Antibody cleavage site binding molecules

Abstract

The present disclosure provides pharmaceutical compositions comprising an antibody with ADCC activity, a T cell redirecting antibody, or a cell expressing a chimeric receptor for use in combination with administration of an antigen binding molecule capable of binding a target antigen, wherein the primary molecule comprises a protease cleavable linker, the antigen binding molecule cleaved of the linker has binding capacity for the target antigen, the variable region of the antibody with ADCC activity or the T cell redirecting antibody and the extracellular binding domain of the cell expressing the chimeric receptor bind to a cell expressing the target antigen by binding to the antigen binding molecule cleaved of the linker produced upon cleavage of the cleavable linker.

Description

Technical Field

[0001] This disclosure relates to antibodies with ADCC activity, T-cell redirection antibodies, chimeric receptors, cells expressing chimeric receptors, and disease treatment methods utilizing the cells or antibodies. In particular, it relates to therapies utilizing the ADCC activity of antibodies, CAR-T therapy utilizing the cells, and T-cell redirection antibody therapy. Background Technology

[0002] Antibody drugs are medications that use immunoglobulins or their analogues, which are part of the living immune system, as their main components (Non-Patent Literature 1 and Non-Patent Literature 2). Compared to existing small molecule compounds, antibody drugs have large molecular weights and can recognize complex molecules, thus exhibiting high target specificity and fewer unexpected side effects. Furthermore, while foreign substances in the blood are generally taken up by cells and broken down via endocytosis, antibodies possess a mechanism for antibody recovery through specific receptors FcRn and the antibody's Fc region, resulting in long-lasting retention in the blood and the ability to achieve prolonged efficacy with a single administration. Moreover, since antibody drugs are prepared as recombinant proteins, their function can be altered using genetic engineering.

[0003] For example, the constant region of an antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC) by binding to FcγR on NK cells or macrophages. When an antibody with such a constant region is used, the constant region adds an alteration that enhances the binding to FcγR, which is known to induce stronger cytotoxicity (Non-Patent Literature 3).

[0004] Furthermore, conventional antibodies typically recognize and bind to only 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) can be developed (Non-Patent Literature 11). These antibodies 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, employing cytotoxicity as their anti-tumor mechanism (Non-Patent Literature 12, 13, 14). Unlike antibodies that use NK cells or macrophages as effector cells and mobilize ADCC as their anti-tumor mechanism, T-cell retargeting 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. This creates an intercellular bridge between T cells and cancer antigen-expressing cells, allowing T cells to act as effector cells and induce strong cytotoxicity (T-cell dependent cellular cytotoxicity; TDCC) on cancer antigen-expressing cells (Non-Patent Literature 4, 12, 13, 14).

[0005] Furthermore, recent developments have focused on anti-tumor therapies that leverage the high specificity of antibodies for antigen recognition on effector cells, demonstrating significant efficacy in some cancers. In this therapy, known as chimeric antigen receptor (CAR) adoptive immunotherapy, an extracellular domain with antigen-binding capabilities, primarily derived from antibody scFv, is artificially fused with an intracellular signal transduction domain to form a CAR, which is expressed on effector cells such as T cells. When these CAR-expressing T cells (CAR-T cells) are transplanted into cancer patients, the intracellular domains are activated when tumor antigens are recognized by the CAR, inducing cytotoxicity in effector cells and exerting the therapeutic effect by killing tumor cells (Non-Patent Literature 5).

[0006] Clinical trials of cancer immunotherapy using CAR-T cells (Non-Patent Literature 10) have demonstrated its effectiveness in treating hematopoietic malignancies such as leukemia or lymphoma. In 2017, the CD19-based CAR-T inhibitors Kymriah (registered trademark) (Novartis, tisagenlecleucel, CTL-019, CD3ζ-CD137) and Yescarta (registered trademark) (KiTE, axicabtageneciloleucel, CD3ζ-CD28) were approved as drugs in the United States, and in 2019 in Japan.

[0007] In addition, methods have been proposed that utilize single-domain antibodies derived from camelids, targeting diverse antigens, and simplifying antibody preparation. In summary, antibody drugs offer numerous advantages and are therefore suitable for a wide range of diseases, including tumors, autoimmune diseases, and infectious diseases (Non-Patent Literature 6).

[0008] On the other hand, the limitations of antibody drugs are also pointed out. One of these is the issue of the lesion site specificity of the antigen. Surface antigens that become antibody targets can also be expressed in normal tissues outside the lesion site. Although the expression level of the antigen is lower than at the lesion site, the antibody effect can have side effects on the normal tissue expressing the antigen.

[0009] One possible solution to this problem is to identify lesion sites that target 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 can be shown that lesion sites can be identified via protease activity (Non-Patent Literature 7).

[0010] A study was reported on detecting lesions by recognizing protease cleavage products generated at the lesion site using antibodies (Non-Patent Document 8). This study reported that the cleavage products of the IdeS protease expressed by actinomycetes could be specifically recognized in vivo using antibodies that specifically bind to the cleavage products (Non-Patent Document 8).

[0011] In the field of chronic diseases, there are also studies on developing reagents for detecting lesions of osteoarthritis of the knee, and developing antibodies that recognize type II collagen cleaved by activated MMP (matrix metalloproteinase) in the lesion site of osteoarthritis of the knee (non-patent literature 9).

[0012] As an example of applying existing protease-activated antibody technology to treatment, one can cite "Probody (registered trademark) technology" as an example. This technology expands tissue specificity and the therapeutic window by conferring sensitivity to proteases expressed or activated to an increased degree at lesion sites such as cancerous or inflamed tissues on antibodies. Figure 1 ).

[0013] "Probody (registered trademark)" is a molecule formed by linking the masking peptide of the antigen-binding site of an antibody and a cleaving peptide sequence of a protease expressed at the lesion site (Non-Patent Document 15). The antigen-binding site of an antibody is masked by the masking peptide in its uncleaved state, thus preventing it from binding to the antigen. The cleaving peptide sequence of Probody (registered trademark) is cleaved by a protease expressed at the target lesion site, thereby dissociating the masking peptide and generating an antibody molecule with antigen-binding activity, making it possible to bind to the specific antigen 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.

