cleavage.

[0204] [D1-2] A pharmaceutical composition comprising a primary molecule for use in combination with the administration of a secondary molecule.

[0205] The primary molecule contains a protease-cleavable linker and has the ability to bind to the target antigen after linker cleavage.

[0206] The secondary molecule has the ability to bind to the primary molecule after linker cleavage, and can bind to cells expressing the target antigen by binding to the primary molecule after linker cleavage.

[0207] [D1-3] The pharmaceutical composition according to [D1-1] or [D1-2], wherein the primary molecule is an antigen-binding molecule and the secondary molecule is a bispecific antibody, a chimeric receptor, or an IgG antibody characterized by enhanced antibody-dependent cytotoxicity.

[0208] [D1-4] The pharmaceutical composition according to any one of [D1-1] to [D1-3] is the pharmaceutical composition according to any one of [A1-1] to [A1-21], [B1-1] to [B1-20], and [C1-1] to [C1-20]. Brief Description of the Drawings

[0209] [Figure 1] Figure 1 illustrates the concept of antibody technology (Probody (registered trademark)) that expands tissue specificity and therapeutic dose range (therapeutic window) by conferring sensitivity to proteases expressed at lesion sites such as cancerous or inflamed tissues.

[0210] [Figure 2] Figure 2 is a schematic diagram showing the TDCC activity induced by an antibody that specifically recognizes an antigen generated by a protease cleavage linker.

[0211] [Figure 3] Figure 3 is a schematic diagram showing the ADCC activity induced by an antibody that specifically recognizes an antigen generated by a protease cleavage linker.

[0212] [Figure 4] Figure 4 is a schematic diagram showing the TDCC activity induced by an antibody that specifically recognizes an antigen generated by a protease cleavage linker.

[0213] [Figure 5] Figure 5 is a schematic diagram showing the ADCC activity induced by antibodies that specifically recognize antigens generated by protease cleavage linkers.

[0214] [Figure 6] Figure A in Figure 6 is a schematic diagram showing CAR-T cell-induced cytotoxicity by specifically recognizing protease-cleaved antigen-binding molecules.

[0215] [Figure 6] Figure B in Figure 6 is a schematic diagram showing CAR-T cell-induced cytotoxicity by specifically recognizing antigens exposed by protease cleavage.

[0216] [Figure 7] Figure 7 shows antibodies with inserted protease cleavage sequences (partial sequences of type II collagen) in vitro (in) protease (MMP13).The results of in vitro cleavage. Starting from the left lane, corresponding to wells 1-5, well 1 shows MWM, wells 2 and 3 show the pre-reaction and post-reaction of antigen-binding molecules without cleavage sequences, respectively, and wells 4 and 5 show the pre-reaction and post-reaction of antigen-binding molecules containing cleavage sequences with the addition of MMP13, respectively.

[0217] [Figure 8] Figure 8 shows the results of in vitro cleavage of antibodies recognizing tumor antigens by protease (IdeS). Starting from the left lane, corresponding to wells 6-10, well 6 shows MWM, wells 7 and 8 show the pre-reaction and post-reaction of antigen-binding molecules without cleavage sequences, respectively, and wells 9 and 10 show the pre-reaction and post-reaction of antigen-binding molecules containing cleavage sequences with the addition of IdeS, respectively. Specification 13 / 139 pages 16 CN 121818917 A

[0218] [Figure 9] Figure 9 (left) shows the results of treatment of antigen-binding molecules (antibodies) with inserted protease cleavage sequences (partial sequences of type II collagen) by tumor cell lines. Figure 9 (right) shows the results of treating antigen-binding molecules without the inserted protease cleavage sequence (partial sequence of type II collagen) with tumor cell lines.

[0219] [Figure 10] Figure 10 shows the Biacore assay results of antigen-binding molecules with the linker (partial sequence of type II collagen) cleaved by protease binding to anti-cleavage linker anti-CD3 bispecific antibody.

[0220] [Figure 11] Figure 11 shows the Biacore assay results of antigen-binding molecules (IgG1) cleaved by protease binding to anti-cleavage linker anti-CD3 bispecific antibody.

[0221] [Figure 12] Figure 12 shows the Biacore assay results of antigen-binding molecules with the linker (partial sequence of type II collagen) cleaved by protease binding to ADCC activity enhancing antibody.

[0222] [Figure 13] Figure 13 shows the Biacore assay results of antigen-binding molecules (IgG1) cleaved by protease binding to ADCC activity enhancing antibody.

[0223] [Figure 14] Figure 14 shows the Jurkat reporter gene analysis results using an antigen-binding molecule (anti-GPC3 antibody, IgG1) with a linker (a partial sequence of type II collagen) cleaved by a protease and an anti-linker anti-CD3 bispecific antibody.

[0224] [Figure 15] Figure 15 shows the Jurkat reporter gene analysis results using an antigen-binding molecule (anti-GPC3 antibody, IgG1) with a linker (a partial sequence of type II collagen) cleaved by a protease and an anti-linker anti-CD3 bispecific antibody.

[0225] [Figure 16] Figure 16 shows the Jurkat reporter gene analysis results with enhanced ADCC activity against an antigen-binding molecule with a linker (a partial sequence of type II collagen) cleaved by a protease.

[0226] [Figure 17] Figure 17 shows the results of Jurkat reporter gene analysis using antigen-binding molecules with enhanced ADCC activity against protease-cleaved linkers (partial sequences of type II collagen).

[0227] [Figure 18] Figure 18 shows the results of cytotoxicity analysis via human PBMCs using an anti-linker anti-CD3 bispecific antibody that recognizes protease-cleaved linkers (partial sequences of type II collagen) and an anti-GPC3 antibody with inserted protease-cleaving sequences.

[0228] [Figure 19] Figure 19 is a schematic diagram showing the configuration order of the vector construct and the structural elements of the 5' to 3' end framework units.

[0229] [Figure 20] Figure 20 shows the evaluation results of PC-10 cytotoxicity against MMP-cleaved linkers. The proportion of surviving cancer cells is calculated as the proportion of CD45- fraction cells in live cells. The horizontal axis represents the concentration of added antigen-binding molecules.

[0230] [Figure 21] Figure 21 shows the evaluation results of KYSE70 cytotoxicity against MMP-cleaved linkers that are almost unable to cleave them. The proportion of surviving cancer cells is calculated as the proportion of CD45- fractionated cells in living cells. The horizontal axis represents the concentration of added antigen-binding molecules. Detailed Description

[0231] Other features and advantages of this disclosure will become apparent from the following detailed description. However, based on this detailed description, those skilled in the art will of course make various changes and modifications within the spirit and scope of this disclosure, and therefore it should be understood that the detailed description and specific examples showing preferred embodiments of this disclosure are for illustrative purposes only.

[0232] Hereinafter, embodiments of this disclosure will be described with reference to the accompanying drawings.

[0233] The terms “substantially,” “about,” or “approximately” indicate a reasonable amount of deviation of the modified term that does not significantly change the final result, i.e., within the permissible error range of a particular value as determined by those skilled in the art. For example, “about,” according to practice in the art, indicates a permissible standard deviation. Alternatively, “about” may indicate a maximum ±20% of a value, preferably a maximum ±10%, more preferably a maximum ±5%, and even more preferably a maximum ±1%. Alternatively, specifically, in biological systems or processes, the term "about" in this specification (pages 14 / 139, CN 121818917 A) means within a single digit, preferably within twice, a given value. When a particular value is stated within the scope of this specification and the claims, unless otherwise stated, the term "about" implicitly means within the permissible range of error for that particular value in this context.

[0234] In the English translation of the scope of this specification and the claims, the singular forms "a," "an," and "the" include plural indicative terms unless expressly stated otherwise. Additionally, the term "or," unless expressly stated otherwise, should generally be used within the scope of meaning including "and / or."

[0235] The numerical ranges listed by endpoints in this disclosure include all numbers and fractions contained within the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). Additionally, it should be understood that all numbers and fractions may also be modified by the term "about". However, when it is explicitly stated that the numerical range represents an integer, it can be understood that the numerical range is a limiting list of integers contained within the range. Such a case, for example, 1 to 5 or 1 to 5, is understood to be a limiting list of 1, 2, 3, 4, and 5.

[0236] Further, the definitions and embodiments described in particular paragraphs, as understood by those skilled in the art, are intended to be applied, if appropriate, to other embodiments of the present specification. For example, various aspects of this disclosure are defined in more detail in the following sections. These aspects, unless expressly stated otherwise, may also be combined with any other one or more aspects. In particular, any feature shown as preferred or advantageous may also be combined with any other one or more features shown as preferred or advantageous.

[0237] In this disclosure, there are cases where an antigen-binding molecule comprising a region (“antigen-binding domain”) that binds to an antigen expressed by a target cell and a protease-cleavable linker is referred to as a “primary molecule”. The primary molecule, upon protease cleavage of the linker, releases an antigen-binding fragment that binds to an antigen (“target antigen”) expressed by the target cell or diseased cell. One of the antigen-binding molecules produced by protease cleavage of the linker is referred to as a “linker-cleaved antigen-binding molecule” or “linker-cleaved antigen-binding molecule”, comprising an antigen-binding domain and a portion of the cleaved linker. There are cases where a polypeptide that bridges target cells and effector cells and induces cytotoxicity is referred to as a “secondary molecule”. Examples of secondary molecules include, for instance, antibodies with ADCC activity having an antibody variable region capable of binding a linker-cleaved antigen-binding molecule, T-cell retargeting antibodies having an antibody variable region capable of binding a linker-cleaved antigen-binding molecule and an antibody variable region capable of binding a T-cell receptor complex, or chimeric receptors having an extracellular domain capable of binding a linker-cleaved antigen-binding molecule. The linker contained in the antigen-binding molecule contains a protease-cleaving sequence and has a cleavage site that can be cleaved by the protease. There are cases where a linker composed of peptides having a protease-cleaving sequence is referred to as a protease-cleaving linker.

[0238] In one embodiment, the antigen-binding molecule (primary molecule) containing the protease-cleavable linker is an antibody, more specifically, examples include IgG antibodies or heavy chain antibodies containing the protease-cleavable linker, and more preferably, examples include IgG1 antibodies, camel heavy chain antibodies (hcIgG), or shark heavy chain antibodies (IgNAR).

[0239] In one embodiment, the antigen-binding molecule resulting from the protease cleavage of the linker can be, for example, Fv,Fab, Fab', Fab'-SH, F(ab')2, minibody, single-chain antibody molecule (e.g., scFv), VHH, VH, and more specifically, Fab, scFv, VHH, VH.

[0240] The polypeptide in this invention generally refers to peptides and proteins with a length of about 4 amino acids or more. Furthermore, the polypeptide in this invention is generally a polypeptide formed from an artificially designed sequence, but is not particularly limited; for example, it may also be a polypeptide derived from a biological source. Additionally, it may be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, etc. Further, fragments of the aforementioned polypeptides are also included in the polypeptides of this invention.

[0241] In this specification, amino acids, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V, are represented by single-letter codes, three-letter codes, or both. When representing an amino acid at a specific position, it is appropriate to use a combination of numbers representing the specific position and single-letter or three-letter codes for the amino acid. For example, in CN 121818917 A, page 15 / 139 of the specification, the amino acid 37V in a single-domain antibody represents Val at position 37, indicated by the Kabat number.

[0242] To alter the amino acid sequence of peptides such as antibodies, site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR, or other known methods, can be appropriately employed. Additionally, several known methods can be used to alter the amino acid sequence by replacing it with an amino acid other than the natural one (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100(11), 6353-6357). For example, cell-free translation systems such as Clover Direct (Protein Express) can be appropriately used, where the complementary succinate repressor tRNA of the UAG codon (succinate codon), one of the stop codons, contains tRNA that binds to non-natural amino acids. In this specification, substitutions may be exemplified as changes, but are not limited thereto.

[0243] In this specification, the term "and / or" used to indicate a change in the position of an amino acid includes all suitable combinations of "and" and "or". Specifically, for example, "replacing the amino acid at position 37, position 45, and / or position 47" includes changes in the position of the following amino acids: (a) position 37, (b) position 45, (c) position 47, (d) positions 37 and 45, (e) positions 37 and 47, (f) positions 45 and 47, and (g) positions 37, 45, and 47.

[0244] In this specification, as a way of indicating a change in amino acids, it is appropriate to use a single-letter or three-letter code for the amino acid before and after the change, with the numbers indicating the specific position listed together. For example, the change referred to as F37V or Phe37Val when adding an amino acid replacement in the variable region or single-domain antibody indicates that the 37th position Phe, as indicated by the Kabat number, is replaced with Val. That is, the number indicates the amino acid position shown in the Kabat number, the single-letter or three-letter code of the amino acid before it indicates the amino acid before the substitution, and the single-letter or three-letter code of the amino acid after it indicates the amino acid after the substitution. Similarly, the change called P238A or Pro238Ala used when adding the substitution of amino acids in the Fc region contained in the antibody constant region indicates that the Pro at position 238 shown in the EU number is replaced with Ala. That is, the number indicates the amino acid position shown in the EU number, the single-letter or three-letter code of the amino acid before it indicates the amino acid before the substitution, and the single-letter or three-letter code of the amino acid after it indicates the amino acid after the substitution.

[0245] In this specification, the term "antibody" is used in the broadest sense, as long as it exhibits the desired antigen-binding activity, including, but not limited to, various antibody structures containing monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, and antibody fragments.

[0246] "Antibody fragment" refers to a molecule other than the complete antibody that contains a portion of the complete antibody that binds to the antigen that binds to the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibodies, single-chain antibody molecules (such as scFv), and multispecific antibodies formed from antibody fragments.

[0247] The terms “full-length antibody,” “complete antibody,” and “all antibody” are used interchangeably in this specification and refer to antibodies having a structure substantially similar to that of natural antibodies, or antibodies having a heavy chain containing an Fc region as defined in this specification.

[0248] The terms “variable region” or “variable domain” refer to the heavy or light chain of an antibody involved in the binding of the antibody to an antigen.The variable domains of the heavy and light chains of the antibody (VH and VL, respectively) typically have similar structures where each variable domain contains 4 conserved framework regions (FRs) and 3 complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A VH or VL domain may adequately provide antigen-binding specificity.

[0249] As used in this specification, the terms “complementarity-determining region” or “CDR” refer to regions that are hypervariable in the sequence and / or form structurally fixed loops (“hypervariable loops”), and / or antigen contact residues (“antigen contacts”), as described in the specification of the variable domains of the antibody, page 16 / 139, CN 121818917 A. Typically, an antibody contains 6 CDRs: 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3). The CDRs illustrated in this specification include the following:

[0250] (a) a supervariable ring generated at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));

[0251] (b) a CDR generated at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0252] (c) Antigen contacts generated at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); and

[0253] (d) Containing HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35b (H1), 47-58 (H2), and 93-101 (H3)Combinations of (a), (b), and / or (c) of (H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).

[0254] Unless otherwise specified, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered according to the above-mentioned Kabat et al.

[0255] “Framework” or “FR” refers to the variable domain residues other than the complementarity-determining region (CDR) residues. The FR of the variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Correspondingly, the sequences of CDR and FR typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0256] In this specification, the terms “constant region” or “constant domain” refer to the portion of the antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) called a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of natural antibodies may also be classified into type 1 of type 2, called kappa (κ) or lambda (λ), based on the amino acid sequence of their constant domains.

[0257] In this specification, the term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the Fc region of the natural sequence and the variant Fc region. In one embodiment, in the case of human IgG1, the Fc region of the heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present. Unless otherwise specified in this specification, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU Index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0258] The “class” of an antibody refers to the type of constant structural domain or constant region possessed by the antibody heavy chain. There are 5 major classes of antibodies.Classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0259] In this specification, "antigen-binding domain" is limited to the region that binds to the target antigen. The antigen-binding domain may also be any domain of any structure that binds to the target antigen. Examples of such domains include the heavy chain variable region (VH) and light chain variable region (VL) of antibodies, single-domain antibodies (sdAbs), the A domain of approximately 35 amino acids contained in the cell membrane protein Avimer (international publication WO2004 / 044011, WO2005 / 040229), Adnectin (international publication WO2002 / 032925), which contains the 10Fn3 domain, a protein-binding domain in the cell membrane glycoprotein fibronectin, and Affibody (international publication WO1995 / ). 001937), the exposed region on the surface of ankyrin repeat (AR) molecules with a structure containing 33 amino acid residues of turn and two antiparallel helical and loop subunits, and the region of four loops on one side of a twisted barrel structure supported by eight highly conserved antiparallel chains in lipid transport protein molecules such as neutrophil gelatinase-associated lipocalin (NGAL) in the central direction, and Anticalin et al. (International Publication WO2003 / 029462), and the variable lymphocyte receptor without immunoglobulin structure in jawless animals such as lampreys and hagfish as part of the acquired immune system. The horseshoe-shaped structure containing the repetitive accumulation of leucine-rich repeat (LRR) modules of the receptor (VLR) is a recessed region of parallel lamellar structures (International Publication WO2008 / 016854), but is not limited thereto.

[0260] As a suitable example of the antigen-binding domain of the present invention, examples can be given of structures formed solely by this antigen-binding domain.Antigen-binding domains that can bind antigens to molecules, and antigen-binding domains that can bind antigens independently after being freed from other peptides. Examples of such antigen-binding domains include single-domain antibodies, scFv, Fv, Fab, Fab', F(ab')2, etc., but are not limited thereto.

[0261] As one suitable example of the antigen-binding domain of the present invention, antigen-binding domains with a molecular weight of 60 kDa or less can be cited. Examples of such antigen-binding domains include single-domain antibodies, scFv, Fab, Fab', etc., but are not limited thereto. Antigen-binding domains with a molecular weight of 60 kDa or less are usually present in the blood as monomers and are highly likely to be cleared by the kidneys (see J Biol Chem, 1988 Oct 15; 263(29): 15064-70).

[0262] On the other hand, as one suitable example of the antigen-binding domain of the present invention, antigen-binding domains with a half-life of 12 hours or less in the blood can be cited. Examples of such antigen-binding domains include single-domain antibodies, scFv, Fab, Fab', etc., but are not limited thereto.

[0263] As one suitable example of the antigen-binding domain of the present invention, a single-domain antibody (sdAb) can be cited.

[0264] In this specification, the term "single-domain antibody" is used to refer to any antibody that can exert antigen-binding activity by its domain alone, without limiting its structure. Compared with conventional antibodies such as IgG antibodies, which exhibit antigen-binding activity by forming a variable region through VH and VL pairing, it is known that single-domain antibodies do not pair with other domains, and single-domain antibodies can exert antigen-binding activity by their own domain structure alone. Single-domain antibodies usually have a low molecular weight and exist in monomeric form.

[0265] Examples of single-domain antibodies include, for example, antigen-binding molecules that inherently lack light chains, such as VHH of camels and VNAR of sharks, or antibody fragments containing all or part of the VH domain or all or part of the VL domain of the antibody, but are not limited thereto. Examples of single-domain antibodies comprising all or part of the VH / VL domain of an antibody fragment include, for example, single-domain antibodies artificially prepared from human antibody VH or human antibody VL as described in U.S. Patent No. 6,248,516 B1, but are not limited thereto. In some embodiments of the present invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0266] In the case where the single-domain antibody is a VHH or a single-domain VH antibody, the CDRs of the single-domain antibody typically include the following:

[0267] (a) Hypervariable rings generated at amino acid residues 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));

[0268] (b) CDRs generated at amino acid residues 31-35b (CDR1), 50-65 (CDR2), and 95-102 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0269] (c) Antigen contacts generated at amino acid residues 30–35b (CDR1), 47–58 (CDR2), and 93–101 (CDR3) (MacCallum et al., J. Mol. Biol. 262: 732–745 (1996)); and

[0270] (d) Combinations of (a), (b), and / or (c) comprising CDR amino acid residues 26–35 (CDR1), 26–35b (CDR1), 49–65 (CDR2), 93–102 (CDR3), or 94–102 (CDR3).

[0271] In the case of a single-domain antibody being a single-domain VL antibody, the CDR of the single-domain antibody typically includes the following:

[0272] (a) a hypervariable ring generated at amino acid residues 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));

[0273] (b) a CDR generated at amino acid residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0274] (c) Antigen contacts generated at amino acid residues 27c-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) (MacCallum et al., J.).Mol. Biol. 262:732-745 (1996)); and

[0275] (d) combinations of (a), (b), and / or (c) containing CDR amino acid residues 46-56 (CDR2), 47-56 (CDR2), and 48-56 (CDR2). Unless otherwise stated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered in this specification according to the above-mentioned Kabat et al.

[0276] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies, or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, for example, camelids and transgenic animals into which a gene capable of producing single-domain antibodies has been introduced. Camelids include, but are not limited to, camels, llamas, alpacas, dromedary camels, and guanacos. Examples of transgenic animals in which genes capable of producing single-domain antibodies have been introduced include those described in International Publication No. WO2015 / 143414 and US Patent Publication No. US2011 / 0123527A1, but are not limited thereto. Humanized single-domain antibodies can also be obtained by replacing the scaffold sequence of a single-domain antibody obtained from an animal with a human germline sequence or a similar sequence. Humanized single-domain antibodies (e.g., humanized VHH) are also one embodiment of the single-domain antibody of the present invention.

[0277] Furthermore, single-domain antibodies can be obtained from a polypeptide library containing single-domain antibodies via ELISA, panning, etc. Examples of peptide libraries containing single-domain antibodies include, for instance, natural antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11). (1691–1701), but not limited to this.

[0278] In this specification, "antigen" is limited to those containing epitopes that bind to an antigen-binding domain. Suitable examples of antigens may include, but are not limited to, peptides, polypeptides, and proteins derived from animals or humans. In this invention, target antigens are antigens used to treat diseases originating from target tissues. Suitable preferred examples may include, for instance, molecules expressed on the surface of target cells (such as cancer cells or inflammatory cells), molecules expressed on the surface of other cells in tissues containing target cells, molecules expressed on the surface of cells that have an immune effect on target cells and tissues containing target cells, macromolecules present in the stroma of tissues containing target cells, etc., but are not limited to these. The antigens shown below can be cited as examples of target antigens.