[0014] Existing technical documents

[0015] Patent documents

[0016] Patent Document 1: WO2009 / 025846

[0017] Patent Document 2: WO2017 / 143094

[0018] Patent Document 3: WO2018 / 097307

[0019] Non-patent literature

[0020] Non-patent literature 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

[0021] Non-patent literature 2: Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. (2005) 59(3), 389-396.

[0022] Non-patent document 3: The impact of Fc engineering on an anti-CD19 antibody: increased Fcgamma receptor affinity enhances B-cell clearing in nonhumanprimates. Zalevsky J, Leung IW, Karki S, Chu SY, Zhukovsky EA, Desjarlais JR, Carmichael DF, Lawrence CE. Blood.2009Apr 16;113(16):3735-43.

[0023] Non-Patent Literature 4: Advances in bispecific biotherapeutics for the treatment of cancer. Biochem Pharmacol. 2012 Nov 1; 84(9): 1105-12

[0024] Non-patent literature 5: Chimeric Antigen Receptor Therapy N Engl J Med 2018;379:64-73

[0025] Non-Patent Literature 6: Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol. 2016; 38(1): 21-8

[0026] Non-Patent Literature 7: Shedding light onto live molecular targets Nat Med. 2003 Jan; 9(1): 123-8

[0027] Non-patent document 8: Structure and specificity of an antibody targeting aproteolytically cleaved IgG hinge Malia TJ1, Teplyakov A, Brezski RJ, Luo J, Kinder M, Sweet RW, Almagro JC, Jordan RE, Gilliland GL.Proteins.2014Aug;82(8):1656-67

[0028] Non-patent document 9: Development of a novel immunoassay for the measurement of type II collagen neoepitope generated by collagenase cleavage Clin ChimActa. 2012 Oct 9; 413 (19-20): 1591-9.

[0029] Non-patent literature 10: Grupp et al., 2013 N Engl J Med 368(16): 1509-18.

[0030] Non-patent literature 11: Kontermann, mAbs 2012; 4: 182-197.

[0031] Non-patent literature 12: Mezzanzanica et al., International journal of cancer 1988;41:609-615.

[0032] Non-patent literature 13: Staerz and Bevan, Proceedings of the National Academy of Sciences of the United States of America 1986; 83: 1453-1457.

[0033] Non-patent literature 14: Staerz et al., Nature 1985; 314: 628-631.

[0034] Non-patent literature 15: Desnoyers LR et al., Sci Transl Med. 2013 Oct 16; 5(207): 207ra144. Invention Overview

[0036] The problem that the invention aims to solve

[0037] Probody (registered trademark) exhibits cytotoxicity in normal tissues due to its high blood retention rate when activated and its antigen-binding activity even in its inactivated state without protease cleavage. Furthermore, while high cytotoxicity is known in T-cell redirection antibody therapy and CAR-T therapy, its activity can also occur in normal cells, causing serious side effects, highlighting the need to improve the safety profile 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 target tumor antigen, which presents significant challenges in terms of economic cost and labor. Moreover, target tumor antigens may respond to treatment with reduced expression or sudden mutations, leading to immune escape and reduced or absent treatment efficacy.

[0039] We investigated potential CAR-T cells and T-cell redirection antibodies, as well as therapeutic approaches, in response to tumor antigens that change recognition during treatment. We look forward to developing safer and cheaper treatments through the development of therapeutic methods that are effective and safe for patients and widely available technologies.

[0040] Methods for solving problems

[0041] To address these challenges, the inventors conducted repeated research and discovered that antibodies, T-cell redirection antibodies, or CAR-T cells with ADCC activity that bind to the newly generated antigen-binding domain after protease cleavage exhibit selective action on therapeutic target cells and are effective in treatment, thus completing this invention. In one aspect of this disclosure, a general-purpose group of therapeutic molecules is disclosed, which comprises 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, pharmaceutical compositions comprising molecules with antigen-binding capacity and pharmaceutical compositions comprising molecules with effector-activating capacity, wherein the pharmaceutical compositions are used to activate effector cells by cleavage by a target tissue-specific protease, thereby bridging target cells expressing antigens and effector cells, for treating diseases originating from target tissues, and the antigen-binding and effector-activating molecules used as active ingredients of the pharmaceutical compositions. Further, a method for manufacturing the pharmaceutical compositions and the antigen-binding and effector-activating molecules used as the active ingredients is provided.

[0043] Furthermore, according to this disclosure, by using a common molecule that 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 therapies depending on the treatment status.

[0044] Furthermore, according to this disclosure, by using multiple common molecules that bind 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 therapies depending on the treatment status.

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

[0046] Another aspect of this disclosure relates to isolated nucleic acid molecules, which are encoding nucleic acid molecules of antibodies, bispecific antibodies, and / or CARs that can be used to target cells expressing target antigens.

[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 molecules or vectors of this disclosure.

[0049] More specifically, one aspect of this disclosure provides the following invention.

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

[0051] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0052] Chimeric receptors contain an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain has the ability to bind antigen-binding molecules after cleavage by the adaptor. By binding to antigen-binding molecules after cleavage by the adaptor, it can bind to cells expressing the above-mentioned target antigens.

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

[0054] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0055] Chimeric receptors contain an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain has the ability to bind antigen-binding molecules after cleavage by the adaptor. By binding to antigen-binding molecules after cleavage by the adaptor, it can bind to cells expressing the above-mentioned target antigens.

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

[0057] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0058] 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.

[0059] Bispecific antibodies can bind to cells expressing target antigens by binding to antigen-binding molecules cleaved by the linker.

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

[0061] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0062] 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.

[0063] Bispecific antibodies can 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 an antigen-binding molecule, wherein...

[0065] The aforementioned antigen-binding molecules contain protease-cleavable linkers, and after being cleaved by proteases, they exhibit binding activity to antigens expressed on the surface of target cells.