[0279] The antigens are the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, α1-antitrypsin, α-V / β-1 antagonist, ANG, Ang. APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artesunate, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-Lymphocyte Stimulating Factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, Osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6 Vgr‑1, BMP‑7(OP‑1), BMP‑8(BMP‑8a, OP‑2), BMPR, BMPR‑IA(ALK‑3),BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, β-NGF, BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, Complement Factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, Calcitonin, cAMP, Carcinoembryonic antigen (CEA), Cancer-associated antigen, Cathepsin A, Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1 CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN‑1, COX, C‑Ret, CRG‑2, CT‑1, CTACK, CTGF, CTLA‑4, PD1, PDL1, LAG3,TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin, tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulator (decay accelerating factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, coagulation factor IIa, coagulation factor VII, coagulation factor VIIIc, coagulation factor IX, fibroblast activation protein (FAP), Fas... FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, Instruction manual 20 / 139 pages 23 CN 121818917 A GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP- 12. CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNFGFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 ring, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL- 6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, Interferon (INF)-α, INF-β, INF-γ, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, Integrin α5 (αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, IP-10, I-TAC, JE, Kallikrein 2. Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, keratinKibstein-Glycerin (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing growth hormone, lymphotoxin β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Muc1, MUC18, Mullerian inhibiting substance, Mug, Musk, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophic Factor-3,-4, or-6, Neurturin, Nerve Growth Factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1 OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, relaxin proinsulin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor,RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β 3. TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1, DR4), TNFRSF10B (TRAIL R2, DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3, DcR1, LIT, TRID), TNFRSF10D (TRAIL R4, DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R) FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A (TNF RI CD120a, p55‑60), TNFRSF1B (TNF RII CD120b, p75‑80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFCTNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL) TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-α conectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand, gp34, TXGP1), TNFSF5 (CD40 ligand, CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70). TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-like receptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase.VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, Oxidized LDL, PCSK9, Prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, High Molecular Weight Kininogen, IL-31, IL-31R, Nav1.1 Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, Factor B, Factor D, Factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, coagulation factor V, coagulation factor Va, coagulation factor VII, coagulation factor VIIa, coagulation factor VIII, coagulation factor VIIIa, coagulation factor IX, coagulation factor IXa, coagulation factor X, coagulation factor Xa, coagulation factor XI, coagulation factor XIa, coagulation factor XII, coagulation factor XIIa, coagulation factor XIII, coagulation factor XIIIa, TFPI, antithrombin III, EPCR, coagulation regulatory protein(thrombomodulin), TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, S1P, and receptors for hormones and growth factors.

[0280] In the examples of the above antigens, although receptors are also described, these receptors can be used as antigens that bind to the antigen-binding domain of the present invention, even if they are present in biological fluids as soluble type 25 CN 121818917 A on page 22 / 139 of the specification. As a non-limiting embodiment of such a soluble receptor, an example is the soluble IL-6R protein described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968).

[0281] In the above examples of antigens, there are membrane-type molecules expressed on the cell membrane and soluble molecules secreted from the cell to the extracellular space. In the case where the antigen-binding domain of the present invention binds to a soluble molecule secreted from the cell, it is suitable for the antigen-binding domain to have neutralizing activity.

[0282] There is no limitation on the solution in which the soluble molecule is present; the soluble molecule can be present in biological fluids, i.e., all fluids filling blood vessels or tissues and between cells in a living body. In a non-limiting embodiment, the soluble molecule bound to the antigen-binding domain of the present invention can be present in extracellular fluid. Extracellular fluid refers to the components of bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, bone marrow fluid, puncture fluid or joint fluid in vertebrates, alveolar fluid (bronchopneumonic lavage fluid), ascites, pleural fluid, cardiac sac fluid, cystic fluid, or aqueous humor (fluid in various glandular cavities resulting from active transport and secretion of cells, and fluid in other body cavities of the digestive tract).

[0283] The term "tumor antigen" refers to an antigen expressed in cancer cells, which is recognized as an antigenic biological molecule whose expression is related to malignant changes in cells. The tumor antigens disclosed herein include tumor-specific antigens (antigens that exist only in tumor cells and are not found in other normal cells) and tumor-associated antigens (antigens that also exist in other organs and tissues or xenogeneic and allogeneic normal cells, or antigens expressed during occurrence and / or differentiation). In addition, abnormal glycans that appear on the cell surface or protein molecules during cell carcinogenesis are also tumor antigens, also known as cancer glycan antigens. In one embodiment of the present invention, the target antigen is a tumor antigen.

[0284] Examples of tumor antigens that can be aptly cited include GPC3 (Int J Cancer. (2003) 103(4), 455-65), which belongs to the GPI-anchored receptor family but is expressed in several cancers, primarily hepatocellular carcinoma, and EpCAM (Proc Natl Acad Sci US A. (1989) 86(1), 27-31), which is expressed in multiple cancers, primarily lung cancer (their polynucleotide sequences are recorded as RefSeq accession number NM_002354.2 and their polypeptide sequences as RefSeq accession number NP_002345.2, respectively), EGFR, CA19-9, CA15-3, and sialic acid. SSEA-1 (SLX), Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), tumor antigen-125 (CA-125), calreticulin, MUC-1, MUC-16, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), and fluid proteins in gross cystic diseases (liquid proteins in giant cystic disease). Proteins including GCDFP-15, HMB-45 antigen, Melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, nerve-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, pyruvate kinase isoenzyme M2 dimer (tumor M2-PK), GD2 (ganglioside G2), EGFRvIII (epidermal growth factor receptor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor γ variable reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of prostate 1), TROP-2, Claudin6, and RNF43a. Abnormal ras protein, or abnormal p53 protein, integrin αvβ3 (CD61), galectin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma virus oncogene), or Ral-B, etc.

[0285] Further examples may also include thyroid-stimulating hormone receptor (TSHR); CD171; CS-1 (CD2 subgroup 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3 (aNeu5Ac(2-8) [Instructions 23 / 139 page 26 CN 121818917 A aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (TnAg); T antigen (TAg); Fms-like tyrosine kinase 3 (FLT3); CD38; CD44v6; B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL- 13Ra2); Interleukin-11 receptor α (IL-11Ra); Interleukin-2 receptor α (IL-2Ra); Prostate stem cell antigen (PSCA); Protease serine 21 (PRSS21); Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific embryogenic antigen-4 (SSEA-4); Neural cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); Proteasome (Macropain) subunit β-9 (LMP2); EphA2; Fucosyl GM1; Sialized Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; High molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 associated (TEM7R); claudin 6 (CLDN6); G protein-coupled receptor C5 family subtype D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; undifferentiated lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenaline receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR γSelective reading frame protein (TARP); Wilms' tumor protein (WT1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); cell surface receptor 2 that binds angiopoietin (Tie 2); melanoma testis antigen-1 (MAD-CT-1); melanoma testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; p53 variant; human telomerase reverse transcriptase (hTERT); sarcoma translocation cleavage site; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); Paired Box Proteins Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myeloma virus oncogene neuroblastoma-derived homologue (MYCN); Ras homologue family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); T cell 3-recognized squamous cell tumor antigen (SART3); Paired Box protein Pax-5 (PAX5); proacrosin-binding protein p32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase ankylosing protein 4 (AKAP-4); synovial sarcoma, X-cleavage site 2 (SSX2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin receptor-like subfamily A member 2 (LILRA2); CD300 molecular-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); Mucin-like hormone receptor-like 2 containing an EGF-like pattern (EMR2); Lymphocyte antigen 75 (LY75); Phosphatidylinositol polysaccharide-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin λ-like polypeptide 1 (IGLL1), etc.

[0286] "MHC antigens" are gene products of the major histocompatibility complex (MHC), among which the glycoproteins expressed on the cell membrane are mainly classified as MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, while MHC class II antigens include HLA-DR, -DQ, and -DP. Additionally, they also include tumor antigens presented by these MHC antigens.Peptides. Complexes of MHC that present tumor antigens such as GP100, MART-1, MAGE-1, or variants of RAS or p53 are also considered to be tumor antigens.

[0287] "Differentiation antigens" are a general term for cell surface molecules that grow and decline along with the differentiation of macrophages, T cells, B cells, etc., from bone marrow stem cells. Differentiation antigens may include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57. (See instruction manual, page 24 / 139, CN 121818917 A) CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD70, CD71, CD73, CD95, CD99, CD102, CD106, CD117, CD122, CD126, CDw130.

[0288] The term "tumor" generally refers to any mass on or inside the body that feels like a lump or has parts of different colors. Tumors can be malignant, characterized by three features: autonomous proliferation, infiltration or metastasis, and cachexia; or benign, characterized only by autonomous proliferation. Malignant tumors, or "cancer," refer to diseases characterized by the uncontrolled growth of abnormal cells. Cancer cells have the potential to spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers described in this disclosure include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and similar cancers. The terms "tumor" and "cancer" are used synonymously in this disclosure, for example, both terms encompass solid and fluid-like, such as diffuse or circulating tumors. The terms "cancer" or "tumor," as used in this disclosure, include precancerous cancers and tumors of the same malignancy.

[0289] Examples of cancers that can be used as anticancer agents or cancer treatment methods described later in this disclosure include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small bowel cancer, and stomach cancer.In addition to cancers such as esophageal cancer, gallbladder cancer, testicular cancer, and ovarian cancer, or cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, there are also sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant Schwann cell tumor, osteosarcoma, and soft tissue sarcoma, or blastomas or germ cell tumors such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatic blastoma, pleuropulmonary blastoma, and retinoblastoma, lymphoma, or leukemia.

[0290] In one embodiment, the tumor antigen associated with the cancer type is a marker expressed by both normal cells and cancer cells, such as a systemic marker, like CD19 on B cells. In a particular embodiment, the tumor antigen of this disclosure is derived from cancer, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and allogeneic cancers. In one embodiment, the tumor antigen is an antigen common to a specific proliferative disease. In one embodiment, the cancer-associated antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, for example, by 1-fold, 2-fold, 3-fold, or more compared to normal cells. In some embodiments, the cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed in normal cells. In one embodiment, the cancer-associated antigen is expressed exclusively on the cell surface of cancer cells in its full length or in fragments (such as MHC / peptides), but is neither synthesized nor expressed on the surface of normal cells. In some embodiments, the chimeric receptor and TRAB of this disclosure comprise CARs and TRABs containing an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the grooves of major histocompatibility complex (MHC) class I molecules and are recognized by T-cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed in all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes are representative of specific cell surface targets for immunotherapy. In the case of human leukocyte antigen (HLA)-A1 or HLA-A2, TCR-like antibodies targeting peptides derived from viruses or tumor antigens have been described (e.g., Sastry et al., J Virol. 2011).85(5): 1935-1942; Sergeeva et al., Bood, 2011 117(16): 4262-4272; Verma et al., J Immunol 2010 184(4): 2156-2165; Willemsen et al., Gene Ther 2001 8(21): 1601-1608; Dao et al., Sci Transl Med 2013 5(176): 176ra33; Tassev et al., Instructions 25 / 139 pages 28 CN 121818917 A Cancer Gene Ther 2012 19(2): 84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0291] An epitope, meaning an antigenic determinant present in an antigen, refers to a site on the antigen that binds to the antigen-binding domain disclosed in this specification. Therefore, an epitope can be defined, for example, based on its structure. Additionally, an epitope can be defined based on the binding activity of the antigen-binding domain that recognizes the epitope to the antigen. In the case where the antigen is a peptide or polypeptide, the epitope can also be determined based on the amino acid residues constituting the epitope. Furthermore, in the case where the epitope is a glycan, the epitope can also be determined based on the specific glycan structure.

[0292] A linear epitope is an epitope that contains an amino acid primary sequence that is recognized. A linear epitope typically contains at least 3, most commonly at least 5, for example about 8 to 10, or 6 to 20 amino acids in its intrinsic sequence.

[0293] In contrast to a linear epitope, a stereoepitope is not an epitope containing an amino acid primary sequence that is a single defining component of the recognized epitope (e.g., the amino acid primary sequence is not necessarily an epitope recognized by an antibody for a specific epitope). Stereotopes, compared to linear epitopes, can contain an increased number of amino acids. Regarding the recognition of stereotopes, the antigen-binding domain recognizes the tertiary structure of a peptide or protein. For example, when a protein molecule folds to form a tertiary structure, the amino acids and / or polypeptide backbone forming the stereotope are arranged side-by-side, allowing the antibody to recognize the epitope. Methods for determining the stereostructure of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin tagging and electromagnetic paramagnetic resonance spectroscopy, but are not limited to these. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0294] The structure of the antigen-binding domain that binds to the epitope is called the complementary site. The epitope and the complementary site are stably bound by hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc., acting between them. The structure between the epitope and the complementary site...The combined force is called affinity. The sum of the binding forces when multiple antigens and multiple antigen-binding domains bind is called affinity. When antibodies containing multiple antigen-binding domains (i.e., multivalent) bind to multiple epitopes, the affinity is higher than the affinity due to the affinity addition effect.

[0295] In a specific embodiment, the antigen-binding domains provided in this specification have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., below 10⁻⁸ M, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M).

[0296] In the following, the method for confirming the binding of an antigen-binding domain of an antigen or an antigen-binding molecule containing an antigen-binding domain to an epitope can be suitably implemented according to the following examples.

[0297] For example, the recognition of a linear epitope present in an antigen molecule by an antigen-binding domain can be confirmed, for example, as follows. For the above purpose, a linear peptide formed by the amino acid sequence constituting the extracellular domain of an antigen is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering methods using a region in the cDNA of an antigen that encodes the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide formed by the amino acid sequence constituting the extracellular domain to the antigen-binding domain of the antigen is evaluated. For example, the binding activity of the antigen-binding domain to the peptide can be evaluated by ELISA using a fixed linear peptide as the antigen. Alternatively, the binding activity to the linear peptide can be determined based on the level of inhibition caused by the linear peptide in the binding of the antigen-binding domain to an antigen-expressing cell. Through these tests, the binding activity of the antigen-binding domain to the linear peptide can be determined.

[0298] Furthermore, the recognition of a stereoeptopy by the antigen-binding domain of a certain antigen can be confirmed as follows. For the above purpose, cells expressing a certain antigen are prepared. For example, when the antigen-binding domain of a certain antigen comes into contact with a cell expressing the antigen, it binds strongly to the cell, but on the other hand, the antigen-binding domain substantially does not bind to linear peptides formed by a fixed amino acid sequence constituting the extracellular domain of the antigen, or to linear peptides formed by denaturing the amino acid sequence constituting the extracellular domain of the antigen using a conventional denaturing agent such as guanidine. Here, substantially not binding means that the binding activity is 80% or less of the binding activity against human antigen-expressing cells, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less.

[0299] In addition, as a method to confirm the antigen-binding activity of the antigen-binding domain, for example, by radiolabeling an antigen...A method for determining Kd values ​​using a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a target antigen-binding domain and its antigen. For example, the binding affinity of an antigen-binding domain against an antigen in solution can be determined by equilibrating the antigen-binding domain with a minimum concentration of (125I) labeled antigen in the presence of a gradually increasing series of unlabeled antigens, followed by capturing the bound antigen with a plate coated with the antigen-binding domain (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)).

[0300] According to another embodiment, Kd is determined using the surface plasmon resonance method of BIACORE (registered trademark). For example, the assay is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RU) of antigen using BIACORE-2000 or BIACORE-3000 (BIAcore, Inc., Piscataway, NJ). In one embodiment, a carboxymethyl dextran biosensor chip (CM5, BIACORE, Inc.) is activated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), according to the supplier's guidelines. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8 before infusion at a flow rate of 5 μl / min to achieve approximately 10 reactive units (RU) of protein binding. Following antigen infusion, 1 M ethanolamine is injected to block unreacted groups. For kinetic determination, serially diluted (0.78 nM to 500 nM) two-fold dilutions of the antigen-binding domain in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20 (registered trademark)) surfactant are injected at approximately 25 °C. The binding rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Langmuir binding model (BIACORE evaluation software version 3.2), simultaneously fitting the sensor maps of binding and dissociation. The equilibrium dissociation constant (Kd) was calculated as the ratio of koff / kon. Furthermore, the apparent dissociation constant (Kd) can also be obtained using equilibrium analysis. These methods refer to the operating manual accompanying BIACORE. For example, see Chen et al., J. Mol. Biol. 293:865-881 (1999) or Methods Enzymol. 2000;323:325-40. Additionally, on surface plasmons...In the resonance assay, the fixed protein mass or the protein mass used for the reaction, temperature, and solution composition can be changed by those skilled in the art. According to the above-described surface plasmon resonance assay, when the ion velocity exceeds 106 M⁻¹ s⁻¹, the ion velocity can be determined by fluorescence extinction technique, which measures the increase or decrease of the fluorescence intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) of the antigen-binding domain at 25 °C in PBS pH 7.2 in the presence of gradually increasing concentrations of antigen, using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or a ThermoSpectronic SLM-AMINCO (registered trademark) series 8000 spectrophotometer using a stirred cuvette).

[0301] Furthermore, the antigen-binding activity of the antigen-binding domain can also be determined by known methods for measuring intermolecular interactions, such as electrochemiluminescence.

[0302] As a method for determining the binding activity of an antigen-binding domain against an antigen on cells expressing that antigen, an example can be the method described in the Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). That is, it can be evaluated using the principle of ELISA or FACS (fluorescence activated cell sorting) with cells expressing the antigen as the antigen.

[0303] In the ELISA form, the binding activity of an antigen-binding domain against an antigen on cells expressing that antigen is quantitatively evaluated by comparing the signal levels generated by the enzyme reaction. That is, the antigen-binding domain to be tested is added to an ELISA plate containing cells expressing the antigen, and an enzyme-labeled antibody that recognizes the antigen-binding domain to be tested is used to detect the antigen-binding domain to be bound to the cells. Alternatively, in FACS, a dilution series of the antigen-binding domain to be tested is prepared, and the binding activity of the antigen-binding domain to a specific antigen-expressing cell can be compared by determining the antibody binding titer against that antigen.

[0304] The binding of the antigen-binding domain to an antigen expressed on the surface of a cell suspended in a buffer solution or the like can be detected by flow cytometry. Devices known as flow cytometry include, for example, the following:

[0305] FACSCanto™ II

[0306] FACSAria™

[0307] FACSArray™

[0308] FACSVantage™ SE

[0309] FACSCalibur™ (both are trade names of BD Biosciences)

[0310] EPICS ALTRA HyPerSort

[0311] Cytomics FC 500

[0312] EPICS XL-MCL ADC EPICS XL ADC

[0313] Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter).

[0314] For example, as an example of a suitable method for determining the binding activity of an antigen-binding domain against an antigen, the following method can be cited. First, staining with a FITC-labeled secondary antibody that recognizes and reacts with cells expressing an antigen. The antigen-binding domain is diluted with an appropriate buffer to prepare it to the desired concentration for use. For example, it can be used at any concentration between 10 μg / ml and 10 ng / ml. Then, the fluorescence intensity and cell number are measured using FACSCalibur (BD Biosciences). The amount of antigen-binding domain binding to the cell is reflected by the fluorescence intensity obtained by analysis using CELL QUEST Software (BD Corporation), i.e., the geometric mean value. That is, by obtaining this geometric mean, the binding activity of the antigen-binding domain, represented by the amount of binding of the antigen-binding domain, can be determined.

[0315] When an antigen-binding domain for a certain antigen shares an epitope with another antigen-binding domain, it can be confirmed by the competition between the two for the same epitope. Competition between antigen-binding domains can be detected by methods such as cross-blocking assays. For example, competitive ELISA assays are preferred cross-blocking assays.

[0316] Specifically, in a cross-blocking assay, an antigen protein coated on a well of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen-binding domain, and then a test antigen-binding domain is added. The amount of the test antigen-binding domain binding to the antigen protein in the well is indirectly related to the binding ability of the candidate competing antigen-binding domain that competes for the same epitope. That is, the greater the affinity of the competing antigen-binding domain for the same epitope, the lower the binding activity of the tested antigen-binding domain to the pore coated with a certain antigen protein.

[0317] The amount of the tested antigen-binding domain bound to the pore via a certain antigen protein can be easily determined by pre-labeling the antigen-binding domain. For example, biotin-labeled antigen-binding domains can be determined by using an avidin peroxidase conjugate and a suitable substrate. Cross-blocking assays using enzymes such as peroxidase are particularly effective.This is referred to as a competitive ELISA assay. The antigen-binding domain can be labeled with other detectable or measurable labeling substances. Specifically, known labels include radioactive labels or fluorescent labels.

[0318] Compared with the binding activity obtained in a control test performed in the absence of a candidate competing antigen-binding domain binder, if the competing antigen-binding domain can block the binding of an antigen-binding domain against an antigen by at least 20%, preferably at least 20-50%, more preferably at least 50%, then the test antigen-binding domain and the competing antigen-binding domain are substantially bound to the same epitope, or are competing antigen-binding domains for binding to the same epitope. Specification 28 / 139 pages 31 CN 121818917 A

[0319] In the case of identifying the structure of an epitope bound by an antigen-binding domain against an antigen, the common epitope between the test antigen-binding domain and the control antigen-binding domain can be evaluated by comparing the binding activity of the test antigen-binding domain and the control antigen-binding domain for a peptide or polypeptide with an amino acid-modified peptide constituting the epitope.

[0320] As a method for determining such binding activity, for example in the above-described ELISA form, it can be determined by comparing the binding activity of the test antigen-binding domain and the control antigen-binding domain to the introduced variant linear peptide. As a method other than ELISA, the binding activity to the variant peptide bound to the column can also be determined by passing the test antigen-binding domain and the control antigen-binding domain through a column bound to the variant peptide, and then quantitatively eluting the antigen-binding domain in the elution buffer. The method of adsorbing the variant peptide and, for example, GST as a fusion peptide onto the column is known.

[0321] In addition, when the identified epitope is a stereoepitaxy, the common epitope between the test antigen-binding domain and the control antigen-binding domain can be evaluated by the following method. First, cells expressing a certain antigen and cells expressing a certain antigen with an introduced variant epitope are prepared. The test antigen-binding domain and the control antigen-binding domain are added to a cell suspension in a suitable buffer such as PBS. Next, for the cell suspension washed with an appropriate buffer, a FITC-labeled antibody recognizing both the test antigen-binding domain and the control antigen-binding domain was added. The fluorescence intensity and cell number of cells stained with the labeled antibody were determined using a FACSCalibur (BD). The concentrations of the test antigen-binding domain and the control antigen-binding domain were appropriately diluted with suitable buffer to prepare the desired concentrations for use. For example, any concentration between 10 μg / ml and 10 ng / ml was used. The amount of the labeled antibody binding to the cells was reflected as the fluorescence intensity obtained by analysis using CELL QUEST Software (BD), i.e., the geometric mean value. That is, by obtaining this geometric mean, the amount of the labeled antibody can be determined.The binding activity of the antigen-binding domain and the control antigen-binding domain is represented by the amount of antibody binding.

[0322] Further, in addition to the above-mentioned ELISA or FACS, the competition of the antigen-binding domain for the same epitope as other antigen-binding domains can also be confirmed by radiolabeled antigen binding assay (RIA), BIACORE (registered trademark) surface plasmon resonance assay, electrochemiluminescence assay, etc.

[0323] The comparison value reflecting the geometric mean of the binding amount of the antigen-binding domain of the tested antigen to the expression cells of the variant antigen (the molecular Δ Geo-Mean value of the variant antigen) obtained by the analysis is compared with the Δ Geo-Mean value reflecting the binding amount of the antigen-binding domain of the tested antigen to the expression cells of the antigen. In this case, the concentration of the antigen-binding domain of the tested antigen used when calculating the Δ Geo-Mean comparison value of the expression cells of the variant antigen and the expression cells of the antigen is particularly preferably prepared to be the same or substantially the same concentration. The antigen-binding domain that recognizes the epitope in the antigen in a prior-confirmed manner is used as the control antigen-binding domain.

[0324] The Δ Geo-Mean comparison value of the tested antigen-binding domain to cells expressing a variant antigen is less than at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the Δ Geo-Mean comparison value of the tested antigen-binding domain to cells expressing a variant antigen, indicating that it "does not substantially bind to cells expressing a variant antigen." The formula for calculating the Δ Geo-Mean value is described in the CELL QUEST Software User's Guide (BD Biosciences). By comparing the comparison values, if they are substantially equivalent, the epitopes of the tested antigen-binding domain and the control antigen-binding domain can be assessed as identical.

[0325] In this specification, the term "transport portion" refers to the portion of an antigen-binding molecule other than the antigen-binding domain. The transport portion of the present invention is typically a peptide or polypeptide composed of amino acids. As a specific embodiment, the transport portion of the antigen-binding molecule is linked to the antigen-binding domain via a cleavage site. The transport portion of the present invention can be a series of peptides or polypeptides linked by amide bonds, or it can be a complex formed by multiple peptides or polypeptides through covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0326] In some embodiments of the present invention, the antigen-binding activity of the antigen-binding molecule after cleavage is higher than that of the antigen-binding molecule before cleavage. In other words, the antigen-binding portion removed by cleavage of the cleavage molecule has higher antigen-binding activity.The antigen-binding activity of the antigen-binding domain of a molecule is inhibited by the inhibitory domain. Methods for confirming that the antigen-binding activity of the antigen-binding domain is inhibited by the inhibitory domain include FACS (fluorescence activated cell sorting), ELISA (Enzyme-Linked Immunosorbent Assay), ECL (electrogenated chemiluminescence), SPR (Surface Plasmon Resonance) (Biacore), and BLI (Bio-Layer Interferometry) (Octet). In some embodiments of the present invention, the binding activity of the antigen-binding molecule after cleavage is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 times greater than that of the antigen-binding molecule before cleavage. In some more specific embodiments of the present invention, when the antigen-binding activity of the antigen-binding domain before cleavage is determined by one of the methods described above, no binding of the antigen-binding domain to the antigen is found.

[0327] In some embodiments of the present invention, the comparison of antigen-binding activity can be performed by comparing the antigen-binding activity before and after cleavage. That is, compared with the antigen-binding activity measured using antigen-binding molecules before cleavage, the antigen-binding activity measured using antigen-binding molecules after cleavage is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 times greater. In some more specific embodiments, when antigen-binding activity of antigen-binding molecules before cleavage is measured using one of the methods described above, no binding of the antigen-binding domain to the antigen is found.

[0328] In some embodiments of the present invention, since the linker of the antigen-binding molecule is cleaved by a protease, in such an approach, the comparison of antigen-binding activity can be made by comparing the antigen binding of the antigen-binding molecule before and after protease treatment.The activity is measured using protease-treated antigen-binding molecules. That is, the antigen-binding activity measured using protease-treated antigen-binding molecules is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 times greater than that measured using untreated antigen-binding molecules. In some more specific embodiments, when the antigen-binding activity of untreated antigen-binding molecules is measured using one of the methods selected above, no binding of the antigen-binding domain to the antigen is found.

[0329] In this invention, antigen-binding molecules before linker cleavage have a longer half-life in the blood compared to antigen-binding molecules after linker cleavage. In order to extend the half-life of the antigen-binding molecule, in some embodiments of the present invention, the antigen-binding molecule before cleavage is designed to have a longer half-life in blood. Examples of embodiments for extending the half-life in blood include, for example, having a large molecular weight of the antigen-binding molecule before cleavage, or having FcRn binding, or having albumin binding, or being PEGylated, but are not limited thereto.

[0330] In the present invention, the comparison of half-life is preferably compared with the half-life in human blood. When it is difficult to determine the half-life in human blood, the half-life in human blood can be predicted based on the half-life in the blood of mice (e.g., normal mice, transgenic mice expressing human antigens, transgenic mice expressing human FcRn, etc.) or monkeys (e.g., cynomolgus monkeys, etc.).

[0331] As an embodiment for extending the half-life of the antigen-binding molecule in blood, examples include, for example, endowing the antigen-binding molecule before cleavage with FcRn binding. In order to have FcRn binding, there are methods that typically provide an FcRn binding region in the antigen-binding molecule before cleavage. As an FcRn binding region, it refers to a region that has the ability to bind to FcRn. Any structure can be used as long as it has the ability to bind to FcRn.

[0332] After being taken into the cell via the FcRn salvage pathway by including an FcRn binding region, it returns to the plasma. For example, the plasma retention of IgG molecules is relatively long (slow disappearance) because FcRn is known to function as a salvage receptor for IgG molecules. IgG molecules taken into the endosome via endocytosis bind to FcRn expressed in the endosome under acidic conditions. IgG molecules that cannot bind to FcRn enter the lysosome.(lysosome) is decomposed at this site, but IgG molecules bound to FcRn migrate to the cell surface, dissociate from FcRn under neutral conditions in plasma, and return to the plasma.

[0333] The FcRn binding region is preferably a region that directly binds to FcRn. As a preferred example of the FcRn binding region, the Fc region of an antibody can be cited. However, regions that can bind to polypeptides with the ability to bind to FcRn, such as albumin or IgG, can bind to FcRn indirectly via albumin or IgG. Therefore, the FcRn binding region of the present invention can also be such a region that binds to polypeptides with the ability to bind to FcRn.

[0334] The binding activity of the FcRn binding region of the present invention to FcRn, especially to human FcRn, as described in the aforementioned binding activity paragraph, can be determined by methods known to those skilled in the art, and the conditions can be suitably determined by those skilled in the art. The binding activity of human FcRn can be evaluated using KD (dissociation constant), apparent KD (apparent dissociation constant), dissociation rate kd (dissociation rate), or apparent kd (apparent dissociation). These can be determined using methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard plot, flow cytometry, etc. can be used.

[0335] The conditions for determining the binding activity of the FcRn binding region to FcRn can be suitably selected by those skilled in the art and are not particularly limited. For example, it can be determined under conditions of MES buffer and 37°C as described in WO2009 / 125825. In addition, the determination of the binding activity of the FcRn binding region to FcRn of the present invention can be performed using methods known to those skilled in the art, such as Biacore (GE Healthcare).

[0336] The pH used as the measurement condition can be used to assess the binding affinity of the FcRn binding region to FcRn at any pH range of 4.0 to 6.5. Preferably, to determine the binding affinity of the FcRn binding region to human FcRn, a pH range of 5.8 to 6.0, close to the early intracellular pH in vivo, can be used. The temperature used as the measurement condition can also be used to assess the binding affinity of the FcRn binding region to FcRn at any temperature range of 10°C to 50°C. More preferably, a temperature range of 15°C to 40°C is used to determine the binding affinity of the FcRn binding region to human FcRn. Preferably, temperatures such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,Any temperature between 20°C and 35°C, such as 31, 32, 33, 34, and 35°C, can also be used to determine the binding affinity between the FcRn binding region and FcRn. The temperature of 25°C is a non-limiting example of the present invention.

[0337] As an example of the FcRn binding region, the Fc region of an IgG antibody can be cited, but it is not limited thereto. When using the Fc region of an IgG antibody, its type is not limited, and the Fc regions of IgG1, IgG2, IgG3, IgG4, etc. can be used.

[0338] In addition, as long as the Fc region of a natural IgG antibody has FcRn binding ability, a variant Fc region with one or more amino acids replaced can of course be used. For example, positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, and 308 of the EU number selected from the Fc region of the IgG antibody can be used. The Fc region is a variant of the amino acid sequence in which at least one amino acid at positions 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is replaced with another amino acid. Instructions for Use, Page 31 / 139, 34, CN 121818917 A

[0339] More specifically, the following can be used:

[0340] The amino acid substitution at position 237 (Gly) is replaced with that of Met;

[0341] The amino acid substitution at position 238 (Pro) is replaced with that of Ala;

[0342] The amino acid substitution at position 239 (Ser) is replaced with that of Lys;

[0343] The amino acid substitution at position 248 (Lys) is replaced with that of Ile;

[0344] The amino acid substitution at position 250 (Thr) is replaced with that of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr;

[0345] The amino acid substitution at position 252 (Met) is replaced with that of Phe, Trp, or Tyr;

[0346] The amino acid substitution at position 254 (Ser) is replaced with that of Thr;

[0347] The Arg at position 255 is replaced with an amino acid of Glu;

[0348] The Thr at position 256 is replaced with an amino acid of Asp, Glu, or Gln;

[0349] The 257th Pro position is replaced with an amino acid substitution of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;

[0350] The 258th Glu position is replaced with an amino acid substitution of His;

[0351] The 265th Asp position is replaced with an amino acid substitution of Ala;

[0352] The 270th Asp position is replaced with an amino acid substitution of Phe;

[0353] The 286th Asn position is replaced with an amino acid substitution of Ala or Glu;

[0354] The 289th Thr position is replaced with an amino acid substitution of His;

[0355] The 297th Asn position is replaced with an amino acid substitution of Ala;

[0356] The 298th Ser position is replaced with an amino acid substitution of Gly;

[0357] The 303rd Val position is replaced with an amino acid substitution of Ala;

[0358] The 305th Val position is replaced with an amino acid substitution of Ala;

[0359] The 307th Thr is replaced with an amino acid substitution of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;

[0360] The 308th Val is replaced with an amino acid substitution of Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;

[0361] The 309th Leu or Val is replaced with an amino acid substitution of Ala, Asp, Glu, Pro, or Arg;

[0362] The 311th Gln is replaced with an amino acid substitution of Ala, His, or Ile;

[0363] The 312th Asp is replaced with an amino acid substitution of Ala or His;

[0364] The 314th Leu is replaced with an amino acid substitution of Lys or Arg;

[0365] The 315th Asn is replaced with an amino acid substitution of Ala or His;

[0366] The 317th Lys position is replaced with an amino acid substitution of Ala;

[0367] The 325th Asn position is replaced with an amino acid substitution of Gly;

[0368] The 332nd Ile position is replaced with an amino acid substitution of Val;

[0369] The 334th Lys position is replaced with an amino acid substitution of Leu;

[0370] The 360th Lys position is replaced with an amino acid substitution of His;

[0371] The 376th Asp position is replaced with an amino acid substitution of Ala;

[0372] The 380th Glu position is replaced with an amino acid substitution of Ala;

[0373] The 382nd Glu position is replaced with an amino acid substitution of Ala;

[0374] The 384th Asn or Ser position is replaced with an amino acid substitution of Ala;

[0375] The 385th Gly position is replaced with an amino acid substitution of Asp or His;

[0376] The 386th Gln position is replaced with an amino acid substitution of Pro; Specification 32 / 139Page 35 CN 121818917 A

[0377] The 387th Pro position is replaced with an amino acid substitution of Glu;

[0378] The 389th Asn position is replaced with an amino acid substitution of Ala or Ser;

[0379] The 424th Ser position is replaced with an amino acid substitution of Ala;

[0380] The 428th Met position is replaced with an amino acid substitution of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;

[0381] The 433rd His position is replaced with an amino acid substitution of Lys;

[0382] The 434th Asn position is replaced with an amino acid substitution of Ala, Phe, His, Ser, Trp, or Tyr; and

[0383] The 436th Tyr or Phe position is replaced with an amino acid substitution of His;

[0384] The variant Fc region with at least one amino acid substitution.

[0385] Alternatively, the following can be used: EU numbers selected from the Fc region of an IgG antibody:

[0386] Met for the 237th amino acid;

[0387] Ala for the 238th amino acid;

[0388] Lys for the 239th amino acid;

[0389] Ile for the 248th amino acid;

[0390] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr for the 250th amino acid;

[0391] Phe, Trp, or Tyr for the 252nd amino acid;

[0392] Thr for the 254th amino acid;

[0393] Glu for the 255th amino acid;

[0394] Asp, Glu, or Gln for the 256th amino acid;

[0395] The amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;

[0396] The amino acid at position 258 is His;

[0397] The amino acid at position 265 is Ala;

[0398] The amino acid at position 270 is Phe;

[0399] The amino acid at position 286 is Ala or Glu;

[0400] The amino acid at position 289 is His;

[0401] The amino acid at position 297 is Ala;

[0402] The amino acid at position 298 is Gly;

[0403] The amino acid at position 303 is Ala;

[0404] The amino acid at position 305 is Ala;

[0405] The amino acid at position 307 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;

[0406] The amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;

[0407] The amino acid at position 309 is Ala, Asp, Glu, Pro, or Arg;

[0408] The amino acid at position 311 is Ala, His, or Ile;

[0409] The amino acid at position 312 is Ala or His;

[0410] The amino acid at position 314 is Lys or Arg;

[0411] The amino acid at position 315 is Ala or His;

[0412] The amino acid at position 317 is Ala;

[0413] The amino acid at position 325 is Gly; Specification 33 / 139 Page 36 CN 121818917 A

[0414] The amino acid at position 332 is Val;

[0415] The amino acid at position 334 is Leu;

[0416]

[0417] The amino acid at position 360 is His;

[0418] The amino acid at position 376 is Ala;

[0419] The amino acid at position 380 is Ala;

[0420] The amino acid at position 382 is Ala;

[0421] The amino acid at position 384 is Ala;

[0422] The amino acid at position 385 is Asp or His;

[0423] The amino acid at position 386 is Pro;

[0424] The amino acid at position 387 is Glu;

[0425] The amino acid at position 389 is Ala or Ser;

[0426] The amino acid at position 424 is Ala;

[0427] The amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;

[0428] Lys of amino acid at position 433;

[0429] Ala, Phe, His, Ser, Trp, or Tyr of amino acid at position 434; and

[0430] His of amino acid at position 436;

[0431] Fc region of at least one amino acid.

[0432] The antigen-binding molecule may also have FcRn binding in the antigen-binding domain. As an embodiment in which the half-life in blood of the antigen-binding molecule before cleavage is longer than the half-life in blood of the antigen-binding domain, it is of course possible that the antigen-binding domain does not have FcRn binding, or even if the antigen-binding domain has FcRn binding, it may have a weaker FcRn binding than the antigen-binding molecule before cleavage.

[0433] In addition, as an embodiment for prolonging the half-life in blood, there is a method for binding the antigen-binding molecule before cleavage to albumin. Albumin is not excreted by the kidneys and has FcRn binding properties, thus its half-life in the blood is as long as 17–19 days (J Clin Invest).August 1953; 32(8): 746-768). It was reported that the protein bound to albumin increased in size and became possible to bind indirectly to FcRn, thus increasing the half-life in the blood (Antibodies 2015, 4(3), 141-156).

[0433] Further, as an embodiment of prolonging the half-life in the blood, there is a method for PEGylating the antigen-binding molecule before cleavage. It is believed that by PEGylating the protein, the protein increases in size and at the same time, by inhibiting the degradation by proteases in the blood, the half-life in the blood of the protein is prolonged (J Pharm Sci. 2008 Oct; 97(10): 4167-83).

[0434] In some embodiments of the present invention, the antigen-binding molecule before cleavage contains the antibody Fc region. As a specific embodiment, the antigen-binding molecule before cleavage contains the CH2 domain and CH3 domain of a human IgG antibody. As one specific embodiment, the antigen-binding molecule prior to linker cleavage comprises a portion extending from Cys226 of the human IgG1 antibody heavy chain or from Pro230 to the carboxyl terminus of the heavy chain. However, the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region may or may not be present.

[0435] In some embodiments of the present invention, the antigen-binding molecule prior to linker cleavage comprises an antibody constant region. In a preferred embodiment, the antigen-binding molecule prior to linker cleavage comprises an IgG antibody constant region. In a preferred embodiment, the antigen-binding molecule prior to linker cleavage comprises a human IgG antibody constant region. Specification 34 / 139 pages 37 CN 121818917 A

[0436] In still some embodiments of the present invention, the antigen-binding molecule prior to linker cleavage comprises a region having a structure substantially similar to the antibody heavy chain constant region and a region having a structure substantially similar to the antibody light chain, which is bonded to the region by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0437] The linker of the antigen-binding molecule is specifically cleaved by a protease at a rate of about 0.001 to 1500 × 10⁴ M⁻¹ S⁻¹ or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10⁴ M⁻¹ S⁻¹.

[0438] In this specification, the term "protease" refers to an enzyme such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, usually referring to an endopeptidase. The protease used in this disclosure is limited to enzymes capable of cleaving protease cleavage sequences, and there is no particular limitation on its type.In some embodiments, a target tissue-specific protease is used. A target tissue-specific protease may refer to any of the following, for example:

[0439] (1) a protease expressed at a higher level in the target tissue than in normal tissue,

[0440] (2) a protease having higher activity in the target tissue than in normal tissue,

[0441] (3) a protease expressed at a higher level in the target cells than in normal cells,

[0442] (4) a protease having higher activity in the target cells than in normal cells.

[0443] In more specific embodiments, a cancer tissue-specific protease or an inflammatory tissue-specific protease may be used.

[0444] The term “target tissue” in this specification means tissue containing at least one target cell. In some embodiments of the invention, the target tissue is cancer tissue. In some embodiments of the invention, the target tissue is inflammatory tissue.

[0445] The term “cancer tissue” means tissue containing at least one cancer cell. Thus, it may be referred to as, for example, all cell types that contain cancer cells and blood vessels and are involved in the formation of tumors containing cancer cells and endothelial cells. In this specification, a tumor is referred to as a foci of tumor tissue. The term “tumor” is generally used to refer to benign or malignant growths.

[0446] In this specification, “inflammatory tissue” may be exemplified by, for example, the following:

[0447] • Joints of rheumatoid arthritis or osteoarthritis

[0448] • Lungs (alveolar) of bronchial asthma or COPD

[0449] • Digestive organs of inflammatory bowel disease or Crohn's disease or ulcerative colitis

[0450] • Fibrotic tissue of fibrotic diseases of the liver, kidneys, and lungs

[0451] • Tissue causing rejection in organ transplantation

[0452] • Blood vessels and heart (myocardium) of arteriosclerosis or heart failure

[0453] • Visceral fat of metabolic syndrome

[0454] • Skin tissue of atopic dermatitis or other dermatitis

[0455] • Spinal nerves of herniated discs or chronic low back pain

[0456] In some types of target tissues, proteases that are specifically expressed or specifically activated, or proteases that are considered to be associated with the disease state of the target tissue (target tissue-specific proteases) are known. For example, international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846 disclose proteases that are specifically expressed in cancer tissues. In addition, J Inflamm (Lond). 2010; 7:45., Nat Rev Immunol. 2006 Jul; 6(7):541-50., Nat Rev Drug Discov. 2014 Dec; 13(12):904-27..、Respir Res . 2016 Mar 4; 17: 23 .、Dis Model Mech . 2014 Feb; 7(2): 193-203 .、Biochim Biophys Acta . 2012 Jan; 1824(1): 133-45. disclosed proteases that are considered to be associated with inflammation.

[0457] In addition to proteases that are specifically expressed in target tissues, there are also proteins that are specifically activated in target tissues. For example, there are cases where proteases are expressed in an inactive form and then become activated. In many tissues, there are substances that inhibit activated proteases, and the activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer . 2003 Jul; 3(7): 489-501). In target tissues, there are cases where activated proteases are released from inhibition and specifically activated. The determination of activated proteases can be performed using an antibody that recognizes the activated protease (PNAS 2013 Jan 2; 110 (1): 93-98), or by using a method that fluorescently labels the peptide that the protease recognizes, which is quenched before cleavage but glows after cleavage (Nat Rev Drug Discov. 2010 Sep; 9(9): 690-701. doi: 10.1038 / nrd3053.).