[0066] The aforementioned IgG antibody contains an antibody variable region that is active in binding to antigen-binding molecules after the linker has been cleaved by proteases.

[0067] The aforementioned IgG antibodies can 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] 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.

[0070] IgG contains an antibody variable region that is active in binding to antigen-binding molecules after protease cleavage of the linker.

[0071] IgG antibodies can bind to target cells by binding to antigen-binding molecules that have been cleaved by the linker.

[0072] [7] The pharmaceutical composition according to any one of [1] to [6], wherein the antigen-binding molecule after linker cleavage binds to the antigen K D Value, relative to the K value of the antigen-binding molecule against the antigen before cleavage. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) 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 antigen-binding molecule 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 in the aforementioned 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 of the antibody or an antigen-binding fragment thereof.

[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 comprises a protease-cleaving sequence.

[0079]

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

[12] , wherein the protease-cleavable linker comprises a peptide having a protease-cleaving sequence having any one of sequence numbers 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] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0083] Chimeric receptors contain an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain has the ability to bind antigen-binding molecules after cleavage by the adaptor. By binding to antigen-binding molecules after cleavage by the adaptor, it can bind to cells expressing the above-mentioned target antigens.

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

[0085] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0086] Chimeric receptors contain an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain has the ability to bind antigen-binding molecules after cleavage by the adaptor. By binding to antigen-binding molecules after cleavage by the adaptor, it can bind to cells expressing the above-mentioned target antigens.

[0087] [A1-3] The pharmaceutical composition according to [A1-1] or [A1-2], wherein the antigen-binding molecule after linker cleavage is K-type of the antigen. D Value, relative to the K value of the antigen-binding molecule against the antigen before cleavage. D The ratio of values ​​(K) D (After cutting) / KD (Before cutting) is less than 0.1 or less than 0.01.

[0088] [A1-4] The pharmaceutical composition according to any one of [A1-1] to [A1-3], wherein the antigen-binding molecule 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 linker 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 linker 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 aforementioned 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 the 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 portion of the VHH or 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 connector, a portion of the connector, or a portion containing the connector.

[0097] [A1-13] The pharmaceutical composition according to any one of [A1-1] to [A1-12], wherein the extracellular binding domain of the chimeric receptor is K-type of the antigen-binding molecule after cleavage by the linker. D Value, relative to the K value of the antigen-binding molecule before cleavage of the linker. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) 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 one of sequence numbers 1 to 725.

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

[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, germ cell tumor 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 the protease-cleaved antigen-binding molecule, and the extracellular binding domain binds to the antigen-expressing cell by binding to the antigen-binding molecule cleaved by the adaptor.

[0108] [A2-2] According to the chimeric receptor described in [A2-1], the extracellular binding domain recognizes a protease-cleaved linker, a portion of the linker, or a portion containing the linker.

[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] According to the chimeric receptor described in [A2-4], wherein the co-stimulatory molecules are 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, which encodes any one of [A2-1] to [A2-7] chimeric receptors.

[0115] [A2-9] Vector, which contains the nucleic acid described in [A2-8].

[0116] [A2-10] cells, which contain the vector described in [A2-8].

[0117] [A2-11] The cells described in [A2-9] are T cells.

[0118] [A2-12] Based on the cells described in [A2-11], wherein the aforementioned T cells are CD4+ cells. + or CD8 + T cells.

[0119] [A2-13] The cells described in [A2-11], wherein the T cells are regulatory T cells (Treg) or follicular regulatory T cells (TFR).

[0120] [A3-1] Antigen-binding molecules, which are antigen-binding molecules containing protease-cleavable linkers.

[0121] The antigen-binding molecule after cleavage by the linker has the ability to bind to the antigen. Through the binding of the extracellular binding domain of the chimeric receptor to the antigen-binding molecule after cleavage by the linker, it can bind to the target cells expressing the antigen.

[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 the sequence numbers 1 to 725.

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

[0124] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0125] 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.

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

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

[0128] Antigen-binding molecules contain proteases that can cleave linkers, which, after cleavage, enable them to bind to target antigens.

[0129] 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.

[0130] Bispecific antibodies can bind to cells expressing target antigens by binding to antigen-binding molecules cleaved by the linker.

[0131] [B1-3] The pharmaceutical composition according to [B1-1] or [B1-2], wherein the antigen-binding molecule after linker cleavage is K-type of the antigen. D Value, relative to the K value of the antigen-binding molecule against the antigen before cleavage. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) is less than 0.1 or less than 0.01.

[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 with the linker cleaved by a protease comprises the aforementioned 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 containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is the VL, VH or antigen-binding fragment 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 containing a protease-cleavable linker, and the antigen-binding molecule after linker cleavage is the 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 cleavage of the linker 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 containing the linker.

[0139] [B1-11] A pharmaceutical composition according to any one of [B1-1] to [B1-10], wherein the bispecific antibody targets the K-type antigen-binding molecule after linker cleavage. D Value, relative to the K value of the bispecific antibody against the antigen-binding molecule before cleavage of the linker. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) 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 adapter further comprises a flexible adapter.

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

[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] is used for 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] is used in bispecific antibody therapy.

[0149] [B2-1] Bispecific antibody comprising 1) a first antibody variable region having binding activity against molecules expressed on the surface of T cells; and 2) a second antibody variable region having binding activity against antigen-binding molecules containing protease-cleaved linkers after the linkers have been cleaved by proteases.

[0150] After the antigen-binding molecule is cleaved by the protease, it becomes active in binding to the antigen expressed on the surface of the target cell. Bispecific antibodies can bind to the target cell by binding to the antigen-binding molecule after it has been cleaved by the protease.

[0151] [B2-2] The bispecific antibody described in [B2-1] contains CD3 as the molecule expressed on the surface of T cells.

[0152] [B2-3] The bispecific antibody according to [B2-1], wherein the antibody variable region, which has binding activity to molecules expressed on the surface of T cells, 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 Fcγ receptor.

[0155] [B2-6] A bispecific antibody according to any one of [B1-1] to [B1-5], wherein the protease-cleavable linker 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 a protease-cleaving sequence having any one of sequence numbers 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, containing nucleic acid as described in [B3-1].

[0161] [B3-3] cells, comprising the vector as described in [B3-2].

[0162] [B3-4] A method for manufacturing bispecific antibodies includes culturing cells as described in [B3-3] and recovering bispecific antibodies from the culture supernatant.

[0163] [C1-1] A pharmaceutical composition comprising an IgG antibody characterized by enhanced antibody-dependent cellular cytotoxicity (ADCC) for use in combination with an antigen-binding molecule.

[0164] The aforementioned antigen-binding molecules contain protease-cleavable linkers, and after being cleaved by proteases, they exhibit binding activity to antigens expressed on the surface of target cells.

[0165] The aforementioned IgG antibody contains an antibody variable region that is active in binding to antigen-binding molecules after the linker has been cleaved by proteases.

[0166] The aforementioned IgG antibodies can bind to target cells by binding to antigen-binding molecules cleaved by the linker.

[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 cytotoxicity (ADCC).

[0168] 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.

[0169] IgG contains an antibody variable region that is active in binding to antigen-binding molecules after protease cleavage of the linker.

[0170] IgG antibodies can bind to target cells by binding to antigen-binding molecules that have been cleaved by the linker.

[0171] [C1-3] The pharmaceutical composition according to [C1-1] or [C1-2], wherein the antigen-binding molecule after linker cleavage is K-type of the antigen. D Value, relative to the K value of the antigen-binding molecule against the antigen before cleavage. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) 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, and the antigen-binding molecule with the linker cleaved by a protease comprises the aforementioned 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 the VL, VH or 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 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 cleavage of the linker 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 connector, a portion of the connector, or a portion containing the connector.

[0179] [C1-11] The pharmaceutical composition according to any one of [C1-1] to [C1-10], wherein the IgG antibody targets the K-type antigen-binding molecule after linker cleavage. D Value, relative to the K value of IgG antibody against antigen-binding molecules before linker cleavage. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) 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 linker 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 linker comprises a peptide having a protease-cleaving sequence having any one of sequence numbers 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 the treatment or prevention of antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytic capacity (ADCP).

[0186] [C1-17] The pharmaceutical composition according to any one of [C1-1] to [C1-16] is used for the 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] The pharmaceutical composition according to [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]IgG antibody is an IgG antibody containing a variable region that is active in binding to antigen-binding molecules containing protease-cleavable linkers, after the linkers have been cleaved by proteases.

[0191] Antigen-binding molecules, after being cleaved by proteases, have binding activity to antigens expressed on the surface of target cells. IgG antibodies can bind to target cells by binding to antigen-binding molecules cleaved by the linker.

[0192] [C2-2] The antibody-dependent cytotoxicity (ADCC) of the IgG antibody described in [C2-1] is enhanced.

[0193] [C2-3] The IgG antibody according to [C2-1] or [C2-2] contains an Fc region with increased binding activity to the Fcγ 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 one of sequence numbers 1 to 725.

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

[0197] [C3-1] nucleic acid, which encodes any one of [C2-1] to [C2-6] IgG antibody.

[0198] [C3-2] Vector, containing nucleic acid as described in [C3-1].

[0199] [C3-3] cells, comprising the vector as described in [C3-2].

[0200] [C3-4] A method for manufacturing IgG antibodies includes culturing cells as described in [C3-3] and recovering IgG antibodies 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] The primary molecule contains a protease-cleavable linker, which, after cleavage, enables binding to the target antigen.

[0203] The secondary molecule has the ability to bind to the primary molecule after the adapter is cleaved, and through binding to the primary molecule after the adapter is cleaved, it can bind to cells expressing the above-mentioned target antigen.

[0204] [D1-2] Pharmaceutical composition, which is 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, which, after cleavage, enables binding to the target antigen.

[0206] The secondary molecule has the ability to bind to the primary molecule after the adapter is cleaved, and through binding to the primary molecule after the adapter is cleaved, it can bind to cells expressing the above-mentioned target antigen.

[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], wherein it is any one of [A1-1] to [A1-21], [B1-1] to [B1-20], and [C1-1] to [C1-20]. Brief description of the attached diagram

[0210] [ Figure 1 ] Figure 1 Probody (a registered trademark) is a concept that demonstrates how antibody technology can expand tissue specificity and therapeutic window by conferring sensitivity to proteases that are expressed at elevated levels in lesions such as cancerous or inflamed tissues.

[0211] [ Figure 2 ] Figure 2This is a schematic diagram illustrating the TDCC activity induced by antibodies that specifically recognize antigens and are generated by protease cleavage of the linker.

[0212] [ Figure 3 ] Figure 3 This is a schematic diagram illustrating the ADCC activity induced by antibodies that specifically recognize antigens and are generated by protease cleavage of the linker.

[0213] [ Figure 4 ] Figure 4 This is a schematic diagram illustrating the TDCC activity induced by antibodies that specifically recognize antigens and are generated by protease cleavage of the linker.

[0214] [ Figure 5 ] Figure 5 This is a schematic diagram illustrating the ADCC activity induced by antibodies that specifically recognize antigens and are generated by protease cleavage of the linker.

[0215] [ Figure 6-1 ] Figure 6-1 This is a schematic diagram illustrating CAR-T cell cytotoxicity induced by specific recognition of antigen-binding molecules cleaved by proteases.

[0216] [ Figure 6-2 ] Figure 6-2 This is a schematic diagram illustrating CAR-T-induced cytotoxicity through specific recognition of antigens exposed by protease cleavage.

[0217] [ Figure 7 ] Figure 7 This display shows the results of in vitro cleavage of antibodies containing an inserted protease cleavage sequence (a partial sequence of type II collagen) by the protease (MMP13). Starting from the left lane, corresponding to wells 1-5, well 1 shows the MWM, wells 2 and 3 show the pre-reaction and post-reaction results for antigen-binding molecules without the cleavage sequence, respectively, and wells 4 and 5 show the pre-reaction and post-reaction results for antigen-binding molecules containing the cleavage sequence with the addition of MMP13, respectively.