[0458] From one perspective, the term “target tissue-specific protease” can refer to any of the following:

[0459] (i) a protease expressed at a higher level in a target tissue than in a normal tissue,

[0460] (ii) a protease having higher activity in a target tissue than in a normal tissue,

[0461] (iii) a protease expressed at a higher level in a target cell than in a normal cell,

[0462] (iv) a protease having higher activity in a target cell than in a normal cell.

[0463] Without limiting the interpretation of these, specific proteases may be exemplified as, for example, cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartic proteases (cathepsin D, E, K, O, etc.), serine proteases (including Matriptase (including MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, protease 3, thrombin, kallikrein, trypsin-like enzymes, chymase-like enzymes), and metalloproteinases (including both membrane-bound (MMP14-17 and MMP24-25) and secretory (MMP1-13 and MMP18-23 and MMP26-28) metalloproteinases).(MMP1-28), A disintegrin and metalloproteinase (ADAM), ADAM proteases with thrombospondin motif (ADAMTS), transmembrane peptidases (meprin α, meprin β), CD10 (CALLA), prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), β-secretase (BACE), fibroblast activation protein α (FAP), granzyme B, guanidino-benzoic acid esterase (GB), heparin, Neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, ostuain 2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), bone morphogenetic protein 1 (BMP-1), active protein C, blood coagulation-related proteases (coagulation factor VIIa, coagulation factor IXa, coagulation factor Xa). Coagulation factor XIa, coagulation factor XIIa), HtrA1, lactoferrin, Marapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, calpain 2, glutamate carboxypeptidase 2, AMSH-like protease, AMSH, γ secretase, anti-fibrinolytic enzyme A (APCE), Decysin 1, N-acetylated α-linked acid dipeptidase-like 1 (NAALADL1), furin, etc.

[0464] From another perspective, target tissue-specific proteases may refer to cancer tissue-specific proteases or inflammatory tissue-specific proteases.

[0465] Examples of cancer tissue-specific proteases include those disclosed in International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, which are specifically expressed in cancer tissues.

[0466] The higher the specificity of the expression of the cancer tissue-specific protease in the cancer tissue of the treatment target, the more side effects are obtained. The cancer tissue-specific protease is preferably at a concentration in cancer tissue that is more than 5 times higher than that in normal tissue, more preferably more than 10 times higher, even more preferably more than 100 times higher, particularly preferably more than 500 times higher, and most preferably more than 1000 times higher. In addition, the activity of the cancer tissue-specific protease in cancer tissue is preferably more than 2 times higher than that in normal tissue, more preferably more than 3 times, more than 4 times, more than 5 times, more than 10 times higher, even more preferably more than 100 times higher, particularly preferably more than 500 times higher, and most preferably more than 1000 times higher.

[0467] In addition, the cancer tissue-specific protease can bind to the cell membrane of cancer cells or it can be secreted outside the cell without binding to the cell membrane. In cases where cancer tissue-specific proteases do not bind to the cell membrane of cancer cells, in order to make the cytotoxicity caused by immune cells specific to cancer cells, cancer tissue-specific proteases are preferably present inside or near cancer tissue. "Near cancer tissue" in this specification means within the range where the cleavage sequence of the cancer tissue-specific protease is cleaved and the antigen-binding domain exerts antigen-binding activity. However, it is preferably within the range where normal cells are not harmed as much as possible.

[0468] From another viewpoint, a cancer tissue-specific protease is any of the following:

[0469] (i) a protease expressed at a higher level in cancer tissue than in normal tissue,

[0470] (ii) a protease having higher activity in cancer tissue than in normal tissue,

[0471] (iii) a protease expressed at a higher level in cancer cells than in normal cells,

[0472] (iv) a protease having higher activity in cancer cells than in normal cells.

[0473] A cancer tissue-specific protease may be a single protease or a combination of two or more proteases. The number of types of cancer tissue-specific proteases may be appropriately determined by those skilled in the art, taking into account the type of cancer to be treated.

[0474] From the above viewpoint, among the proteases exemplified above, the most preferred cancer tissue-specific proteases are serine proteases and metalloproteinases, more preferably mastriptase (containing MT-SP1), urokinase (uPA), and metalloproteinases, and even more preferably MT-SP1, uPA, MMP2, and MMP9.

[0475] The higher the specificity of the expression of the inflammation tissue in the treatment subject, the more effective the side effects are. The inflammation tissue-specific protease is preferably expressed at a concentration at least 5 times higher in the inflammation tissue than in the normal tissue, more preferably at least 10 times higher, even more preferably at least 100 times higher, and most preferably at least 100 times higher.More than 500 times, most preferably more than 1000 times higher. Furthermore, the inflammatory tissue-specific protease preferably exhibits activity in inflammatory tissue that is more than twice higher than its activity in normal tissue, more preferably more than 3 times, 4 times, 5 times, or 10 times higher, further preferably more than 100 times higher, particularly preferably more than 500 times higher, and most preferably more than 1000 times higher.

[0476] Additionally, the inflammatory tissue-specific protease may bind to the cell membrane of inflammatory cells or may be secreted extracellularly without binding to the cell membrane. In the case where the inflammatory tissue-specific protease does not bind to the cell membrane of inflammatory cells, in order to make the cytotoxicity caused by immune cells specific to inflammatory cells, the inflammatory tissue-specific protease is preferably present inside or near the inflammatory tissue. "Near the inflammatory tissue" in this specification refers to the range within which the inflammatory tissue-specific protease cleaves its sequence and the antigen-binding domain exerts its antigen-binding activity. However, it is preferably within a range that does not harm normal cells as much as possible.

[0477] From another viewpoint, the inflammatory tissue-specific protease is any one of the following:

[0478] (i) a protease expressed at a higher level in inflammatory tissue than in normal tissue,

[0479] (ii) a protease having higher activity in inflammatory tissue than in normal tissue,

[0480] (iii) a protease expressed at a higher level in inflammatory cells than in normal cells,

[0481] (iv) a protease having higher activity in inflammatory cells than in normal cells.

[0482] The inflammatory tissue-specific protease may be a single protease or a combination of two or more proteases. The type of inflammatory tissue-specific protease may be appropriately determined by those skilled in the art, taking into account the symptoms of the patient being treated.

[0483] From the above viewpoint, among the proteases exemplified above, the metalloproteinase described on page 37 / 139 of the specification, 40 CN 121818917 A, is preferably a metalloproteinase, and more preferably ADAMTS5, MMP2, MMP7, MMP9, and MMP13 among the metalloproteinases.

[0484] The protease cleavage sequence is a specific amino acid sequence that is specifically recognized by the target tissue-specific protease when the antigen-binding molecule is hydrolyzed by the target tissue-specific protease in aqueous solution.

[0485] From the viewpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed in or more specifically activated in the target tissue / cells of the treatment subject.

[0486] Examples of specific protease cleavage sequences include those specifically expressed in cancer tissues as illustrated above, such as those disclosed in International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846.Target sequences that are specifically hydrolyzed by proteases, inflammatory tissue-specific proteases, etc., may also be used. Artificially modified sequences, such as those with appropriate amino acid alterations, may also be used in target sequences that have been specifically hydrolyzed by known proteases. Additionally, protease cleavage sequences may be sequences identified using methods known to those skilled in the art, as described in Nature Biotechnology 19, 661-667 (2001).

[0487] Furthermore, naturally occurring protease cleavage sequences may also be used. For example, sequences cleaved by proteases in proteins whose molecular form has been altered by protease cleavage, such as TGFβ which is changed to a latent form by protease cleavage, may also be used.

[0488] Examples of protease cleavage sequences may include International Publication Nos. WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, Japanese Patent Application No. 2019-105464, US Patent Publication No. US2016 / 0289324, US Patent Publication No. US2016 / 0311903, PNAS (2000) 97:7754-7759, and Biochemical Journal (2010). The sequences shown in 426:219-228, Beilstein J Nanotechnol. (2016) 7:364-373, but not limited thereto.

[0489] The protease cleavage sequence is preferably an amino acid sequence that has been specifically hydrolyzed by a suitable target tissue-specific protease, as described above. The amino acid sequence that has been specifically hydrolyzed by a target tissue-specific protease preferably includes the following amino acid sequences.

[0490] LSGRSDNH (can be cleaved by MT-SP1, uPA)

[0491] PLALAG (can be cleaved by MMP2, MMP9)

[0492] VPLSLTMG (can be cleaved by MMP7)

[0493] The following sequences may also be used as protease cleavage sequences.

[0494] TSTSGRSANPRG (can be cut by MT-SP1, uPA)

[0495] ISSGLLSGRSDNH (can be cut by MT-SP1, uPA)

[0496] AVGLLAPPGGLSGRSDNH (can be cut by MT-SP1, uPA)

[0497] GAGVPMSMRGGAG (can be cut by MMP1)

[0498] GAGIPVSLRSGAG ​​(can be cut by MMP2)

[0499] GPLGIAGQ (can be cut by MMP2)

[0500] GGPLGMLSQS (can be cut by MMP2)

[0501] PLGLWA (can be cut by MMP2)

[0502] GAGRPFSMIMGAG (can be cut by MMP3)

[0503] GAGVPLSLTMGAG (can be cut by MMP7) Specification 38 / 139 pages 41 CN 121818917 A

[0504] GAGVPLSLYSGAG (can be cut by MMP9)

[0505] AANLRN (can be cut by MMP11)

[0506] AQAYVK (can be cut by MMP11)

[0507] AANYMR (can be cut by MMP11)

[0508] AAALTR (can be cut by MMP11)

[0509] AQNLMR (MMP11 cleavable)

[0510] AANYTK (MMP11 cleavable)

[0511] GAGPQGLAGQRGIVAG (MMP13 cleavable)

[0512] PRFKIIGG (prourokinase cleavable)

[0513] PRFRIIGG (prourokinase cleavable)

[0514] GAGSGRSAG (uPA cleavable)

[0515] SGRSA (uPA cleavable)

[0516] GSGRSA (uPA cleavable)

[0517] SGKSA (uPA cleavable)

[0518] SGRSS (uPA cleavable)

[0519] SGRRA (uPA cleavable)

[0520] SGRNA (uPA cleavable)

[0521] SGRKA (uPA cleavable)

[0522] QRGRSA (tPA cleavable)

[0523] GAGSLLKSRMVPNFNAG (thipsin B cleavable)

[0524] TQGAAA (thipsin B cleavable)

[0525] GAAAAA (thipsin B cleavable)

[0526] GAGAAG (thipsin B cleavable)

[0527] AAAAAG (thipsin B cleavable)

[0528] LCGAAI (thipsin B cleavable)

[0529] FAQALG (thipsin B cleavable)

[0530] LLQANP (thipsin B cleavable)

[0531] LAAANP (thipsin B cleavable)

[0532] LYGAQF (thipsin B cleavable)

[0533] LSQAQG (thipsin B cleavable)

[0534] ASAASG (cleavable by cathepsin B)

[0535] FLGASL (cleavable by cathepsin B)

[0536] AYGATG (cleavable by cathepsin B)

[0537] LAQATG (cleavable by cathepsin B)

[0538] GAGSGVVIATVIVITAG (cleavable by cathepsin L)

[0539] APMAEGGG (cleavable by transmembrane peptidase α and transmembrane peptidase β)

[0540] EAQGDKII (cleavable by transmembrane peptidase α and transmembrane peptidase β)

[0541] LAFSDAGP (cleavable by transmembrane peptidase α and transmembrane peptidase β)

[0542] YVADAPK (cleavable by transmembrane peptidase α and transmembrane peptidase β) Specification 39 / 139 Page 42 CN 121818917 A

[0543] RRRRR (cleavable by furin)

[0544] RRRRRR (cleavable by furin)

[0545] GQSSRHRRAL (cleavable by furin)

[0546] SSRHRRALD (cleavable by TGFβ)

[0547] RKSSIIIRMRDVVL (cleavable by plasminogen)

[0548] SSSFDKGKYKKGDDA (cleavable by streptokinase)

[0549] SSSFDKGKYKRGDDA (cleavable by streptokinase)

[0550] IEGR (cleavable by coagulation factor Xa)

[0551] IDGR (cleavable by coagulation factor Xa)

[0552] GGSIDGR (cleavable by coagulation factor Xa)

[0553] GPQGIAGQ (cleavable by collagenase)

[0554] GPQGLLGA (cleavable by collagenase)

[0555] GIAGQ (cleavable by collagenase)

[0556] GPLGIAG (can be cleaved by collagenase)

[0557] GPEGLRVG (can be cleaved by collagenase)

[0558] YGAGLGVV (can be cleaved by collagenase)

[0559] AGLGVVER (can be cleaved by collagenase)

[0560] AGLGISST (can be cleaved by collagenase)

[0561] EPQALAMS (can be cleaved by collagenase)

[0562] QALAMSAI (can be cleaved by collagenase)

[0563] AAYHLVSQ (can be cleaved by collagenase)

[0564] MDAFLESS (can be cleaved by collagenase)

[0565] ESLPVVAV (can be cleaved by collagenase)

[0566] SAPAVESE (can be cleaved by collagenase)

[0567] DVAQFVLT (can be cleaved by collagenase)

[0568] VAQFVLTE (can be cleaved by collagenase)

[0569] AQFVLTEG (can be cleaved by collagenase)

[0570] PVQPIGPQ (can be cleaved by collagenase)

[0571] LVPRGS (can be cleaved by thrombin).

[0572] In some embodiments, the protease cleavage sequence is at least cleaved by cysteine ​​protease. In some embodiments, the protease cleavage sequence is at least cleaved by metalloproteinase. In some embodiments, the protease cleavage sequence is at least cleaved by Matriptase. In some embodiments, the protease cleavage sequence is at least cleaved by MT-SP1. In some embodiments, the protease cleavage sequence is at least cleaved by uPA. In some embodiments, the protease cleavage sequence is at least cleaved by Matriptase and uPA. In some embodiments, the protease cleavage sequence is at least cleaved by MT-SP1 and uPA.

[0573] In one embodiment, the protease cleavage sequence is selected from the group consisting of PLALAG, VPLSLTMG, GAGVPMSMRGGAG, GAGIPVSLRSGAG, GPLGIAGQ, GGPLGMLSQS, PLGLWA, GAGRPFSMIMGAG, GAGVPLSLTMGAG, GAGVPLSLYSGAG, AANLRN, AQAYVK, AANYMR, AAALTR, AQNLMR, AANYTK, and GAGPQGLAGQRGIVAG. Instructions for Use, pages 40 / 139, CN 121818917 A

[0574] In one embodiment, the protease cleavage sequence is selected from the group consisting of GPQGIAGQ, GPQGLLGA, GIAGQ, GPLGIAG, GPEGLRVG, YGAGLGVV, ALGVVER, AGLGISST, EPQALAMS, QALAMSAI, AAYHLVSQ, MDAFLESS, ESLPVVAV, SAPAVESE, DVAQFVLT, VAQFVLTE, AQFVLTEG, and PVQPIGPQ, which can be cleaved by collagenase.

[0575] Sequences shown in sequence numbers 1 to 725 may also be used as protease cleavage sequences.

[0576] The following sequence can also be used as the protease cleavage sequence:

[0577] X1-X2-X3-X4-X5-X6-X7-X8

[0578] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H,Amino acids I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0579] The following sequence can also be used as the protease cleavage sequence:

[0580] X1-X2-X3-X4-X5-X6-X7-X8

[0581] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0582] The following sequence can also be used as the protease cleavage sequence:

[0583] X1-X2-X3-X4-X5-X6-X7-X8

[0584] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E,Amino acids of F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0585] The following sequence can also be used as the protease cleavage sequence:

[0586] X1-X2-X3-X4-X5-X6-X7-X8

[0587] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0588] The following sequence can also be used as the protease cleavage sequence:

[0589] X1-X2-X3-X4-X5-X6-X7-X8 Specification 41 / 139 pages 44 CN 121818917 A

[0590] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0591] The following sequence can also be used as the protease cleavage sequence:

[0592] X1-X2-X3-X4-X5-X6-X7-X8

[0593] Wherein, X1 to X8 each represent one amino acid, X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 being an amino acid selected from A, D, E, F, G, H, ... Amino acids of I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0594] The following sequence can also be used as the protease cleavage sequence:

[0595] X1-X2-X3-X4-X5-X6-X7-X8

[0596] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0597] The following sequence can also be used as the protease cleavage sequence:

[0598] X1-X2-X3-X4-X5-X6-X7-X8

[0599] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R,X6 consists of amino acids selected from S, T, V, W, and Y; X7 consists of amino acids selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 consists of amino acids selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0600] The following sequence can also be used as the protease cleavage sequence:

[0601] X1-X2-X3-X4-X5-X6-X7-X8

[0602] Wherein, X1 to X8 each represent one amino acid, X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; X8 is an amino acid selected from H, V and Y.

[0603] The following sequence can also be used as the protease cleavage sequence:

[0604] X1-X2-X3-X4-X5-X6-X7-X8

[0605] Wherein, X1 to X8 each represent one amino acid, X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X8 is an amino acid selected from H, P, V and Y. Instructions 42 / 139 pages 45 CN 121818917 A

[0606] As a protease cleavage sequence, the following sequence may also be used:

[0607] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0608] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, Amino acids I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G.

[0609] The following sequence may also be used as the protease cleavage sequence:

[0610] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0611] Where X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 ...1 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, E, F, H, I, K, L, M, N Amino acids D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G.

[0612] The following sequence can also be used as the protease cleavage sequence:

[0613] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0614] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from R and G.

[0615] The following sequence can also be used as the protease cleavage sequence:

[0616] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0617] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K,Amino acids L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G.

[0618] The following sequence can also be used as the protease cleavage sequence:

[0619] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0620] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, X6 consists of amino acids selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 consists of amino acids selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 consists of amino acids selected from A, D, E, F, G, H, I, K, L, M, N, P, AQ, R, S, T, V, W, and Y; X9 consists of amino acids selected from R and G.

[0621] The following sequence can also be used as the protease cleavage sequence:

[0622] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0623] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 ...Amino acids E, F, K, M, N, P, Q, R, S, and W; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G.

[0624] The following sequence can also be used as the protease cleavage sequence:

[0625] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0626] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, X6 is an amino acid selected from S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, L, M, P, Q, V, W, and Y; X9 is an amino acid selected from R and G.

[0627] The following sequence can also be used as the protease cleavage sequence:

[0628] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0629] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; X9 is an amino acid selected from R and G.

[0630] The following sequence may also be used as the protease cleavage sequence:

[0631] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0632] Wherein, X1 to X9 each represent one amino acid, X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; X8 is an amino acid selected from H, V and Y; X9 is an amino acid selected from R and G.

[0633] The following sequence can also be used as the protease cleavage sequence:

[0634] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0635] Wherein, X1 to X9 each represent one amino acid, X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X8 is an amino acid selected from H, P, V and Y; X9 is an amino acid selected from R and G.

[0636] In addition to using the above protease cleavage sequence, new protease cleavage sequences can also be obtained through new screening. For example, by changing the interaction between the cleavage sequence and the active residues and recognition residues of the enzyme, new protease cleavage sequences can be explored based on the results of crystal structure analysis of known protease cleavage sequences. In addition, by adding amino acid variations to known protease cleavage sequences and confirming the interaction with the protease, new protease cleavage sequences can be explored. As another example, peptide libraries can be displayed using in vitro display methods such as phage display and ribosome display, or peptide arrays immobilized on chips or beads can be used. (See specification 44 / 139, page 47, CN 121818917 A). By confirming interactions with proteases, sequences cleaved by proteases can be explored. Methods for confirming the interaction between protease cleavage sequences and proteases can be performed in vitro or in vivo.

[0637] The protease cleavage sequence disclosed herein may be specifically modified (cleaved) by a protease at a rate of about 0.001 to 1500 × 10⁴ M⁻¹ S⁻¹ or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10⁴ M⁻¹ S⁻¹.

[0638] Confirmation of methods for protease cleavage

[0639] As a method for evaluating the protease substrate or protease cleavage sequence described in this specification, the method described in Mol Cell Proteomics. June 2014; 13(6): 1585-97. doi: 10.1074 / mcp.M113.033308. Epub April 4, 2014.

[0640] The type or concentration of the protease used in the evaluation, the treatment temperature, or the treatment time can be appropriately selected. For example, PBS containing 1000 nM human uPA, PBS containing 1000 nM mouse uPA, PBS containing 500 nM human MT-SP1, or PBS containing 500 nM mouse MT-SP1 can be used, and the treatment can be performed at 37°C for 1 hour.

[0641] Alternatively, serum (containing human serum or mouse serum) can be used instead of the solution containing the protease to treat the peptide array, and the fluorescence value measured from the chip can be used.

[0642] The type or concentration of the serum used in the evaluation, the treatment temperature, or the treatment time can be appropriately selected. For example, human serum diluted to 80% concentration can be used as the treatment solution, and the treatment can be performed at 37°C overnight.

[0643] As a method for qualitatively confirming whether the protease cleavage sequence contained in the polypeptide has been cleaved by the protease, it can be confirmed by performing SDS-PAGE (polyacrylamide gel electrophoresis) on a solution containing the polypeptide containing the protease cleavage sequence and determining the molecular weight of the fragment. Alternatively, it can be confirmed by comparing the molecular weight of the untreated polypeptide and the protease-treated polypeptide.

[0644] In this specification, the term "cleaved" refers to the state of polypeptide cleavage resulting from the action of a protease, alteration of the protease cleavage sequence, and / or reduction of the cysteine-cysteine ​​disulfide bonds in the protease cleavage sequence. In this specification, the term "uncleaved" refers to the state in which the portions on both sides of the protease cleavage sequence in the polypeptide remain connected even without protease cleavage and / or without reduction of the cysteine-cysteine ​​disulfide bonds in the protease cleavage sequence.