[0218] [ Figure 8 ] Figure 8 This displays the results of in vitro cleavage of antibodies recognizing tumor antigens by the protease IdeS. Starting from the left lane, corresponding to wells 6-10, well 6 shows the MWM (Multiple Microscopic Membrane), wells 7 and 8 show the pre-reaction and post-reaction results for antigen-binding molecules without the cleavage sequence, respectively, and wells 9 and 10 show the pre-reaction and post-reaction results for antigen-binding molecules containing the cleavage sequence with the addition of IdeS, respectively.

[0219] [ Figure 9 ] Figure 9(Left) Shows the results of treatment with tumor cell lines with antigen-binding molecules (antibodies) containing an inserted protease cleavage sequence (partial sequence of type II collagen). 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.

[0220] [ Figure 10 ] Figure 10 Biacore assay results show the binding of antigen-binding molecules that have been cleaved by proteases (partial sequences of type II collagen) to anti-cleavage linker anti-CD3 bispecific antibodies.

[0221] [ Figure 11 ] Figure 11 Biacore assay results show the binding of protease-cleaved antigen-binding molecules (IgG1) to anti-cleavage linker anti-CD3 bispecific antibodies.

[0222] [ Figure 12 ] Figure 12 Biacore assay results show the binding of antigen-binding molecules whose linkers (partial sequences of type II collagen) have been cleaved by proteases to antibodies that enhance ADCC activity.

[0223] [ Figure 13 ] Figure 13 Biacore assay results show the binding of protease-cleaved antigen-binding molecules (IgG1) to antibodies that enhance ADCC activity.

[0224] [ Figure 14 ] Figure 14 Jurkat reports gene analysis results using an antigen-binding molecule (anti-GPC3 antibody, IgG1) with a linker (partial sequence of type II collagen) cleaved by protease and an anti-linker anti-CD3 bispecific antibody.

[0225] [ Figure 15 ] Figure 15 Jurkat reports gene analysis results using an antigen-binding molecule (anti-GPC3 antibody, IgG1) with a linker (partial sequence of type II collagen) cleaved by protease and an anti-linker anti-CD3 bispecific antibody.

[0226] [ Figure 16 ] Figure 16 Jurkat reports gene analysis results using antigen-binding molecules with enhanced ADCC activity against protease-cleaved linkers (partial sequences of type II collagen).

[0227] [ Figure 17 ] Figure 17Jurkat reports gene analysis results using antigen-binding molecules with enhanced ADCC activity against protease-cleaved linkers (partial sequences of type II collagen).

[0228] [ Figure 18 ] Figure 18 The results are based on cytotoxicity analysis performed via human PBMCs using an anti-protonator anti-CD3 bispecific antibody that recognizes an antigen-binding molecule that has been cleaved by a protease (a partial sequence of type II collagen) and an anti-GPC3 antibody that has an inserted protease-cleaved sequence.

[0229] [ Figure 19 ] Figure 19 A schematic diagram showing the arrangement order of the constituent elements of the carrier structure and the frame unit from the 5' end to the 3' end.

[0230] [ Figure 20 ] Figure 20 The results represent the evaluation of PC-10 cytotoxicity with a cleavable MMP cutterhead. The proportion of surviving cancer cells is calculated as the proportion of CD45-fractionated cells among viable cells. The horizontal axis represents the concentration of the added antigen-binding molecule.

[0231] [ Figure 21 ] Figure 21 Evaluation results of KYSE70 cytotoxicity for cells that can barely cleave the MMP cleavage linker. The proportion of surviving cancer cells is calculated as the proportion of CD45-fractionated cells in viable cells. The horizontal axis represents the concentration of the added antigen-binding molecule. Detailed Implementation

[0232] 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 naturally make various changes and modifications within the scope and spirit of this disclosure, and therefore it should be understood that the detailed description and specific examples illustrating the preferred embodiments of this disclosure are for illustrative purposes only.

[0233] The following describes the implementation scheme of this disclosure with reference to the accompanying drawings.

[0234] The terms “substantially,” “about,” or “approximately” indicate a reasonable amount of deviation of the modified term that does not significantly alter the final result; that is, within the permissible error range for 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 of ±20%, preferably ±10%, more preferably ±5%, and even more preferably ±1% of a value. Or, particularly, in biological systems or processes, this term indicates within a single digit of a value, preferably within twice the value. When a particular value is described within the scope of this specification and claims, unless otherwise stated, the term “about” implicitly means within the permissible error range for that particular value in this context.

[0235] When used in the English translation of this specification and the scope of the claims, the singular forms “a,” “an,” and “the” include plural indicative terms unless expressly stated otherwise. Additionally, the term “or” should be noted to generally be used within the scope of meaning “and / or” unless expressly stated otherwise.

[0236] The numerical ranges listed by endpoints in this disclosure include all numbers and fractions contained within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). Furthermore, 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, the numerical range can be understood as a restrictive list of integers contained within the range. Such cases, for example, 1 to 5 or 1 to 5, are understood as a restrictive list of 1, 2, 3, 4, and 5.

[0237] Furthermore, the definitions and embodiments described in particular paragraphs, as understood by those skilled in the art, are intended, if appropriate, to apply to other embodiments of this specification. For example, various aspects of this disclosure are defined in more detail in the following sections. These so-called aspects may also be combined with any other one or more aspects unless expressly stated otherwise. 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.

[0238] In this disclosure, an antigen-binding molecule comprising a region (“antigen-binding domain”) that binds to an antigen expressed on 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 expressed on the target cell or diseased cell (“target antigen”). 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 the antigen-binding domain and a portion of the cleaved linker. 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 with protease cleavage sequences is called a protease cleavage linker.

[0239] In one embodiment, the antigen-binding molecule (primary molecule) containing a protease-cleavable linker is an antibody, more specifically, IgG antibodies or heavy chain antibodies containing a protease-cleavable linker may be cited as examples, and more preferably, IgG1 antibodies, camel heavy chain antibodies (hcIgG) or shark heavy chain antibodies (IgNAR) may be cited as examples.

[0240] In one implementation, the antigen-binding molecules resulting from the cleavage of the linker by the protease can be exemplified by 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.

[0241] In this invention, polypeptides generally refer to peptides and proteins with a length of about four amino acids or more. Furthermore, the polypeptides in this invention are generally polypeptides formed from artificially designed sequences, but are not particularly limited; for example, they can also be polypeptides derived from biological sources. Additionally, they can be any type of natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. Further, fragments of the aforementioned polypeptides are also included in the polypeptides of this invention.

[0242] In this specification, amino acids represented 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, and 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, the amino acid so-called amino acid 37V contained in a single-domain antibody represents Val at position 37, indicated by Kabat numbering.

[0243] To alter the amino acid sequence of peptides such as antibodies, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR can be appropriately employed. Additionally, several known methods can be used to change 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. Examples of substitutions can be cited in this specification, but are not limited to these.

[0244] In this specification, the terms “and / or” used to indicate changes in the position of amino acids include 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.

[0245] In this specification, as a way to indicate amino acid changes, it is appropriate to use a description that lists the amino acid before and after the change using single-letter or three-letter codes, both before and after the number indicating a specific position. For example, when adding an amino acid substitution in the variable region or single-domain antibody, the change referred to as F37V or Phe37Val indicates that the 37th position of Phe, as shown in the Kabat number, is replaced with Val. That is, the number indicates the amino acid position indicated by 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, when adding an amino acid substitution in the Fc region included in the constant region of the antibody, the change referred to as P238A or Pro238Ala indicates that the 238th position of Pro, as shown in the EU number, is replaced with Ala. That is, the number indicates the amino acid position indicated by 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.

[0246] 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.

[0247] "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 antibody, single-chain antibody molecules (such as scFv), and multispecific antibodies formed from antibody fragments.

[0248] The terms “full-length antibody,” “complete antibody,” and “all antibody” are used interchangeably in this specification and refer to antibodies that have a structure substantially similar to that of natural antibodies, or antibodies that have a heavy chain containing an Fc region as defined in this specification.

[0249] The term "variable region" or "variable domain" refers to a domain of the heavy or light chain of an antibody that participates in the binding of the antibody to an antigen. The variable domains of the antibody's heavy and light chains (VH and VL, respectively) typically have similar structures, each containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may adequately provide antigen-binding specificity.

[0250] As used in this specification, the term "complementarity-determining region" or "CDR" refers to the regions of the antibody's variable domains that are hypervariable in the sequence and / or form structurally fixed loops ("hypervariable loops"), and / or antigen contact residues ("antigen contacts"). Typically, an antibody contains six CDRs: three in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3). The CDRs illustrated in this specification include the following:

[0251] (a) Hypervariable rings 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));

[0252] (b) CDRs 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));

[0253] (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

[0254] (d) Combinations of (a), (b), and / or (c) containing HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).

[0255] 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. in this specification.

[0256] "Framework" or "FR" refers to the variable domain residues outside the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Correspondingly, the sequences of the CDR and FR usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0257] In this specification, the terms "constant region" or "constant domain" refer to the portion of an antibody other than its 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 one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of their constant domains.

[0258] In this specification, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. This term includes the Fc region of the native sequence and variant Fc regions. In one embodiment, in the case of human IgG1, the heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues in 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.

[0259] The "class" of an antibody refers to the type of constant structural domain or constant region possessed by the antibody heavy chain. There are five main classes of antibodies: 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 constant structural domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0260] In this specification, "antigen-binding domain" is limited to the region that binds to the target antigen. However, any domain of any structure that binds to the target antigen may also be used. 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 publications 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), which scaffolds the IgG-binding domain that constitutes a 58-amino acid bundle of protein A, and the region DARPins (Designed Ankyrin Repeat: AR) exposed on the molecular surface of ankyrin repeat (AR) sequences, which have a structure of 33 amino acid residues of turn and two antiparallel helical and loop subunits. Anticalin et al. (International Publication WO2003 / 029462) and neutrophil gelatinase-associated lipocalin (NGAL) are among the lipid transport protein molecules that support a twisted barrel-shaped structure in the central direction with eight highly conserved antiparallel chains in the central direction. This is also found in the horseshoe-shaped structure of the horseshoe-shaped structure in jawless animals such as lampreys and hagfish, where a repeating leucine-rich repeat sequence (LRR) module accumulates repeatedly, forming a recessed region of parallel sheet structures. These are examples of the complexes found in these proteins, including those in the neutrophil gelatinase-associated lipocalin (NGAL) molecule (International Publication WO2002 / 020565), and four loop regions on one side of the central direction supporting a twisted barrel-shaped structure.

[0261] Suitable examples of the antigen-binding domain of the present invention include antigen-binding domains that function solely as antigen-binding molecules, and antigen-binding domains that can independently function as antigen-binding molecules after being freed from other self-linked peptides. Examples of such antigen-binding domains include single-domain antibodies, scFv, Fv, Fab, Fab', F(ab')2, etc., but are not limited thereto.

[0262] As one suitable example of the antigen-binding domain of the present invention, an antigen-binding domain with a molecular weight of less than 60 kDa can be cited. Examples of such antigen-binding domains include single-domain antibodies, scFv, Fab, Fab', but are not limited thereto. Antigen-binding domains with a molecular weight of less than 60 kDa are generally 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).

[0263] From another perspective, as a suitable example of the antigen-binding domain of the present invention, an antigen-binding domain with a half-life of less than 12 hours in blood can be cited. Examples of such antigen-binding domains include single-domain antibodies, scFv, Fab, Fab', etc., but are not limited thereto.

[0264] One suitable example of the antigen-binding domain of the present invention is a single-domain antibody (sdAb).