[0645] Furthermore, by quantifying the amount of protease-treated fragments separated by electrophoresis methods such as SDS-PAGE, the protease cleavage sequence can be evaluated, as well as the cleavage rate of molecules incorporating the protease cleavage sequence can be assessed. As a non-limiting method for evaluating the cleavage rate of molecules incorporating the protease cleavage sequence, the following methods can be cited. For example, the cleavage rate of antibody variants incorporating protease cleavage sequences was assessed using recombinant human u-plasminogen activator / urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human Matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010). The mixture was prepared with huPA 40 nM or hMT-SP1 3 nM and antibody variant 100 μg / mL PBS, reacted at 37°C for 1 hour, and then subjected to capillary electrophoresis immunoassay. While the capillary electrophoresis immunoassay can use Wes (Protein Simple), it is not limited to this method and can be used as an alternative to capillary electrophoresis immunoassay.The method can also be used, such as separation by SDS-PAGE, followed by detection by Western blotting, but is not limited to these methods. Anti-human λ chain HRP-labeled antibodies (abcam; ab9007) can be used to detect the light chains before and after cleavage, but any antibody can be used to detect the cleaved fragment. The areas of each peak obtained after protease treatment are output using the Wes software (Compass for SW; Protein Simple). The cleavage rate (%) of the antibody variant can be calculated using the formula (cleavage light chain peak area) × 100 / (cleavage light chain peak area + uncleaved light chain peak area). By calculating the cleavage rate using the above method, for example, the cleavage rates of antibody variants with different cleavage sequences introduced can be compared in vivo, or the cleavage rates of the same antibody variant can be compared between different animal models such as normal mouse models or tumor transplantation models (page 45 / 139, CN 121818917 Type A).

[0646] For example, a linker comprising any of the protease cleavage sequences exemplified by sequence numbers 1 to 725 is useful as a protease substrate hydrolyzed by protease action. That is, in this invention, a linker that is a protease substrate exemplified in this specification can be used. This linker, for example, when incorporated into an antigen-binding molecule, can be used as a library for selecting substances with specific properties according to the purpose. Specifically, in order to selectively cleave the antigen-binding molecule by a protease locally present at the lesion site, the sensitivity of the protease can be evaluated. After being administered to a organism, the antigen-binding molecule containing the linker has the potential to reach the lesion site after contact with various proteases. Therefore, it is desirable to have sensitivity to proteases located at the lesion site and to have the highest possible tolerance to proteases other than those located thereon. In order to select the desired protease cleavage sequence according to the purpose, the tolerance of the protease can be known if a comprehensive analysis of the sensitivity of each protease substrate to various proteases is performed beforehand. Based on the obtained protease tolerance profile, a protease cleavage sequence with the necessary sensitivity and tolerance can be found.

[0647] Alternatively, antigen-binding molecules incorporating protease cleavage sequences can reach the lesion not only through the enzymatic action of the protease but also through various environmental loads such as pH changes, temperature, and redox stress. Even with such external factors, protease cleavage sequences with desired properties can be selected based on information comparing the tolerance of various protease substrates.

[0648] In one embodiment of the invention, a flexible linker is further added to either or both ends of the protease cleavage sequence. The flexible linker at one end of the protease cleavage sequence may be referred to as a first flexible linker, and the flexible linker at the other end may be referred to as a second flexible linker. In a particular embodiment, the protease cleavage sequence and the flexible linker comprise one of the following formulas:

[0649] (Protease cleavage sequence)

[0650] (First flexible linker) - (Protease cleavage sequence)

[0651] (Protein cleavage sequence) - (Second flexible linker)

[0652] (First flexible linker) - (Protein cleavage sequence) - (Second flexible linker)

[0653] The flexible linker in this embodiment is preferably a peptide linker. The first and second flexible linkers are independent and arbitrary, and are the same or different flexible linkers containing at least one flexible amino acid (Gly, etc.). For example, the protein cleavage sequence contains a sufficient number of residues (arbitrarily selected from amino acids such as Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., especially Gly, Ser, Asp, Asn, Ala, and more especially Gly and Ser, especially Gly, etc.) to obtain the desired protease accessibility.

[0654] Flexible linkers suitable for use at both ends of the protease cleavage sequence generally improve the protease accessibility to the protease cleavage sequence and improve the protease cleavage efficiency. Suitable flexible linkers can be easily selected, and can be chosen from different lengths, starting from 1 amino acid (Gly, etc.) to 20 amino acids, 2 amino acids to 15 amino acids, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and from 3 amino acids to 12 amino acids. In some embodiments of the present invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0655] Examples of flexible linkers include, for example, glycine polymers (G)n, glycine-serine polymers (e.g., comprising (GS)n, (GSGGS)n and (GGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the prior art, but not limited thereto.

[0656] Among them, glycine and glycine-serine polymers are of interest because these amino acids are relatively unstructured and can easily function as neutral linkers between components.

[0657] Examples of flexible joints formed from glycine-serine polymers include, for example:

[0658] Ser Specification 46 / 139 pages 49 CN 121818917 A

[0659] Gly・Ser(GS)

[0660] Ser・Gly(SG)

[0661] Gly・Gly・Ser(GGS)

[0662] Gly・Ser・Gly(GSG)

[0663] Ser・Gly・Gly(SGG)

[0664] Gly・Ser・Ser(GSS)

[0665] Ser・Ser・Gly(SSG)

[0666] Ser・Gly・Ser(SGS)

[0667] Gly・Gly・Gly・Ser(GGGS)

[0668] Gly・Gly・Ser・Gly(GGSG)

[0669] Gly・Ser・Gly・Gly(GSGG)

[0670] Ser・Gly・Gly・Gly(SGGG)

[0671] Gly・Ser・Ser・Gly(GSSG)

[0672] Gly・Gly・Gly・Gly・Ser(GGGGS)

[0673] Gly・Gly・Gly・Ser・Gly(GGGSG)

[0674] Gly・Gly・Ser・Gly・Gly(GGSGG)

[0675] Gly・Ser・Gly・Gly・Gly(GSGGG)

[0676] Gly・Ser・Gly・Gly・Ser(GSGGS)

[0677] Ser・Gly・Gly・Gly・Gly(SGGGG)

[0678] Gly・Ser・Ser・Gly・Gly(GSSGG)

[0679] Gly・Ser・Gly・Ser・Gly(GSGSG)

[0680] Ser・Gly・Gly・Ser・Gly(SGGSG)

[0681] Gly・Ser・Ser・Ser・Gly(GSSSG)

[0682] Gly・Gly・Gly・Gly・Gly・Ser(GGGGGS)

[0683] Ser・Gly・Gly・Gly・Gly・Gly(SGGGGG)

[0684] Gly・Gly・Gly・Gly・Gly・Gly・Ser(GGGGGGS)

[0685] Ser・Gly・Gly・Gly・Gly・Gly・Gly(SGGGGGG)

[0686] (Gly・Gly・Gly・Gly・Ser(GGGGS)) n

[0687] (Ser・Gly・Gly・Gly・Gly(SGGGG)) n etc., but not limited thereto.

[0688] In this specification, "association" can be interpreted as, for example, the state in which two or more polypeptide regions interact. Generally, an associative complex is formed between the polypeptide regions that are targeted, consisting of hydrophobic bonds, hydrogen bonds, ionic bonds, etc. As a common example of association, it is known that in antibodies representing natural antibodies, a paired structure is maintained by non-covalent bonds between the heavy chain variable region (VH) and the light chain variable region (VL).

[0689] In this specification, "interface" generally refers to the associative surface during association (interaction), and the amino acid residues forming the interface are generally one or more amino acid residues contained in the polypeptide region that provides the association, preferably referring to amino acid residues that are close to each other and participate in the interaction during association. This interaction specifically includes the formation of an interface between amino acid residues that are close to each other during association.Non-covalent bonds such as hydrogen bonds, electrostatic interactions, and salt bridges.

[0690] In this specification, "amino acid residues forming an interface" refers to amino acid residues contained in the polypeptide region constituting the interface. The polypeptide region constituting the interface, by way of example, refers to a polypeptide region that selectively binds within or between molecules of antibodies, ligands, receptors, substrates, etc. Examples of amino acid residues forming an interface may include, for example, amino acid residues that are close to each other during association, but are not limited thereto. Amino acid residues that are close to each other during association can be identified, for example, by analyzing the stereostructure of the polypeptide and investigating the amino acid sequence of the polypeptide region that forms the interface during the association of the polypeptide.

[0691] In some embodiments of the present invention, the antigen-binding domain VHH associates with the inhibitory domain VL. Examples of amino acid residues in VHH that are associated with the association of VL may refer to amino acid residues that form the interface between VHH and VL. Furthermore, examples of amino acid residues in VHH that are associated with VL can be, for example, amino acid residues at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125), but are not limited thereto. The activity of VHH is inhibited by promoting the association between VHH and VL. Meanwhile, examples of amino acid residues in VL that are associated with VHH can refer to amino acid residues that form the interface between VHH and VL.

[0692] To promote the association between VHH and VL, the amino acid residues in VHH that are associated with VL can be altered. Examples of such amino acid substitutions can be, for example, F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, or / and S47W, but are not limited thereto. Furthermore, the residues in VHH can be left unchanged, and VHH with amino acid residues from the original 37V, 44G, 45L, or / and 47W can be used.

[0693] Furthermore, as long as the purpose of promoting association between VHH and VL can be achieved, the amino acids in VHH can be left unchanged, but the amino acid residues in VL that are associated with VHH association can be changed. Furthermore, amino acid changes can be introduced in both VHH and VL.

[0694] In order to change the amino acid sequence of the polypeptide, site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR and other known methods can be appropriately used. In addition, amino acid changes by replacing amino acids with amino acids other than natural amino acids can also be achieved by several known methods (Annu. Rev. Biophys. Biomol).Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. U.SA (2003) 100 (11), 6353-6357). For example, cell-free translation systems such as Clover Direct (Protein Express) may also be suitably used, whose complementary succinct repressor tRNA for one of the stop codons, the UAG codon (succinct codon), contains tRNA that binds to non-natural amino acids.

[0695] In some other embodiments of the invention, using VHH as the antigen-binding domain and using VH or VHH as the repressor domain allows the antigen-binding domain and the repressor domain to associate. To promote the association of the antigen-binding domain VHH and the repressor domain VH or VHH, it is possible to identify and modify the amino acid residues in the VHH of the antigen-binding domain that are associated with the association of the repressor domain VH or VHH. In addition, it is possible to identify and modify amino acid residues in the inhibitory domain VH or VHH that are associated with the antigen-binding domain VHH.

[0696] In addition, when using a single-domain antibody other than VHH as the antigen-binding domain, it is also possible to identify and modify amino acid residues in the antigen-binding domain or inhibitory domain.

[0697] In a particular embodiment, the protease cleavage sequence is located in the antibody constant region within the antigen-binding molecule. In this case, it is preferable that the protease cleavage sequence is located within the antibody constant region so that the antigen-binding domain is freed upon cleavage by the protease. In a specific embodiment, the protease cleavage sequence is located within the antibody heavy chain constant region contained in the antigen-binding molecule, more specifically, on the antigen-binding domain side of the 140th (EU number) amino acid in the antibody heavy chain constant region, preferably on the antigen-binding domain side of the 122nd (EU number) amino acid in the antibody heavy chain constant region. In other specific embodiments, the protease cleavage sequence is located within the constant region of the antibody light chain contained in the antigen-binding molecule, more specifically, on the side of the antigen-binding domain adjacent to amino acid position 130 (Kabat number) in the constant region of the antibody light chain, preferably on the side adjacent to amino acid position 113 (Kabat number) in the constant region of the antibody light chain.

[0698] In a particular embodiment, the protease cleavable linker is located near the boundary between the variable region and the constant region or near the boundary between CH1 and CH2 within the constant region. Near the boundary between the variable region and the constant region refers to the area before the site connecting VH and CH1.Afterwards, or before and after the site connecting VL and CL, the sites that do not significantly affect the secondary structure of the antigen-binding domain include the elbow hinge region (from position 109 (EU number) to position 140 (EU number)). The vicinity of the boundary between CH1 and CH2 refers to the sites before and after the site connecting CH1 and CH2 that do not significantly affect the secondary structure of the antigen-binding domain, including the upper hinge region (from position 215 (EU number) to position 220 (EU number)) and the lower hinge region (from position 221 (EU number) to position 230 (EU number)).

[0699] In a more specific embodiment, the protease-cleavable linker is located near the boundary between the antigen-binding domain and the antibody constant region within the antigen-binding molecule. The vicinity of the boundary between the antigen-binding domain and the antibody constant region may refer to the vicinity of the boundary between the antigen-binding domain and the antibody heavy chain constant region, or the vicinity of the boundary between the antigen-binding domain and the antibody light chain constant region. In the case where the antigen-binding domain is a single-domain antibody composed of VH or where VHH is linked to the constant region of the antibody heavy chain, the boundary between the antigen-binding domain and the constant region of the antibody can refer to the area between amino acid position 101 (Kabat number) of the single-domain antibody and amino acid position 140 (EU number) of the antibody heavy chain constant region, preferably between amino acid position 109 (Kabat number) of the single-domain antibody and amino acid position 122 (EU number) of the antibody heavy chain constant region. In the case where the antigen-binding domain is a single-domain antibody composed of VH or where VHH is linked to the constant region of the antibody light chain, the boundary between the antigen-binding domain and the constant region of the antibody light chain can refer to the area between amino acid position 101 (Kabat number) of the single-domain antibody and amino acid position 130 (Kabat number) of the antibody light chain constant region, preferably between amino acid position 109 (Kabat number) of the single-domain antibody and amino acid position 113 (Kabat number) of the antibody light chain constant region. In the case where the antigen-binding domain is a single-domain antibody made of VL, the area near the boundary between the antigen-binding domain and the antibody constant region refers to the region before and after the site connecting VHH and CH2, which does not significantly affect the secondary structure of the antigen-binding domain, including the lower hinge region, starting from position 96 (Kabat number) of the single-domain antibody, preferably starting from position 104 (Kabat number) of the single-domain antibody.

[0700] In other embodiments of the invention, the cleavage site / protease cleavage sequence is located on the variable region side of amino acid position 140 (EU number) in the antibody heavy chain constant region, preferably on the variable region side of amino acid position 122 (EU number) in the antibody heavy chain constant region. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced into the antibody.The cleavage site / protease cleavage sequence is located at any position in the sequence from amino acid 118 (EU number) to amino acid 140 (EU number) in the antibody heavy chain constant region. In other more specific embodiments, the cleavage site / protease cleavage sequence is located on the variable region side of amino acid 130 (Kabat number) in the antibody light chain constant region, preferably on the variable region side of amino acid 113 (Kabat number) or amino acid 112 (Kabat number) in the antibody light chain constant region. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid 108 (Kabat number) to amino acid 131 (Kabat number) in the antibody light chain constant region.

[0701] In one embodiment, the cleavage site / protease cleavage sequence is located near the boundary between the antibody VL and the antibody constant region. The boundary between the antibody VL and the antibody light chain constant region can refer to the area between the 96th amino acid (Kabat number) of the antibody VL and the 130th amino acid (EU number) (Kabat number 130) of the antibody light chain constant region, preferably between the 104th amino acid (Kabat number) of the antibody VL and the 113th amino acid (EU number) (Kabat number 113) of the antibody light chain constant region, or between the 105th amino acid (Kabat number) of the antibody VL and the 112th amino acid (EU number) (Kabat number 112) of the antibody light chain constant region. In the case where antibody VL and antibody heavy chain constant region are linked, the boundary between antibody VL and antibody heavy chain constant region can refer to the area between amino acid position 96 (Kabat number) of antibody VL and amino acid position 140 (EU number) of antibody heavy chain constant region, preferably between amino acid position 104 (Kabat number) of antibody VL and amino acid position 122 (EU number) of antibody heavy chain constant region, or between amino acid position 105 (Kabat number) of antibody VL and amino acid position 122 (EU number) of antibody heavy chain constant region.

[0702] In one embodiment, a cleavage site / protease cleavage sequence is introduced near the CH2 / CH3 interface of antibody heavy chain constant region. Here, the area near CH2 / CH3 interface is the region from position 335 (EU number) to position 345 (EU number).

[0703] Multiple cleavage sites / protease cleavage sequences can be provided in the ligand-binding molecule, for example, they can be provided at multiple positions selected from within the antibody constant region, within antibody VH, within antibody VL, near the boundary between antibody VH and antibody constant region, and near the boundary between antibody VL and antibody constant region. Furthermore, anyone skilled in the art who has access to this invention can replace antibody VH and antibody...The molecular morphology of the antibody VH and antibody VL and the constant region of the antibody is modified, which does not exceed the scope of the present invention.

[0704] The term "IgG antibody-like molecule" as used in this specification is used to define a portion having a structure substantially similar to a constant domain or constant region of an IgG antibody, and a portion having a structure substantially similar to a variable domain or variable region of an IgG antibody, and a molecule having a stereostructure substantially similar to an IgG antibody. However, the term "IgG antibody-like molecule" in this specification is not limited to those molecules that maintain a structure similar to an IgG antibody and exert antigen-binding activity.

[0705] When the antigen-binding molecule is an IgG antibody-like molecule, an embodiment in which antigen-binding domains are respectively provided in the portions corresponding to the two variable regions of an IgG antibody is an embodiment that is understandable to those skilled in the art. An embodiment in which the antigen-binding domains incorporated in both arms have the same antigen-binding specificity or different antigen-binding specificities is also an embodiment that is understandable to those skilled in the art and is clear without exceeding the scope of the present invention.

[0706] In this specification, the term "specificity" refers to the property that a molecule on one side of the specifically bound molecule does not substantially bind to molecules other than molecules on one or more of the target molecules. It is also used when the antigen-binding domain is specific to an epitope contained in a specific antigen. Additionally, it is used when the antigen-binding domain is specific to a specific epitope among multiple epitopes contained in an antigen. Here, substantial non-binding can be confirmed by the method described in the binding activity section. The binding activity of a specific binding molecule against molecules other than the target molecules refers to a binding activity showing 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less, of the binding activity against the target molecules.

[0707] As used in this specification, “treatment” (and its grammatical derivatives, such as “performing treatment”, “treating”, etc.) refers to a clinical intervention intended to alter the natural course of the treated individual, which can be implemented for preventative purposes or during the clinical course of a disease. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological effects caused by the disease, prevention of metastasis, slowing of disease progression, recovery or mitigation from the disease state, and a relieved or improved prognosis. In some embodiments, the pharmaceutical compositions of the present invention are used to delay the onset of a disease or postpone its progression.

[0708] The pharmaceutical compositions of the present invention generally refer to agents for the treatment or prevention of a disease, or for examination or diagnosis. In the present invention, when the pharmaceutical composition is used in combination with the administration of other ingredients, the pharmaceutical composition may be administered simultaneously, separately, or continuously with the administration of other ingredients. The pharmaceutical compositions of the present invention may also contain other ingredients as components.

[0709] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be used non-orally as an injectable form of a sterile solution or suspension of water or other pharmaceutically permissible liquids. For example, they can be formulated by mixing with pharmaceutically permissible carriers or solvents, specifically sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dosage form required by generally accepted pharmaceutical practices. The amount of active ingredient in these formulations is set to an appropriate volume that yields the indicated range.

[0710] The sterile compositions for injection can be prepared using a carrier such as distilled water for injection according to conventional formulation methods. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other excipients (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable cosolvents such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbitol 80(TM), HCO-50, etc.) can be used.

[0711] Examples of oily liquids include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can be used as cosolvents. In addition, it can be formulated with buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The formulated injection is usually filled into a suitable ampoule.

[0712] The pharmaceutical compositions of the present invention are preferably administered by non-oral administration. Administration includes, for example, injectable, nasal, pulmonary, and transdermal formulations. It can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0713] The method of administration can be appropriately selected according to the patient's age and symptoms. The dosage of the pharmaceutical composition of the present invention can be set to, for example, a range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, a dosage of, for example, 0.001 to 100000 mg per patient can be set, but the present invention is not necessarily limited to these values. The dosage and method of administration vary according to the patient's weight, age, symptoms, etc., and appropriate dosage and method of administration can be set by those skilled in the art considering these conditions.

[0714] The polynucleotides in the present invention are usually loaded (inserted) into a suitable vector and introduced into host cells. The vector is not particularly limited as long as it stably maintains the inserted nucleic acid, for example, if Escherichia coli is used as the host,The preferred vector for cloning is pBluescript vector (manufactured by Stratagene), but various commercially available vectors can be used. In the implementation of this invention, when a vector is used for the purpose of producing polypeptides (e.g., chimeric receptors, IgG antibodies, bispecific antibodies, antigen-binding molecules, etc.), expression vectors are particularly useful. As an expression vector, any vector that expresses polypeptides in vitro, in E. coli, in cultured cells, or in an organism is acceptable, without particular limitation. For example, for expression in vitro, pBEST vector (manufactured by Promega) is preferred; for E. coli, pET vector (manufactured by Invitrogen) is preferred; for cultured cells, pME18S-FL3 vector (GenBank accession number AB009864) is preferred; and for an organism, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) is preferred. The insertion of the DNA into the vector of the present invention can be carried out according to conventional methods, for example, by using a ligase reaction with restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al., (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0715] There are no particular limitations on the host cell described above, and various host cells can be used depending on the purpose. Cells used for expressing polypeptides can include, for example, bacterial cells (such as Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (such as yeast, Aspergillus), insect cells (such as Drosophila S2, Spodoptera SF9), animal cells (such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vector delivery to host cells can be performed using known methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al., (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipid transfection (GIBCO-BRL), and microinjection.

[0716] To enable the secretion of polypeptides expressed in the host cell into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, please refer to the instruction manual, page 51 / 139, document number 54, CN 121818917 A.Appropriate secretion signals can be incorporated into the target polypeptide. These signals can be endogenous or heterologous to the target polypeptide.