[0265] In this specification, the term "single-domain antibody" refers to any antibody that can exert antigen-binding activity solely through its domain, without limiting its structure. Unlike conventional antibodies, such as IgG antibodies, which exhibit antigen-binding activity by forming a variable region through VH and VL pairing, known single-domain antibodies do not pair with other domains and can exert antigen-binding activity independently through their own domain structure. Single-domain antibodies typically have a low molecular weight and exist in monomeric form.

[0266] Examples of single-domain antibodies include, but are not limited to, antigen-binding molecules that inherently lack light chains, such as VHH from camels or VNAR from sharks, or antibody fragments containing all or part of the VH domain or VL domain of the antibody. Examples of single-domain antibodies containing all or part of the VH / VL domain of the antibody include, for example, the single-domain antibodies artificially prepared from human antibody VH or human antibody VL as described in U.S. Patent No. 6,248,516 B1. In some embodiments of the present invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0267] In the case of a single-domain antibody being a VHH or a single-domain VH antibody, the CDR of a single-domain antibody typically includes the following:

[0268] (a) The supervariable 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));

[0269] (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));

[0270] (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

[0271] (d) Combinations of (a), (b), and / or (c) containing CDR amino acid residues 26-35 (CDR1), 26-35b (CDR1), 49-65 (CDR2), 93-102 (CDR3), or 94-102 (CDR3).

[0272] In the case where the single-domain antibody is a single-domain VL antibody, the CDR of the single-domain antibody typically includes the following:

[0273] (a) Hypervariable rings 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));

[0274] (b) CDRs 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));

[0275] (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

[0276] (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.

[0277] 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 camels and transgenic animals to which a gene capable of producing single-domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedary camels, and guanacos, but are not limited to these. Examples of transgenic animals to which a gene capable of producing single-domain antibodies has been introduced include the transgenic animals described in International Publication No. WO2015 / 143414 and US Patent Publication No. US2011 / 0123527A1, but are not limited to these. 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 species sequence or a similar sequence. Humanized single-domain antibodies (e.g., humanized VHH) are also an embodiment of the single-domain antibody of the present invention.

[0278] In addition, single-domain antibodies can be obtained from peptide libraries 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.

[0279] In this specification, "antigen" is limited to those containing epitopes that bind to an antigen-binding domain. Suitable examples of antigens 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 include, but are not limited to, 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 response to target cells and tissues containing target cells, macromolecules present in the stroma of tissues containing target cells, etc. The antigens shown below can be cited as examples of target antigens.

[0280] The following molecules serve as antigens: 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, β-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, BMIP-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 AA) 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, CCL2 1. 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, C D7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, C D27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, C D52, 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, CMVUL, 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 accelerator factor). 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, eotaxin1) 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, 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, GDNF, GFAP, 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, HCMVUL, 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 loop, HLA, HLA-DR, HM1.24, HMFGPEM, 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, Keratinocyte growth factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Potential TGF-1, Potential TGF-1bp1, 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, METALLOPRO TEASES, 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, Mucin (Muc1), MUC18, Mullerian inhibiting substance, Mug, Musk, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophic factor-3,-4, or-6, neurotrophic 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, PCN A, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, relaxin prokinase, 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, RSVFgp, 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-β RI (ALK-5), TGF-β RII, TGF-β RIIb, TGF-β RIII, TGF-β 1. TGF-β 2. TGF-β3, TGF-β4, TGF-β5, Thrombin, Thymus 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 Apo-2, 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 (BAFFR), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RICD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbRTNF RIII, TNFC R), TNFRSF4 (OX40ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (FasApo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF23 (DcTRAILR1 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 (LIGHTHVEM 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, TRAILR, 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 LewisY-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 antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor factor), WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, W NT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HM GB1, 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 kininogenFactors including: weightkininogen, 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 factors VIII, VIIIa, IX, IXa, X, Xa, XI, XIa, XII, XIIa, XIII, XIIIa, TFPI, antithrombin III, EPCR, 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.

[0281] In the examples of the antigens described above, 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 exist in a soluble form in biological fluids. As a non-limiting example 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).

[0282] In the examples of antigens described above, there are membrane-bound molecules expressed on the cell membrane and soluble molecules secreted from the cell into 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 that the antigen-binding domain has neutralizing activity.

[0283] There are no restrictions on the solution in which soluble molecules can exist; soluble molecules can exist in biological fluids, that is, all fluids filling blood vessels or tissues and between cells in a living body. In one non-limiting embodiment, soluble molecules that bind to the antigen-binding domain of the present invention can exist 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 (fluids in various glandular cavities resulting from active transport and secretion of cells, and fluids in other body cavities such as the digestive tract).

[0284] The term "tumor antigen" refers to an antigen expressed in cancer cells, a biomolecule with antigenicity that is recognized as being associated with malignant changes in the cell. The tumor antigens disclosed herein include tumor-specific antigens (antigens present only in tumor cells and not found in other normal cells) and tumor-associated antigens (antigens also present in other organs and tissues or in xenogeneic and allogeneic normal cells, or antigens expressed during occurrence and / or differentiation). Additionally, abnormal glycans appearing 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 invention, the target antigen is a tumor antigen.

[0285] 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 liver cancer, and EpCAM (Proc Natl Acad Sci US), which is expressed in multiple cancers, primarily lung cancer. A.(1989)86(1), 27-31 (the polynucleotide sequence is recorded as RefSeq accession number NM_002354.2 and the polypeptide sequence as RefSeq accession number NP_002345.2, respectively), EGFR, CA19-9, CA15-3, 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), liquid protein of gross cystic diseases (liquid protein of giant cystic disease). Cystic disease fluid protein (GCDFP-15), HMB-45 antigen, Melan-A protein (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 type dimer (tumor M2-PK), GD2 (ganglioside G) 2) 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, 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.

[0286] Furthermore, examples could 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)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (TnAg); T antigen (T Ag); 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); pterin A receptor 2 (EphA2); fucosylGM1 ); Sialidized 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); X open reading frame 61 (CXORF61); CD97; CD179a; undifferentiated lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); the 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); TCRγ selective reading frame protein (TARP); Wilms' tumor protein (WT1);ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p; 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 protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myeloma virus oncogene neuroblastoma-derived homologue (MYCN); Ras homologue family member C (RhoC); cytochrome P4501B1 (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 Members of the somatic-like subfamily A (LILRA2); CD300 molecule-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.