[0717] In the above-described manufacturing method, the polypeptide is recovered when it is secreted into a culture medium. When the polypeptide is generated in a cell, the cell is first dissolved, and then the polypeptide is recovered.

[0718] For the recovery and purification of the polypeptide from recombinant cell cultures, known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography can be used.

[0719] In addition, as antigen-binding domains used in some embodiments of the present invention, examples of single-domain antibodies can be cited, which inhibit antigen-binding activity by associating with a specific VL, or with a specific VH, or with a specific VHH. The present invention also relates to methods for screening such single-domain antibodies.

[0720] As VL / VH / VHHs that inhibit the antigen-binding activity of single-domain antibodies, VL / VH / VHHs with known sequences can be used, such as those sequences registered in the IMGT or Kabat databases. Alternatively, sequences of new VL / VH / VHHs identified from human antibody libraries, etc., can also be used. By combining these sequences to prepare proteins and measuring binding activity using the above method, VL / VH / VHHs that inhibit the binding activity of single-domain antibodies can be selected.

[0721] In one embodiment of the present invention, a method for screening single-domain antibodies that inhibit antigen-binding activity by associating with a specific VL is provided, comprising the following steps:

[0722] (a) obtaining a single-domain antibody having target antigen-binding activity;

[0723] (b) associating the single-domain antibody obtained in step (a) with a specific VL;

[0724] (c) confirming that the binding activity of the single-domain antibody associated with the specific VL in step (b) is weakened or lost for the antigen.

[0725] In this invention, "reduced binding activity" refers to a decrease in binding activity to the target antigen compared to before association, regardless of the degree of reduction.

[0726] In one embodiment of the present invention, a method for screening single-domain antibodies that inhibit antigen binding activity by association with a specific VH is provided, comprising the following steps:

[0727] (a) obtaining a single-domain antibody having target antigen binding activity;

[0728] (b) associating the single-domain antibody obtained in step (a) with a specific VH;

[0729] (c) confirming that the single-domain antibody associated with the specific VH in step (b) has reduced or lost binding activity to the antigen.

[0730] In this invention, "weakened binding activity" refers to a decrease in binding activity to the target antigen compared to before association, regardless of the degree of decrease.

[0731] In one embodiment of the present invention, a method for screening single-domain antibodies that inhibit antigen binding activity by association with a specific VHH is provided, comprising the following steps:

[0732] (a) obtaining a single-domain antibody having target antigen binding activity;

[0733] ​​(b) associating the single-domain antibody obtained in step (a) with a specific VHH;

[0734] (c) confirming that the binding activity of the single-domain antibody associated with the specific VHH in step (b) to the antigen is weakened or lost.

[0735] In this invention, "weakened binding activity" refers to a decrease in binding activity to the target antigen compared to before association, regardless of the degree of decrease.

[0736] As an example of a method for a single-domain antibody to associate with a specific VL / VH / VHH, examples can be given, for instance, a method of designing a molecule containing both VH and VL in a complete antibody, Fab, Fab', (Fab)2, or other antibody or antibody fragment, and using the sequence of the single-domain antibody to replace the sequence of either VH or VL, thereby expressing a polypeptide having that sequence.

[0737] In addition to screening single-domain antibodies that inhibit antigen-binding activity by associating with a specific VL, or with a specific VH, or with a specific VHH, the present invention also relates to a method for manufacturing a single-domain antibody that inhibits antigen-binding activity by promoting association of the single-domain antibody with a specific VL / VH / VHH, promoting association with a specific VL, or promoting association with a specific VH, or promoting association with a specific VHH.

[0738] In one embodiment of the present invention, a method for manufacturing a single-domain antibody that inhibits antigen-binding activity by associating with a specific VL is provided, comprising the steps of:

[0739] (a) preparing a variant single-domain antibody that replaces amino acid residues in the single-domain antibody that are associated with the antibody VL, thereby maintaining the binding activity of the single-domain antibody against a target antigen.

[0740] In a particular embodiment, a method for manufacturing a single-domain antibody that inhibits antigen-binding activity by associating with a specific VL is provided, further comprising the steps of:

[0741] (b) associating the variant single-domain antibody prepared in step (a) with a specific VL;

[0742] (c) confirming that the antigen-binding activity of the variant single-domain antibody associated with the VL is weakened or lost.

[0743] In the present invention, "weakened binding activity" means a reduction in binding activity against a target antigen compared to before association, regardless of the degree of reduction.

[0744] In one embodiment of the present invention, a method for manufacturing a single-domain antibody that inhibits antigen-binding activity by associating with a specific VH is provided, comprising the steps of:

[0745] (a) preparing a variant single-domain antibody that replaces amino acid residues in the single-domain antibody that are associated with the antibody VH, thereby maintaining the binding activity of the single-domain antibody against the target antigen.

[0746] In a particular embodiment, a method for manufacturing a single-domain antibody that inhibits antigen-binding activity by associating with a specific VH is provided, further comprising the steps of:

[0747] (b) associating the variant single-domain antibody prepared in step (a) with the specific VH;

[0748] (c) confirming that the antigen-binding activity of the variant single-domain antibody associated with the VH is reduced or lost.

[0749] In the present invention, "reduced binding activity" means a decrease in binding activity against the target antigen compared to before association, regardless of the degree of reduction.

[0750] In one embodiment of the present invention, a method for manufacturing a single-domain antibody that inhibits antigen-binding activity by associating with a specific VHH is provided, comprising the steps of:

[0751] (a) preparing a variant single-domain antibody that replaces amino acid residues in the single-domain antibody that are associated with the antibody VHH, thereby maintaining the binding activity of the single-domain antibody against a target antigen.

[0752] In a particular embodiment, a method for manufacturing a single-domain antibody that inhibits antigen-binding activity by associating with a specific VHH is provided, further comprising the steps of:

[0753] (b) associating the variant single-domain antibody prepared in step (a) with a specific VHH;

[0754] (c) confirming that the antigen-binding activity of the variant single-domain antibody associated with the VHH is reduced or lost.

[0755] In the present invention, "reduced binding activity" means a decrease in binding activity against a target antigen compared to before association, regardless of the degree of reduction.

[0756] The step of associating a single-domain antibody with a specific VL / VH / VHH can be performed by designing an antibody or antibody fragment containing both VH and VL, such as a complete antibody, Fab, Fab', (Fab)2, etc., and replacing one of the sequences of VH and VL with the sequence of the single-domain antibody, and expressing a polypeptide having that sequence.

[0757] According to a certain embodiment of the present invention, a single-domain antibody that inhibits or loses antigen-binding activity by associating with a specific VL / VH / VHH of the present invention can be obtained from a library containing a plurality of fusion polypeptides that link the single-domain antibody with a first association support domain.

[0758] As an embodiment of the “library” in this specification, it is possible to provide a single-domain antibody that can effectively obtain antigen-binding activity by associating with a specific VL / VH / VHH.A library of single-domain antibodies that inhibit or lose antigen-binding activity through VHH association.

[0759] In this specification, "library" refers to a group of multiple fusion peptides, or nucleic acids or polynucleotides encoding these fusion peptides, each having a different sequence. The multiple fusion peptides contained in the library are not single sequences, but fusion peptides with different sequences from each other.

[0760] In this specification, "different sequences from each other" in the context of multiple fusion peptides with different sequences means that the sequences of each fusion peptide in the library are different from each other. More preferably, it means that the sequences of the single-domain antibody portions of each fusion peptide in the library are different. That is, the number of different sequences in the library reflects the number of independent clones with different sequences in the library, also known as "library size". Typical phage display libraries are 10⁶ to 10¹², and the library size can be increased to 10¹⁴ by using known techniques such as ribosome display. However, the actual number of phage particles used in the panning selection of phage display is usually 10 to 10,000 times larger than the library size. This excess factor is also called the "library equivalent number," indicating that there may be 10 to 10,000 times more clones having the same amino acid sequence. Therefore, "different sequences" in this invention means that the sequences of each polypeptide in the library are different from each other, except for the library equivalent number, and more specifically, there are 10⁶ to 10¹⁴ molecules, preferably 10⁷ to 10¹² molecules, of polypeptides with different sequences.

[0761] In addition, the "multiple" in the so-called library formed mainly by multiple fusion polypeptides in this invention means that, for example, the polypeptides, polynucleotide molecules, vectors, or viruses of this invention are usually a collection of two or more types of the substance. For example, if two or more substances are different from each other in terms of a specific property, it means that there are two or more types of the substance. As an example, variant amino acids observed at specific amino acid positions in the amino acid sequence can be cited. For example, there are multiple polypeptides of this invention that are substantially the same, preferably with the same sequence, except for specific variant amino acids at very diverse amino acid positions exposed on the surface. In another example, if two or more polynucleotide molecules of the present invention are substantially identical, preferably identical, except for the bases of specific variant amino acids encoding a wide variety of amino acid positions exposed on the surface, then there are multiple polynucleotide molecules of the present invention.

[0762] The screening method for fusion peptides using binding activity as an indicator can also suitably use a panning method using a phage vector. A gene encoding a single-domain antibody and a gene encoding the CH1 domain or light chain constant region of an IgG antibody can be linked in a suitable manner to form a fusion peptide. By inserting the gene encoding the fusion peptide into a phage vector, a phage expressing the fusion peptide on its surface can be obtained. After contacting this phage with the desired antigen, the antigen-binding phage is recovered.The phage can be recovered to obtain DNA encoding a fusion polypeptide with the target binding activity. This operation can be repeated as needed to concentrate the fusion polypeptide with the desired binding activity.

[0763] In addition to phage display, as a technique for obtaining fusion polypeptides by panning a library, techniques using cell-free translation systems, techniques for presenting fusion polypeptides on the surface of cells or viruses, and techniques using emulsification are known. For example, as a technique using cell-free translation systems, the following can be used: ribosome display method, which forms a complex of mRNA and translated protein by removing the stop codon via ribosomes; cDNA display method, which uses compounds such as puromycin to covalently bond the gene sequence with the translated protein; mRNA display method; CIS display method, which forms a complex of gene and translated protein by using proteins that bind to nucleic acids. In addition to phage display, other techniques for displaying fusion peptides on cell or virus surfaces include E. coli display, Gram-positive bacteria display, yeast display, mammalian cell display, and virus display. As for techniques using emulsification, in vitro virus display methods can be used, which involve including genes and translation-related molecules in the emulsion. These methods are well known (Nat Biotechnol. 2000 Dec; 18 (12): 1287-92, Nucleic Acids Res. 2006; 34 (19): e127, Proc Natl Acad Sci US A. 2004 Mar 2; 101 (9): 2806-10, Proc Natl Acad Sci US A. 2004 Jun 22; 101 (25): 9193-8, Protein Eng Des Sel. 2008 Apr; 21 (4): 247-55, Proc Natl Acad Sci US A. 2000 Sep 26; 97 (20): 10701-5, MAbs. 2010 Sep-Oct; 2 (5): 508-18, Methods Mol Biol.). 2012; 911:183-98).

[0764] In other embodiments of the present invention, a library comprising a plurality of fusion peptides linked to a single-domain antibody and a constant region of the light chain of an IgG antibody is provided. This library contains single-domain antibodies that inhibit or lose antigen-binding activity by associating with specific VL / VH / VHH, and a method for screening single-domain antibodies from this library that inhibit or lose antigen-binding activity by associating with specific VL / VH / VHH is provided.

[0765] "Antigen binding activity below a certain value" can refer to antigen binding activity that is below a certain benchmark when measured by the method exemplified in this specification. "Antigen binding activity above a certain value" can similarly refer to antigen binding activity that is above a certain benchmark when measured by the method exemplified in this specification. Compared with fusion peptides whose antigen binding activity is below a certain value, fusion peptides whose antigen binding activity is above a certain value bind to antigens more strongly.

[0766] Hereinafter, some embodiments using the CH1 domain of an IgG antibody as the first association support domain and the CL domain of an IgG antibody as the second association support domain will be described.

[0767] Fusion peptides containing a target single-domain antibody can be screened from a library containing multiple fusion peptides linked to a single-domain antibody and the CH1 domain of an IgG antibody.

[0768] Some embodiments of the present invention provide a library comprising a plurality of fusion peptides linked to a single-domain antibody and an IgG antibody CH1 domain, wherein the single-domain antibody comprises a library of single-domain antibodies that inhibit or lose antigen-binding activity by associating with a specific VL / VH / VHH, and a method for screening from the library fusion peptides comprising single-domain antibodies that inhibit or lose antigen-binding activity by associating with a specific VL / VH / VHH.

[0769] In a particular embodiment, a method is provided for screening from a library comprising a plurality of fusion peptides linked to a single-domain antibody and an IgG antibody CH1 domain fusion peptides fusion peptides comprising single-domain antibodies that inhibit or lose antigen-binding activity by associating with a specific VL. Specifically, a method for screening single-domain antibodies is provided, comprising the following steps:

[0770] (a) displaying the fusion peptide of the library in this invention in vitro;

[0771] (b) preparing a specific VL and IgG antibody light chain constant region fusion pair;

[0772] (c) associating the fusion peptide displayed in step (a) with the pair prepared in step (b), and selecting a fusion peptide that does not bind to the antigen or whose antigen-binding activity is below a certain value when the single-domain antibody is associated with the VL;

[0773] (d) selecting a fusion peptide in which the single-domain antibody contained in the fusion peptide selected in step (c) binds to the antigen or whose antigen-binding activity is above a certain value when it is not associated with the VL.

[0774] The mating body prepared in step (b) above also contains a protease cleavage sequence. In step (d) above, the association between the single-domain antibody and the VL can be eliminated by protease treatment, confirming the antigen-binding activity of the single-domain antibody in the state where the single-domain antibody and VL do not associate. The protease cleavage sequence in the mating body, as long as it is eliminated during cleavage...The location of the protease cleavage sequence is not limited except for the association between the single-domain antibody and VL. Examples of protease cleavage sequence locations include the area near the boundary between the VL of the conjugate and the constant region of the IgG antibody light chain, preferably between amino acid position 96 (Kabat number) of VL and amino acid position 130 (EU number) (Kabat number 130) of the constant region of the antibody light chain, and more preferably between amino acid position 104 (Kabat number) of VL and amino acid position 113 (EU number) (Kabat number 113) of the constant region of the antibody light chain.

[0775] Alternatively, instead of using a conjugate containing a protease cleavage sequence, a protease cleavage sequence can be introduced into the fusion peptide in the library, and the association between the single-domain antibody and VL can be eliminated by protease cleavage of the fusion peptide. The location of the protease cleavage sequence in the fusion peptide is not limited as long as it eliminates the association between the single-domain antibody and VL during cleavage and maintains the antigen-binding activity of the single-domain antibody after cleavage. As an example of the location of the protease cleavage sequence, it may be located near the boundary between the single-domain antibody and the CH1 domain of the IgG antibody in the fusion polypeptide.

[0776] Further, in step (d) above, the full length of the fusion polypeptide selected in step (c) or the portion containing the single-domain antibody may be displayed again to confirm the antigen-binding activity of the single-domain antibody in the state where the single-domain antibody does not associate with VL.

[0777] In a particular embodiment, a method is provided for screening fusion polypeptides containing single-domain antibodies that inhibit or lose antigen-binding activity by associating with specific VH from a library containing a single-domain antibody and a constant region of the light chain of an IgG antibody. Specifically, a method for screening fusion peptides containing single-domain antibodies is provided, comprising the following steps:

[0778] (a) displaying the fusion peptide of the library of the present invention in vitro;

[0779] (b) preparing a specific VH and IgG antibody CH1 domain fusion mating body;

[0780] (c) associating the fusion peptide displayed in step (a) with the mating body prepared in step (b), and selecting a fusion peptide that does not bind to the antigen or whose antigen-binding activity is below a certain value when the single-domain antibody is associated with the VH;

[0781] (d) selecting a fusion peptide in which the single-domain antibody contained in the fusion peptide selected in step (c) binds to the antigen or whose antigen-binding activity is above a certain value when it is not associated with the VH.

[0782] The conjugate prepared in step (b) above further comprises a protease cleavage sequence. In step (d) above, the association between the single-domain antibody and the VH can be eliminated by protease treatment, confirming that the single-domain antibody does not associate with the VH.The antigen-binding activity of the single-domain antibody in the associated pair is maintained. The location of the protease cleavage sequence in the pair is not limited as long as it eliminates the association between the single-domain antibody and VH upon cleavage. Examples of protease cleavage sequence locations include near the boundary between the VH and CH1 domains of the IgG antibody in the pair, preferably between amino acid position 101 (Kabat number) of VH and amino acid position 140 (EU number) of the antibody heavy chain constant region, and more preferably between amino acid position 109 (Kabat number) of VH and amino acid position 122 (EU number) of the antibody heavy chain constant region.

[0783] Alternatively, instead of using an associated pair containing a protease cleavage sequence, a protease cleavage sequence can be introduced into the fusion peptide in the library, and the association between the single-domain antibody and VH can be eliminated by protease cleavage of the fusion peptide. The location of the protease cleavage sequence in the fusion peptide is not limited as long as it eliminates the association between the single-domain antibody and VH upon cleavage and maintains the antigen-binding activity of the single-domain antibody after cleavage. As an example of the location of the protease cleavage sequence, it may be located near the boundary between the single-domain antibody and the constant region of the light chain of the IgG antibody in the fusion polypeptide.

[0784] Further, in step (d) above, the full length of the fusion polypeptide selected in step (c) or the portion containing the single-domain antibody may be displayed again to confirm the anti-antigen binding activity of the single-domain antibody in the state where the single-domain antibody does not associate with VH.

[0785] The amino acids contained in the amino acid sequence described in this invention may also be subject to post-translational modifications (e.g., the modification of N-terminal glutamine by pyroglutamylation to pyroglutamic acid is a modification known to those skilled in the art), and even such post-translational modifications of amino acids are naturally included in the amino acid sequence described in this invention.

[0786] Methods for preparing antibodies with the desired binding activity are known to those skilled in the art. In this invention, an antigen-binding molecule that is expressed on the surface of a target cell (pathogenic cell) can be used as an antigen (target antigen). When the target cell is a tumor cell or cancer cell, the antigen is used as a tumor antigen, as illustrated below in this specification, and a method for preparing an antibody that binds to the tumor antigen is described.

[0787] The antibody that binds to the tumor antigen can be obtained using known methods, either a polyclonal antibody or a monoclonal antibody. The antibody is preferably prepared as a monoclonal antibody derived from mammals. Monoclonal antibodies derived from mammals include monoclonal antibodies produced by hybridomas, and monoclonal antibodies produced by genetic engineering methods using host cells transformed with an expression vector containing an antibody gene.

[0788] Hybridomas that produce monoclonal antibodies can be prepared using known techniques, for example, as described below. That is, using tumor...The antigen protein is used as a sensitizing antigen to immunize mammals using conventional immunization methods. The obtained immune cells are fused with known parental cells using conventional cell fusion methods. Then, monoclonal antibody-producing cells are screened using conventional screening methods, and hybridomas that produce antibodies against the tumor antigen are selected.

[0789] Specifically, the preparation of monoclonal antibodies is carried out, for example, as follows. First, the tumor antigen gene is expressed to obtain a tumor antigen protein that can be used as a sensitizing antigen for obtaining antibodies. That is, the gene sequence encoding the tumor antigen is inserted into a known expression vector and transformed into a suitable host cell. The desired human tumor antigen protein is purified from the host cell or from the culture supernatant using known methods. In order to obtain a soluble tumor antigen from the culture supernatant, a protein that lacks, for example, the portion constituting the hydrophobic region in the tumor antigen polypeptide sequence can be used. In addition, purified natural GPC3 protein can also be used as a sensitizing antigen.

[0790] The purified tumor antigen protein can be used as a sensitizing antigen for immunizing mammals. A partial peptide of the tumor antigen can also be used as a sensitizing antigen. At this time, the partial peptide can also be obtained by chemical synthesis from the amino acid sequence of the human tumor antigen. Alternatively, it can be obtained by inserting a partial tumor antigen gene into an expression vector and expressing it. Further, it can also be obtained by using a proteolytic enzyme to decompose the tumor antigen protein, but the region and size of the tumor antigen peptide used as a partial peptide are not particularly limited to a specific form. The number of amino acids constituting the peptide as a sensitizing antigen is preferably at least 5, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0791] In addition, a fusion protein formed by fusing a desired portion of the tumor antigen protein with different peptides can be used as a sensitizing antigen. In order to manufacture a fusion protein used as a sensitizing antigen, for example, the Fc fragment of an antibody or a peptide tag can be preferably used. The vector for expressing the fusion protein is fused with a gene encoding two or more desired polypeptide fragments in a reading frame manner, and the fusion gene is prepared by inserting it into the expression vector as described above. The preparation method of the fusion protein is described in Molecular Cloning, 2nd edition (Sambrook, J et al., Molecular Cloning, 2nd edition, 9.47-9.58 (1989) Cold Spring Harbor Lab. press). As an example, the method for obtaining GPC3 as a sensitizing antigen and the immunization method using it are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0792] The mammals immunized with this sensitizing antigen are not limited to specific animals, but those fused with cells are preferred.The suitability of the parent cells used is considered. Generally, rodents, such as mice, rats, hamsters, or rabbits, monkeys, etc., are suitable.

[0793] The above-mentioned animals are immunized by using a sensitizing antigen according to known methods. For example, as a routine method, immunization is carried out by administering the sensitizing antigen intraperitoneally or subcutaneously to mammals. Specifically, the sensitizing antigen diluted with PBS (phosphate-buffered saline) or physiological saline at an appropriate dilution ratio is mixed with a common adjuvant, such as complete Freund's adjuvant, emulsified as needed, and administered to mammals several times every 4 to 21 days. In addition, a suitable carrier can be used when immunizing with the sensitizing antigen. In particular, in cases where a small molecular weight partial peptide is used as the sensitizing antigen, it is desirable to immunize with the sensitizing antigen peptide that binds to carrier proteins such as albumin and keyhole hemocyanin.

[0794] Alternatively, hybridomas that produce the desired antibodies can also be prepared using DNA immunization, as described below. DNA immunization is an immunization method in which a vector DNA constructed in a manner capable of expressing a gene encoding an antigen protein is administered to an immunized animal, thereby expressing the sensitizing antigen in the immunized animal and providing immune stimulation. Compared with conventional immunization methods that administer protein antigens to immunized animals, DNA immunization is expected to have the following advantages:

[0795] - It can maintain the structure of membrane proteins and provide immune stimulation.

[0796] - It is not necessary to purify the immune antigen.

[0797] In order to obtain the monoclonal antibody of the present invention via DNA immunization, DNA expressing a tumor antigen protein is first administered to an immunized animal. The DNA encoding the tumor antigen can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector and administered to an immunized animal. The expression vector can be a commercially available expression vector such as pcDNA3.1. The method of administering the vector to the organism can be a conventionally used method. For example, DNA immunization is performed by introducing gold particles adsorbed with the expression vector into the cells of an individual immunized animal using a gene gun. Furthermore, the preparation of antibodies that recognize tumor antigens can also be performed using the method described in international publication WO2003 / 104453.

[0798] After immunizing mammals in this way and confirming an increase in the antibody titer binding to tumor antigens in the serum, immune cells are collected from the mammals and provided for cell fusion. Preferred immune cells, in particular, are spleen cells.

[0799] The cells fused with the above-mentioned immune cells are mammalian myeloma cells. Myeloma cells preferably have suitable selection markers for screening. Selection markers refer to traits that allow (or prevent) survival under specific culture conditions. Selection markers include hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter referred to as HGPRT deficiency) or thymidine deficiency.Kinase deficiency (hereinafter referred to as TK deficiency) is well known. Cells with HGPRT or TK deficiency are sensitive to inosine-aminopterin-thymidine (hereinafter referred to as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA and die in HAT-selective medium, but when fused with normal cells, they can continue DNA synthesis using the salvage pathway of normal cells, and therefore can proliferate even in HAT-selective medium.

[0800] HGPRT-deficient or TK-deficient cells can be selected in medium containing 6-thioguanine, 8-azguanine (hereinafter referred to as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that take up these pyrimidine analogs in their DNA will die. On the other hand, these enzyme-deficient cells that do not take up these pyrimidine analogs can survive in selective medium. In addition, a selection marker called G418 resistance provides resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via a neomycin resistance gene. Various myeloma cells suitable for cell fusion are well known.

[0801] Such myeloma cells may be suitably used, for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. Specification 58 / 139 pages 61 CN 121818917 A

[0802] Basically, the above-mentioned cell fusion of immune cells and myeloma cells is carried out according to known methods, such as the method of Köhler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46), etc.

[0803] More specifically, for example, the above-mentioned cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter. The fusion promoter used is, for example, polyethylene glycol (PEG), Sendai virus (HVJ), etc., in order to further improve the fusionEfficiency can be improved by adding adjuvants such as dimethyl sulfoxide as needed.

[0804] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is preferable to use an immune cell to myeloma cell ratio of 1 to 10. As a culture medium for the above-mentioned cell fusion, for example, RPMI1640 medium or MEM medium suitable for the proliferation of the above-mentioned myeloma cell line can be used; in addition, conventional culture medium used for such cell culture can be used; furthermore, serum supplements such as fetal bovine serum (FCS) can be appropriately added.

[0805] Cell fusion is performed by uniformly mixing specific amounts of the above-mentioned immune cells and myeloma cells in the above-mentioned culture medium, usually by adding a PEG solution (e.g., an average molecular weight of about 1000 to 6000) preheated to about 37°C at a concentration of 30 to 60% (w / v). By slowly mixing the mixture, the desired fused cells (hybridoma) are formed. Then, a suitable culture medium as exemplified above is added sequentially, and the operation of centrifugation and removal of supernatant is repeated to remove cell fusion agents that are detrimental to hybridoma growth.

[0806] The hybridomas thus obtained can be selected by culturing in a conventional selection medium, such as HAT medium (containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium is continued until sufficient time (typically several days to several weeks) is required for the cells other than the desired hybridoma (non-fusion cells) to die. Then, screening and monoclonalization of hybridomas producing the desired antibodies are performed using a conventional limiting dilution method.

[0807] The hybridomas thus obtained can be selected using a selection medium corresponding to the selection markers of myeloma used for cell fusion. For example, cells with HGPRT or TK deficiencies are selected by culturing in HAT medium (containing hypoxanthine, aminopterin, and thymidine). That is, in cases where HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively proliferate in HAT medium. Culture in the HAT medium is continued until sufficient time is required for the cells other than the desired hybridoma (non-fusion cells) to die. Specifically, after several days to several weeks of culture, desired hybridomas can be selected. Then, using conventional limiting dilution methods, the hybridomas that produce the desired antibodies can be screened and monoclonalized.

[0808] The screening and monoclonalization of the desired antibodies can be appropriately carried out using screening methods based on known antigen-antibody reactions. For example, monoclonal antibodies that bind to GPC3 can bind to GPC3 expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence-activated cell sorting). FACS is a system that uses laser analysis to determine the binding of antibodies to the cell surface by measuring the fluorescence emitted by each cell.

[0809] To screen hybridomas that produce the monoclonal antibodies of the present invention via FACS, cells expressing GPC3 are first prepared. Preferred cells for screening are mammalian cells that forcibly express the desired tumor antigen. By using non-transformed mammalian cells as host cells as a control, the binding activity of the antibody to the tumor antigen on the cell surface can be selectively detected. That is, by selecting hybridomas that produce antibodies that do not bind to host cells but bind to GPC3-forcibly-expressing cells, hybridomas producing monoclonal antibodies against the tumor antigen can be obtained.

[0810] Alternatively, the binding activity of the antibody to immobilized tumor antigen-expressing cells can be assessed based on the ELISA principle. For example, GPC3-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is brought into contact with the immobilized cells in the wells, and the antibody binding to the immobilized cells is detected. In cases where the monoclonal antibody is from mice, the antibody binding to the cells can be detected using anti-mouse immunoglobulin antibodies. Hybridomas selected through such screening that produce the desired antibody with the ability to bind to the antigen can be screened using limiting dilution methods, etc.

[0811] The hybridoma thus prepared to produce monoclonal antibodies can be passaged in conventional culture medium. Furthermore, the hybridoma can be stored long-term in liquid nitrogen.

[0812] The hybridoma can be cultured using conventional methods, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be propagated by introducing it into a suitable mammal, and the monoclonal antibody can be obtained from its ascites. The former method is suitable for obtaining high-purity antibodies.

[0813] Antibodies encoded by antibody genes cloned from antibody-producing cells such as the hybridoma can also be suitably utilized. The cloned antibody gene is inserted into a suitable vector and introduced into a host to express the antibody encoded by the gene. Methods for isolating antibody genes, introducing vectors, and transforming host cells have been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). The method for manufacturing recombinant antibodies as described below is also known.

[0814] For example, cDNA encoding the variable region (V region) of an antibody is obtained from hybridoma cells that produce antibodies that bind to tumor antigens. For this purpose, total RNA is usually extracted from the hybridoma first. Methods for extracting mRNA from cells can be, for example, the following methods: - guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159).

[0815] The extracted mRNA can be purified using an mRNA purification kit (manufactured by GE Healthcare BioSciences) or the like.Alternatively, commercially available kits are available for directly extracting total mRNA from cells, such as the QuickPrep mRNA purification kit (manufactured by GE Healthcare BioSciences). Using such kits, mRNA can be obtained from hybridomas. Using reverse transcriptase, cDNA encoding the antibody V region is synthesized from the obtained mRNA. cDNA can be synthesized using a kit such as the AMV reverse transcriptase first-strand cDNA synthesis kit (manufactured by Biochemical Industry Co., Ltd.). In addition, cDNA synthesis and amplification can be appropriately performed using the SMART RACE cDNA amplification kit (manufactured by Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad . Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932). Furthermore, during such cDNA synthesis, suitable restriction enzyme sites, as described later, can be introduced at both ends of the cDNA.

[0816] The target cDNA fragment is purified from the obtained PCR product and then ligated to the vector DNA. Recombinant vectors are prepared in this way, and the desired recombinant vector can be prepared from E. coli colonies that form the colonies after selection of colonies such as *E. coli*. Then, the presence of the target cDNA base sequence in the recombinant vector is confirmed by known methods, such as dideoxynucleotide chain termination.

[0817] To obtain the gene encoding the variable region, the 5'-RACE method using primers for variable region gene amplification is simple. First, RNA extracted from hybridoma cells is used as a template to synthesize cDNA, obtaining a 5'-RACE cDNA library. For the synthesis of the 5'-RACE cDNA library, commercially available kits such as the SMART RACE cDNA amplification kit can be used appropriately.

[0818] The obtained 5'-RACE cDNA library is used as a template to amplify the antibody gene by PCR. Primers for mouse antibody gene amplification can be designed based on known antibody gene sequences. Such primers are base sequences that are different for each immunoglobulin subclass. Therefore, it is desirable to determine the subtype in advance using commercially available kits such as the Iso Strip mouse monoclonal antibody isotype kit (Roche Diagnostics).

[0819] Specifically, for example, when the goal is to obtain the gene encoding mouse IgG, the gene encoding heavy chains γ1, γ2a, γ2b, γ3 and light chains κ and λ can be amplified using possible primers. In order to amplify the IgG variable region gene, primers that anneal to the portion of the constant region corresponding to the variable region are generally used for the 3' end primer. On the other hand, primers for the 5' end primer are used using the primers provided in the 5'-RACE cDNA library preparation kit.

[0820] Using the PCR product thus amplified, an immunoglobulin formed by a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to the antigen can be used as an indicator to screen for desired antibodies. For example, when the goal is to obtain an antibody against GPC3, it is preferable that the antibody is specific for binding to GPC3. The antibodies used in this invention can be screened, for example, as described below: Specification 60 / 139 pages 63 CN 121818917 A

[0821] (1) a step of contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with an antigen-expressing cell;

[0822] (2) a step of detecting the binding of the antigen-expressing cell to the antibody; and

[0823] (3) a step of selecting an antibody that binds to the antigen-expressing cell.

[0824] Methods for detecting the binding of antibodies to tumor antigen-expressing cells are well known. Specifically, as described above, the binding of antibodies to tumor antigen-expressing cells can be detected by methods such as FACS. The binding activity of the antibody can be appropriately evaluated using a fixed sample of tumor antigen-expressing cells.

[0825] Antibody screening methods that use binding activity as an indicator can also appropriately employ phage vector panning. In cases where a library is obtained from a polyclonal antibody expression cell population containing antibody genes as heavy and light chain subclasses, phage vector screening is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked by suitable adapter sequences to form single-stranded Fvs (scFvs). By inserting the gene encoding scFv into a phage vector, phages expressing scFv on their surface can be obtained. After contacting the phage with the desired antigen, the DNA encoding the scFv with the desired binding activity can be recovered by recovering the phage bound to the antigen. This operation can be repeated as needed to concentrate the scFv with the desired binding activity.

[0826] After obtaining cDNA encoding the V region of an antibody that binds to the target tumor antigen, the cDNA is digested by recognizing a restriction enzyme inserted at the restriction enzyme sites at both ends of the cDNA. Preferably, the restriction enzyme recognizes and digests a base sequence that occurs infrequently in the base sequence constituting the antibody gene. Furthermore, to insert a single copy of the digested fragment into the vector in the correct orientation, it is preferable to use an insertion of a restriction enzyme that produces sticky ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the digested anti-GPC3 antibody, as described above, into a suitable expression vector. At this point, a chimeric antibody can be obtained by fusing the gene encoding the antibody constant region (C region) with the gene encoding the aforementioned V region in a frame-compliant manner. Here, a chimeric antibody refers to an antibody whose constant region and variable region originate from different sources. Therefore, in addition to xenogeneic chimeric antibodies such as mouse-human, allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. The V region gene is inserted beforehand into an expression vector having the constant region.Therefore, a chimeric antibody expression vector can be constructed. Specifically, for example, a restriction enzyme recognition sequence of a restriction enzyme that digests the V region gene can be suitably configured at the 5' end of an expression vector having DNA encoding the desired constant region (C region). The two, digested with the same combination of restriction enzymes, are fused together in a reading frame to construct a chimeric antibody expression vector.

[0827] To produce a monoclonal antibody, an antibody gene is inserted into an expression vector for expression under the control of an expression control region. The expression control region for expressing the antibody includes, for example, an enhancer or a promoter. In addition, a suitable signal sequence can be added to the amino terminus to cause the expressed antibody to be secreted extracellularly. In the embodiments described later, a peptide having the amino acid sequence MGSCIILFLVATATGVHS is used as the signal sequence, but other suitable signal sequences may be added. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the cleaved polypeptide, as a mature polypeptide, can be secreted extracellularly. Then, by transforming a suitable host cell with the expression vector, recombinant cells expressing DNA encoding an antibody that binds to a target tumor antigen can be obtained.

[0828] To express the antibody gene, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is inserted into different expression vectors. Antibody molecules having both H and L chains can be expressed by simultaneously co-transfecting the same host cell with vectors containing both H and L chains. Alternatively, host cells can be transformed by inserting DNA encoding both H and L chains into a single expression vector (see International Publication WO 94 / 11523).

[0829] Various combinations of host cells and expression vectors for preparing antibodies by introducing the isolated antibody gene into a suitable host are known. These expression systems can be applied to isolate any structural domain containing the variable region of the antibody of the present invention. When using eukaryotic cells as host cells, animal cells, plant cells, or fungal cells can be suitably used. Specifically, examples of animal cells include the following. Instructions for Use, pages 61 / 139, CN 121818917 A

[0830] (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc.

[0831] (2) Amphibian cells: Xenopus laevis oocytes, etc.

[0832] (3) Insect cells: sf9, sf21, Tn5, etc.

[0833] Alternatively, antibody gene expression systems from cells of the genus Nicotiana, such as tobacco (Nicotiana tabacum), are known. Transformation of plant cells can be suitably achieved using cells cultured from callus tissue.

[0834] Further, fungal cells can be utilized using the following cells.

[0835] - Yeasts: Saccharomyces such as Saccharomyces serevisiae, Pichia such as Pichia pastoris

[0836] - Filaments: Aspergillus such as Aspergillus niger.

[0837] In addition, antibody gene expression systems using prokaryotic cells are also known. For example, in the case of using bacterial cells, bacterial cells such as Escherichia coli and Bacillus subtilis can be appropriately used. An expression vector containing the target antibody gene is transformed and introduced into these cells. By culturing the transformed cells in vitro, the desired antibody can be obtained from the culture of the transformed cells.

[0838] In addition to the host cells mentioned above, transgenic animals can also be used for the production of recombinant antibodies. That is, the antibody can be obtained from an animal that has been introduced with a gene encoding the desired antibody. For example, by inserting the antibody gene into the gene encoding a protein that is normally produced in milk in a frame-compliant manner, a fusion gene can be constructed. For example, sheep β-casein can be used as a protein secreted in milk. A DNA fragment containing a fusion gene with an inserted antibody gene is injected into a sheep embryo, and the injected embryo is introduced into a female sheep. The milk produced by the transgenic sheep (or its offspring) born from the sheep that received the embryo can contain a fusion protein of the desired antibody and milk protein. In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic sheep, hormones can be administered to the transgenic sheep (Bio / Technology (1994), 12 (7), 699-702).

[0839] In the case of administering the antigen-binding molecule described in this specification to humans, the domain containing the antibody variable region of the antigen-binding molecule is suitably used for the purpose of reducing the heteroantigenicity to humans, etc., and is derived from an artificially modified recombinant antibody. Recombinant antibodies include, for example, humanized antibodies. These variant antibodies can be suitably manufactured using known methods.

[0840] To prepare the antibody variable region domain in the antigen-binding molecule described in this specification, the antibody variable region typically consists of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). The CDRs are the regions that substantially determine the antibody binding specificity. The amino acid sequences of CDRs are highly diverse. On the one hand, the amino acid sequences constituting the FRs often show high similarity even among antibodies with different binding specificities. Therefore, the binding specificity of one antibody can generally be transferred to other antibodies through CDR transplantation.

[0841] Humanized antibodies are also called reshaped human antibodies. Specifically, it is known to transplant humanized antibodies into human antibodies by grafting the CDR of an animal other than humans, such as a mouse antibody, into a human antibody. General gene recombination methods for obtaining humanized antibodies are also known. Specifically, as a method for grafting a mouse antibody CDR into a human FR, overlap extension PCR is known, for example. In overlap extension PCR, the base sequence encoding the mouse antibody CDR to be grafted is added to the primers used to synthesize the FR of the human antibody. Primers are prepared for each of the four FRs. Generally, in the grafting of mouse CDR into human FR, human FRs with high identity to mouse FRs are selected, which is advantageous in maintaining the function of the CDR. That is, generally, it is preferred to use human FRs composed of amino acid sequences with high identity to the amino acid sequences of FRs adjacent to the mouse CDR to be grafted.

[0842] In addition, the linked base sequences are designed to be linked in a reading frame manner. Human FRs are synthesized separately using their respective primers. As a result, products are obtained by adding DNA encoding the mouse CDR to each FR. The base sequences encoding mouse CDRs of each product were designed to overlap as described on page 65 of the specification (62 / 139, CN 121818917 A). Next, using the human antibody gene as a template, the overlapping CDR portions of the synthesized products were renatured, and a complementary chain synthesis reaction was performed. Through this reaction, human FRs were linked via mouse CDR sequences.

[0843] Finally, the V region gene linking the three CDRs and four FRs was renatured at its 5' and 3' ends, and its full length was amplified by adding primers with appropriate restriction enzyme recognition sequences. The DNA obtained as described above and the DNA encoding the C region of the human antibody were fused in a reading frame conformal manner and inserted into an expression vector to produce a humanized antibody expression vector. By introducing this recombinant vector into a host, establishing recombinant cells, and culturing the recombinant cells, the DNA encoding the humanized antibody was expressed, and the humanized antibody was produced in the culture of these cells (European Patent Publication EP 239400, International Publication WO1996 / 002576).

[0844] Qualitative or quantitative determination and evaluation of the binding activity of the humanized antibody prepared as described above against the antigen allows for the appropriate selection of the FR of the human antibody from which a good antigen-binding site is formed when linked to a CDR. If necessary, amino acid residues of the FR can be substituted to allow the CDR of the reconstructed human antibody to form a suitable antigen-binding site. For example, using the PCR method employed for transplanting mouse CDRs into human FRs, amino acid sequence variations can be introduced into the FR. Specifically, partial base sequence variations can be introduced into primers annealed to the FR. FRs synthesized using such primers contain variations in the base sequence. The binding activity of the amino acid-substituted variant antibody against the antigen is evaluated by the methods described above, allowing for the selection of variant FR sequences with desired properties (Sato, K. et al.).Cancer Res, 1993, 53, 851-856.

[0845] In addition, transgenic animals with a complete antibody gene library containing human antibody genes (refer to international publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) can be used as immunized animals to obtain the desired human antibody by DNA immunization.

[0846] Furthermore, the technique of obtaining human antibodies by panning a human antibody library is also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage using phage display. Phages expressing scFv that bind to the antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be prepared by fusing the V region sequence with the desired human antibody C region sequence in a reading frame conformal manner and inserting it into a suitable expression vector. This expression vector is then introduced into suitable expression cells as illustrated above, and the human antibody is obtained by expressing the gene encoding the human antibody. Such methods are known (see international publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

[0847] A domain comprising an antibody variable region having T-cell receptor complex binding activity

[0848] In this specification, "a domain comprising an antibody variable region having T-cell receptor complex binding activity" means a portion of a T-cell receptor complex antibody comprising a region that specifically binds to and is complementary to a portion or all of the T-cell receptor complex. The T-cell receptor complex may be the T-cell receptor itself or an adaptor molecule that, together with the T-cell receptor, constitutes the T-cell receptor complex. A suitable adaptor is CD3.

[0849] A domain comprising an antibody variable region having T-cell receptor binding activity

[0850] In this specification, "a domain comprising an antibody variable region having T-cell receptor binding activity" means a portion of a T-cell receptor antibody comprising a region that specifically binds to and is complementary to a portion or all of the T-cell receptor. The portion of the T-cell receptor bound by the domain of the present invention may be a variable region or a constant region, preferably an epitope present in a constant region. As a sequence in the constant region, an example is the T cell receptor α sequence with RefSeq accession number CAA26636.1.The sequences of the T cell receptor β chain (RefSeq accession number C25777), the T cell receptor γ1 chain (RefSeq accession number A26659), the T cell receptor γ2 chain (RefSeq accession number AAB63312.1), and the T cell receptor δ chain (RefSeq accession number AAA61033.1). Specification 63 / 139 pages 66 CN 121818917 A

[0851] A domain comprising an antibody variable region having CD3 binding activity

[0852] In this specification, "a domain comprising an antibody variable region having CD3 binding activity" refers to a portion of a CD3 antibody comprising a region that specifically binds to and is complementary to a portion or all of CD3. Preferably, the domain comprises a light chain variable region (VL) of an anti-CD3 antibody and a heavy chain variable region (VH) of an anti-CD3 antibody.

[0853] The structural domain of the antibody variable region of the present invention, which includes CD3-binding activity, may also be a structural domain of an antibody variable region that binds to any epitope if an epitope is present in the γ, δ, or ε chain sequence constituting human CD3. In the present invention, preferably, the structural domains of the light chain variable region (VL) and the heavy chain variable region (VH) of an anti-CD3 antibody that bind to an epitope present in the extracellular structural domain of the ε chain of the human CD3 complex are suitably used. As such a structural domain, in addition to the light chain variable region (VL) and the heavy chain variable region (VH) of the anti-CD3 antibody described in the examples, the CD3-binding structural domains of OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) or various known CD3-binding structural domains containing the light chain variable region (VL) and the heavy chain variable region (VH) of an anti-CD3 antibody may also be suitably used. Additionally, a domain containing an antibody variable region having the desired properties, obtained by immunizing a desired animal with the γ, δ, or ε chain constituting human CD3 using the methods described above, may be suitably used. The domain containing the antibody variable region having CD3 binding activity is called the anti-CD3 antibody of origin, and as described above, a suitable humanized antibody or human antibody may be suitably used. For the structures constituting the γ, δ, or ε chain of CD3, the polynucleotide sequences are described in RefSeq accession numbers NM_000073.2, NM_000732.4, and NM_000733.3, and the polypeptide sequences are described in RefSeq accession numbers NP_000064.1, NP_000723.1, and NP_000724.1.

[0854] Specificity

[0855] Specificity refers to the specific binding of a molecule to one or more of its binding targets.Molecules other than molecules do not show any significant binding state. In addition, the domain containing the antibody variable region can also be used to be specific to a particular epitope among multiple epitopes contained in an antigen. In addition, in the case where the epitope containing the antibody variable region is contained in multiple different antigens, the antigen-binding molecule having the domain containing the antibody variable region can bind to multiple antigens containing the epitope.

[0856] Epitope

[0857] An epitope, meaning an antigenic determinant present in an antigen, refers to a site on the antigen that binds to the domain containing the antibody variable region in the antigen-binding molecule disclosed in this specification. Therefore, for example, an epitope can be defined according to its structure. In addition, the epitope can also be defined according to the binding activity to the antigen in the antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can also be determined according to the amino acid residues constituting the epitope. In addition, when the epitope is a glycan, the epitope can also be determined according to a specific glycan structure.

[0858] A linear epitope is an epitope that contains an amino acid primary sequence that is recognized. Linear epitopes typically contain at least three, most commonly at least five, amino acids, such as about eight to about ten, or six to twenty, amino acids in their intrinsic sequence.

[0859] In contrast to linear epitopes, stereoepitopes are not epitopes whose primary amino acid sequence is a single defining component of the epitope being recognized (e.g., the primary amino acid sequence is not necessarily the epitope recognized by an antibody for that specific epitope). Stereoepitopes, relative to linear epitopes, may contain an increased number of amino acids. Regarding the recognition of stereoepitopes, antibodies recognize the tertiary structure of a peptide or protein. For example, when a protein molecule folds to form a tertiary structure, the amino acids and / or polypeptide backbone forming the stereoepitope are arranged side-by-side, and the antibody can recognize the epitope. Methods for determining the stereostructure of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin tagging and electromagnetic paramagnetic resonance spectroscopy, but are not limited thereto. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.). Instructions 64 / 139 pages 67 CN 121818917 A

[0860] The following are examples of methods for confirming epitope binding by antigen-binding molecules that bind to tumor antigens.

[0861] For example, it can be confirmed that antigen-binding molecules that bind to tumor antigens recognize linear epitopes present in tumor antigen molecules. For the above purpose, a linear peptide formed from the amino acid sequence constituting the extracellular domain of the tumor antigen is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering methods using a region in the cDNA of the tumor antigen that encodes the amino acid sequence corresponding to the extracellular domain. Next, the linear peptide formed from the amino acid sequence constituting the extracellular domain is evaluated.The binding activity of a test antigen-binding molecule with a domain containing an antibody variable region that has binding activity against tumor antigens can be assessed. For example, the binding activity of the antigen-binding molecule against the peptide can be evaluated by an ELISA using a fixed linear peptide as the antigen. Alternatively, the binding activity against the linear peptide can be determined based on the level of inhibition caused by the linear peptide in the binding of the antigen-binding molecule to tumor antigen-expressing cells. The binding activity of the antigen-binding molecule against the linear peptide can be determined through these tests.

[0862] In addition, the recognition of stereoepitaxes by a test antigen-binding molecule having a domain containing an antibody variable region that has binding activity against tumor antigens can be confirmed as follows. For the above purpose, cells expressing tumor antigens are prepared. Examples include when a test antigen-binding molecule having a domain containing an antibody variable region that has binding activity against tumor antigens binds strongly to tumor antigen-expressing cells upon contact, and when the antigen-binding molecule substantially does not bind to linear peptides formed by a fixed amino acid sequence constituting the extracellular domain of the tumor antigen. Here, "substantially non-binding" means a binding activity of less than 80%, typically less than 50%, preferably less than 30%, and particularly preferably less than 15% of the binding activity against human tumor antigen-expressing cells.

[0863] A method for determining the binding activity of a test antigen-binding molecule containing an antigen-binding domain against a tumor antigen against tumor antigen-expressing cells is, for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). That is, it can be evaluated using the principle of ELISA or FACS (fluorescence activated cell sorting) with GPC3-expressing cells as antigens.

[0864] In the ELISA form, the binding activity of a test antigen-binding molecule containing an antigen-binding domain against a target tumor antigen against tumor antigen-expressing cells is quantitatively assessed by comparing the signal levels generated by the enzyme reaction. That is, the test antigen-binding molecule is added to an ELISA plate immobilized with tumor antigen-expressing cells, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the binding activity of the test antigen-binding molecule to tumor antigen-expressing cells can be compared by determining the antibody binding titer against the tumor antigen-expressing cells.

[0865] The binding of the test antigen-binding molecule to the antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected by flow cytometry. As flow cytometry, devices such as the following are known.

[0866] FACSCanto™ II

[0867] FACSAria™

[0868] FACSArray™

[0869] FACSVantage™ SE

[0870] FACSCalibur™ (both are trade names of BD Biosciences)

[0871] EPICS ALTRA HyPerSort

[0872] Cytomics FC 500

[0873] EPICS XL-MCL ADC EPICS XL ADC

[0874] Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)

[0875] For example, as an example of a suitable method for determining the binding activity of the test antigen-binding molecule to the antigen, the following method can be cited on pages 65 / 139 of the specification, CN 121818917 A. First, staining with a FITC-labeled secondary antibody that recognizes the test antigen-binding molecule that reacts with cells expressing the target tumor antigen. The test antigen-binding molecule is diluted with an appropriate buffer to prepare the conjugate at the desired concentration for use. For example, it can be used at any concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using FACSCalibur (BD). The amount of antibody binding to the cell is reflected by the fluorescence intensity obtained by analysis using CELL QUEST Software (BD), i.e., the geometric mean. That is, by obtaining this geometric mean, the binding activity of the test antigen-binding molecule, represented by the amount of the test antigen-binding molecule bound, can be determined.

[0876] When the test antigen-binding molecule shares an epitope with a certain antigen-binding molecule, it can be confirmed by the competition between the two for the same epitope. The competition between antigen-binding molecules can be detected by methods such as cross-blocking assays. For example, competitive ELISA assays are preferred cross-blocking assays.

[0877] Specifically, in the cross-blocking assay, a tumor antigen protein coated on the well of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen-binding molecule, and then the test antigen-binding molecule is added. The amount of test antigen-binding molecules that bind to the tumor antigen protein in the pore is indirectly related to the binding ability of candidate competing antigen-binding molecules that compete for binding to the same epitope. That is, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to the pore coated with the tumor antigen protein.

[0878] The amount of test antigen-binding molecules that bind to the pore via the tumor antigen protein can be easily determined by pre-labeling the antigen-binding molecules. For example, this can be done by using an avidin peroxidase conjugate and a suitable substrate.The biotin-labeled antigen-binding molecule is determined. Cross-blocking assays using enzymes such as peroxidase are specifically called competitive ELISA assays. The antigen-binding molecule can be labeled with other detectable or measurable labeling substances. Specifically, radioactive labeling or fluorescent labeling is known.

[0879] Compared to the binding activity obtained in a control assay performed in the absence of a candidate competing antigen-binding molecule, if the competing antigen-binding molecule can block the binding of the test antigen-binding molecule containing the antigen-binding domain of the tumor antigen by at least 20%, preferably at least 20-50%, more preferably at least 50%, then the test antigen-binding molecule is substantially bound to the same epitope as the competing antigen-binding molecule, or is an antigen-binding molecule competing for binding to the same epitope.

[0880] In the case of identifying the structure of an epitope bound by a test antigen-binding molecule containing an antigen-binding domain targeting a tumor antigen, the common epitope between the test antigen-binding molecule and the control antigen-binding molecule can be assessed by comparing the binding activity of the test antigen-binding molecule and the control antigen-binding molecule for a peptide or polypeptide with an amino acid change introduced into the peptide constituting the epitope.

[0881] As a method for measuring such binding activity, for example in the above-described ELISA form, it can be determined by comparing the binding activity of the test antigen-binding molecule and the control antigen-binding molecule for a linear peptide with introduced mutations. As a method other than ELISA, the binding activity against the mutant peptide bound to the column can also be determined by passing the test antigen-binding molecule and the control antigen-binding molecule through a column bound to a mutant peptide, and then quantitatively eluting the antigen-binding molecule in the elution buffer. The method of adsorbing the mutant peptide with, for example, GST as a fusion peptide onto the column is known.

[0882] In addition, in the case where the identified epitope is a stereoepitaxy, the common epitope between the test antigen-binding molecule and the control antigen-binding molecule can be assessed by the following methods. First, cells expressing tumor antigens and cells expressing epitopes of mutated tumor antigens were prepared. A test antigen-binding molecule and a control antigen-binding molecule were added to a cell suspension containing these cells in a suitable buffer such as PBS. Next, a FITC-labeled antibody that recognizes the test antigen-binding molecule and the control antigen-binding molecule was added to the cell suspension after washing with a suitable buffer. The fluorescence intensity and cell number of the cells stained with the labeled antibody were determined using FACSCalibur (BD). The concentrations of the test antigen-binding molecule and the control antigen-binding molecule were appropriately diluted with a suitable buffer to prepare the desired concentration for use. For example, any concentration between 10 μg / ml and 10 ng / ml was used. The binding amount of the labeled antibody to the cells was reflected by analysis using CELL QUEST Software (BD).The obtained fluorescence intensity, i.e., the geometric mean value. That is, by obtaining this geometric mean, the binding activity of the test antigen-binding molecule and the control antigen-binding molecule, represented by the amount of labeled antibody binding, can be determined.

[0883] The following method can be used to determine whether the cells expressing the variant tumor antigen are substantially not bound in this method. First, the test antigen-binding molecule and the control antigen-binding molecule bound to the cells expressing the variant tumor antigen are stained with labeled antibodies. Then, the fluorescence intensity of the cells is detected. In the case where FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. From the geometric mean of the presence and absence of the antigen-binding molecule, this comparison value (Δ Geo-Mean) is calculated according to the following formula, and the proportion of increase in fluorescence intensity caused by the binding of the antigen-binding molecule can be obtained.

[0884] Δ Geo-Mean = Geo-Mean (in the presence of antigen-binding molecules) / Geo-Mean (in the absence of antigen-binding molecules)

[0885] Fv (variable fragment)

[0886] In this specification, the term "Fv (variable fragment)" refers to the smallest unit of the antigen-binding domain of an antibody formed by the pairing of the antibody's light chain variable region (VL) and heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that in E. coli with an antibody gene inserted downstream of the bacterial signal sequence, the gene could be induced to express and prepared uniformly and sustainably from the periplasmic fraction of E. coli (Science (1988) 240 (4855), 1038-1041). In Fv prepared from the periplasmic fraction, VH and VL are associated in a form that binds to the antigen.

[0887] In this specification, Fv is, for example, an antigen-binding molecule that suitably includes a pair of Fvs, in an antigen-binding molecule comprising:

[0888] (1) a bivalent antigen-binding domain; said bivalent antigen-binding domain being a bivalent scFv, formed by linking one monovalent scFv of the bivalent scFv to a polypeptide constituting the Fc region via a heavy chain Fv fragment constituting the CD3-binding domain, and the other monovalent scFv to another polypeptide constituting the Fc region via a light chain Fv fragment constituting the CD3-binding domain;

[0889] (2) a domain comprising an Fc region, wherein the Fc region is an Fc region in which the amino acids constituting the Fc region of IgG1, IgG2a, IgG3, or IgG4 do not have Fc γ receptor-binding activity; and

[0890] (3) At least a monovalent CD3-binding domain, wherein the light chain Fv fragment and the heavy chain Fv fragment are associated in a manner that binds to CD3 to form the CD3-binding domain.

[0891] scFv, single-chain antibody, or sc(Fv)2

[0892] In this specification, the terms “scFv”, “single-chain antibody”, or “sc(Fv)2” refer to an antibody fragment containing variable regions from both the heavy and light chains within a single polypeptide chain, but lacking constant regions. Typically, single-chain antibodies also contain a polypeptide linker between the VH and VL domains, which allows the formation of desired structures that are thought to allow antigen binding. Pluckthun discusses single-chain antibodies in detail in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg, and Moore (eds.), Springer-Verlag, New York, 269–315 (1994). Similarly, reference can be made to International Publication WO1988 / 001649 and US Patents 4,946,778 and 5,260,203. In a particular embodiment, the single-chain antibody may be bispecific and / or humanized.

[0893] scFv is an antigen-binding domain formed by linking the VH and VL constituting Fv via a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). The peptide linker maintains the VH and VL in a close proximity state. (Specification 67 / 139 pages 70 CN 121818917 A)

[0894] sc(Fv)2 is a single-chain antibody consisting of four variable regions, two VLs and two VHs, linked by a linker such as a peptide linker (J Immunol. Methods (1999) 231(1-2), 177-189). The two VHs and VLs can also be derived from different monoclonal antibodies. For example, the bispecific sc(Fv)2 that recognizes two epitopes present in the same antigen, disclosed in Journal of Immunology (1994) 152(11), 5368-5374, is also suitable for exemplification. sc(Fv)2 can be prepared by those skilled in the art using methods known to them. For example, sc(Fv)2 can be prepared by linking scFv with a linker such as a peptide linker.

[0895] As a configuration of the antigen-binding domain constituting sc(Fv)2 in this specification, examples can be given of: two VH and two VL with the N-terminal side of a single-chain polypeptide as the base point, in the order VH, VL, VH, VL([VH]-linker-[VL]-linker-[VH]-linker-The antibody is characterized by the sequential arrangement of [VL]). The order of the two VH and two VL is not particularly limited to the above configuration, but can be arranged in any order. For example, the following configuration can also be given.

[0896] [VL] adapter [VH] adapter [VH] adapter [VL]

[0897] [VH] adapter [VL] adapter [VL] adapter [VH]

[0898] [VH] adapter [VH] adapter [VL] adapter [VL]

[0899] [VL] adapter [VL] adapter [VH] adapter [VH]

[0900] [VL] adapter [VH] adapter [VL] adapter [VH]

[0901] The molecular morphology of sc(Fv)2 is also described in detail in WO2006 / 132352. Based on these descriptions, those skilled in the art can appropriately prepare the required sc(Fv)2 in order to prepare the antigen-binding molecule disclosed in this specification.

[0902] In addition, the antigen-binding molecule of the present invention can also be conjugated with carrier polymers such as PEG or organic compounds such as anticancer agents. Furthermore, inserting additional sequences into the glycan chain allows for the appropriate addition to achieve the desired effect.

[0903] As the adapter for the variable region binding the antibody, any genetically engineered peptide adapter or a synthetic compound adapter (e.g., the adapter disclosed in Protein Engineering, 9 (3), 299-305, 1996) can be used, but peptide adapters are preferred in the present invention. The length of the peptide adapter is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. A preferred length is 5 amino acids or more (there is no particular upper limit, generally 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. In the case where sc(Fv)2 contains 3 peptide adapters, all peptide adapters can be of the same length, or peptide adapters of different lengths can be used.

[0904] For example, in the case of a peptide linker, the following sequence can be cited:

[0905] Ser

[0906] Gly・Ser

[0907] Gly・Gly・Ser

[0908] Ser・Gly・Gly

[0909] Gly・Gly・Gly・Ser

[0910] Ser・Gly・Gly・Gly

[0911] Gly・Gly・Gly・Gly・Ser

[0912] Ser・Gly・Gly・Gly・Gly

[0913] Gly・Gly・Gly・Gly・Gly・Ser

[0914] Ser・Gly・Gly・Gly・Gly・Gly

[0915] Gly・Gly・Gly・Gly・Gly・Gly・Ser

[0916] Ser・Gly・Gly・Gly・Gly・Gly・Gly Instructions Page 68 / 13971 CN 121818917 A

[0917] (Gly・Gly・Gly・Gly・Ser)n

[0918] (Ser・Gly・Gly・Gly・Gly)n

[0919] [wherein, n is an integer greater than or equal to 1] etc. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to their purpose.

[0920] Synthetic chemical linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as: N-hydroxysuccinimide (NHS), disuccinimide octanoate (DSS), bis(sulfosuccinimide) octanoate (BS3), dithiobis(succinimide propionate) (DSP), dithiobis(sulfosuccinimide propionate) (DTSSP), bis(succinimide succinic acid) ethylene glycol ester (EGS), bis(sulfosuccinimide succinic acid) ethylene glycol ester (sulfon-EGS), disuccinimide tartrate (DST), disulfosuccinimide tartrate (sulfon-DST), bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl] sulfone (sulfon-BSOCOES), etc., all of which are commercially available.

[0921] In the case of connecting four antibody variable regions, three adapters are usually required. All adapters can be the same, or different adapters can be used.

[0922] “Fab” consists of a light chain, a CH1 region of a heavy chain, and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0923] “F(ab')2” and “Fab'” refer to antibody fragments prepared by treating immunoglobulins (monoclonal antibodies) with pepsin or papain, which are protein-degrading enzymes. These fragments are digested before and after the disulfide bond between the two H chains present in the hinge region. For example, IgG can be treated with papain and cleaved upstream of the disulfide bond between the two H chains present in the hinge region to prepare two identical antibody fragments: an L chain composed of VL (L chain variable region) and CL (L chain constant region) and an H chain composed of VH (H chain variable region) and CHγ1 (γ1 region in the H chain constant region), linked by a disulfide bond at the C-terminal region. These two identical antibody fragments are referred to as Fab'.

[0924] "F(ab')2" comprises two light chains and two heavy chains, the two heavy chains containing constant regions of CH1 domains and partial CH2 domains to form interchain disulfide bonds between the two heavy chains. The ...

Claims

1. A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with an antigen-binding molecule, wherein... Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens. Chimeric receptors comprise an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain is capable of binding antigen-binding molecules cleaved by the adaptor, and through binding to these antigen-binding molecules, it can bind to cells expressing the target antigen.

2. A pharmaceutical composition comprising an antigen-binding molecule for use in combination with cells expressing a chimeric receptor, wherein... Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens. Chimeric receptors comprise an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain is capable of binding antigen-binding molecules cleaved by the adaptor, and through binding to these antigen-binding molecules, it can bind to cells expressing the target antigen.

3. A pharmaceutical composition comprising a bispecific antibody for use in combination with an antigen-binding molecule, wherein... Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens. Bispecific antibodies consist of an antibody variable region that binds to antigen-binding molecules cleaved by protease linkers and an antibody variable region that binds to molecules expressed on the surface of T cells. Bispecific antibodies can bind to cells expressing target antigens by binding to antigen-binding molecules cleaved by the linker.

4. A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of a bispecific antibody, wherein... Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens. Bispecific antibodies consist of an antibody variable region that binds to antigen-binding molecules cleaved by protease linkers and an antibody variable region that binds to molecules expressed on the surface of T cells. Bispecific antibodies can bind to cells expressing target antigens by binding to antigen-binding molecules cleaved by the linker.

5. A pharmaceutical composition comprising an IgG antibody characterized by enhanced antibody-dependent cytotoxicity for use in combination with an antigen-binding molecule, wherein... The antigen-binding molecule contains a protease-cleavable linker, which, after being cleaved by the protease, exhibits binding activity to antigens expressed on the surface of target cells. The IgG antibody includes an antibody variable region that is active in binding to antigen-binding molecules after the linker has been cleaved by a protease. The IgG antibody can bind to target cells by binding to antigen-binding molecules cleaved by the linker.

6. A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of an IgG antibody characterized by enhanced antibody-dependent cytotoxicity, wherein... Antigen-binding molecules contain protease-cleavable linkers, which, after being cleaved by proteases, enable them to bind to antigens expressed on the surface of target cells. IgG contains an antibody variable region that is active in binding to antigen-binding molecules after protease cleavage of the linker. IgG antibodies can bind to target cells by binding to antigen-binding molecules that have been cleaved by the linker.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein, antigen-binding molecules after cleavage of the linker bind to the antigen's K D Value, relative to the K value of the antigen-binding molecule against the antigen before cleavage of the linker. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) is 0.1 or less.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein, The antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a single-domain antibody.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein, The antigen-binding molecule comprises a variable region and a constant region of an antibody, as well as a protease-cleavable linker, wherein the antibody is selected from IgG antibodies, IgG antibody-like molecules, or heavy chain antibodies, and the protease-cleaved linker antigen-binding molecule comprises an antigen-binding domain and a portion of the cleaved linker.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein, The protease-cleavable linker of the antigen-binding molecule is located near the boundary between the variable region and the constant region, or near the boundary between CH1 and CH2 within the constant region.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein, The antigen-binding molecule is an antibody or IgG antibody-like molecule containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is the VL, VH, VHH of the antibody or its antigen-binding fragment.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein, The antigen-binding molecule is a single-domain antibody containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is part of the antigen-binding domain and linker of the single-domain antibody.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein, Protease-cleavable adapters contain protease-cleaving sequences.

14. The pharmaceutical composition according to any one of claims 1 to 12, wherein, The protease-cleavable adapter contains a peptide having a protease-cleaving sequence of any sequence number 1 to 725.

15. The pharmaceutical composition according to any one of claims 1 to 14, for the treatment or prevention of cancer.

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