[0287] MHC antigens are the gene products of the major histocompatibility complex (MHC). Glycoproteins expressed on the cell membrane are mainly classified into 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, peptides from tumor antigens presented by these MHC antigens are also included. Complexes of MHC that present tumor antigens such as GP100, MART-1, and MAGE-1, or variants of RAS or p53, are also considered tumor antigens.

[0288] "Differentiation antigens" are a collective term for cell surface molecules that change and evolve along with the differentiation of macrophages, T cells, B cells, and other cells 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, and CD42b. CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, C D61, CD62E, CD62L, CD62P, CD64, CD69, CD70, CD71, CD73, CD95, CD99, CD102, CD106, CD117, CD122, CD126, CDw130.

[0289] The term "tumor" generally refers to any mass or part of different color that is located on or inside the body surface. Tumors can be malignant, characterized by three features: autonomous proliferation, infiltration or metastasis, and cachexia; or benign, characterized only by autonomous proliferation. The term "cancer" refers to a disease 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 are described in this disclosure, including, but 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 related cancers. The terms "tumor" and "cancer" are used synonymously in this disclosure; for example, either term includes both solid and fluid-filled tumors, such as diffuse or circulating tumors. When used in this disclosure, the terms "cancer" or "tumor" include precancerous cancers and tumors of the same malignant nature.

[0290] Examples of cancers that can be used as anticancer agents or cancer treatment methods described below 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 intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, etc., or cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue. Other examples include sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant angioendothelioma, malignant Schwann cell tumor, osteosarcoma, soft tissue sarcoma, etc., or hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, etc. blastoma, retinoblastoma, and other blastomas or germ cell tumors, lymphomas, or leukemia.

[0291] In one embodiment, the cancer-related tumor antigen 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 cancer. 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 inappropriately synthesized by cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed in normal cells. In one embodiment, cancer-associated antigens are exclusively expressed on the cell surface of cancer cells in their full length or fragment (e.g., MHC / peptide), but are neither synthesized nor expressed on the cell 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 the MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the grooves of major histocompatibility complex (MHC) class I molecules via CD8. +T-cell receptor (TCR) recognition on T lymphocytes. MHC class I complex is constitutively expressed in all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent specific types of cell surface targets for immunotherapy. In the case of human leukocyte antigen (HLA)-A1 or HLA-A2, TCR-like antibodies targeting peptides derived from viral 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., Cancer GeneTher 2012 19(2): 84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0292] An epitope, referring to an antigenic determinant present in an antigen, is 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. Alternatively, an epitope can be defined based on the binding activity of the antigen-binding domain that recognizes it. In the case where the antigen is a peptide or polypeptide, the epitope can also be determined based on the amino acid residues constituting it. Furthermore, in the case where the epitope is a glycan, it can be determined based on its specific glycan structure.

[0293] A linear epitope is an epitope that contains an amino acid primary sequence that is identified. Linear epitopes typically contain at least 3, most commonly at least 5, amino acids, such as about 8 to 10 or 6 to 20, within an intrinsic sequence.

[0294] Unlike linear epitopes, stereoepitopes are not epitopes whose primary amino acid sequence is the single defining component of the epitope being recognized (e.g., the primary amino acid sequence is not necessarily the epitope recognized by an antibody targeting a specific epitope). Stereoepitopes, compared to linear epitopes, can contain an increased number of amino acids. Regarding the recognition of stereoepitopes, 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 stereoepitope are arranged side-by-side, allowing the antibody to recognize the epitope. Methods for determining the stereostructure of epitopes include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin tagging and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0295] The structure of the antigen-binding domain that binds to the epitope is called the complementary site. The epitope and complementary site bind stably through hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. The binding force between the epitope and complementary site is called affinity. The sum of the binding forces when multiple antigens bind to multiple antigen-binding domains is called affinity. When antibodies containing multiple antigen-binding domains (i.e., multivalent antibodies) bind to multiple epitopes, affinity is higher than affinity due to affinity addition.

[0296] In certain embodiments, the antigen-binding domains provided in this specification have ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 nM). -8 Below M, for example, 10 -8 M~10 -13 M, for example, 10 - 9 M~10 -13 The dissociation constant (Kd) of M).

[0297] The methods for confirming the binding of an antigen-binding domain to an epitope, as described below, can be appropriately implemented according to the following examples.

[0298] For example, the antigen-binding domain of an antigen can recognize a linear epitope present in an antigen molecule, as can be confirmed as follows. For this purpose, a linear peptide formed from the amino acid sequence constituting the extracellular domain of the antigen is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained via genetic engineering methods using a region in the cDNA of the antigen encoding the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide formed from 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 cells expressing the antigen. Through these experiments, the binding activity of the antigen-binding domain to the linear peptide can be determined.

[0299] 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. Examples include situations where the antigen-binding domain of a certain antigen binds strongly to the cells expressing that antigen upon contact, but substantially does not bind to linear peptides formed from fixed amino acid sequences constituting the extracellular domain of the antigen, or linear peptides formed from amino acid sequences constituting the extracellular domain of the antigen denatured using conventional denaturing agents such as guanidine. Here, "substantially does not bind" means a binding activity of 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less, of the binding activity against human antigen-expressing cells.

[0300] Additionally, as a method to confirm the antigen-binding activity of the antigen-binding domain, one approach is to determine the Kd value using a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the target antigen-binding domain and its antigen. For example, the binding affinity of the antigen-binding domain to 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)).

[0301] According to another embodiment, Kd is determined using the surface plasmon resonance method of BIACORE (registered trademark). For example, the assay using BIACORE-2000 or BIACORE-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RU). In one embodiment, a carboxymethylated 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 injection at a flow rate of 5 μl / min to achieve protein binding of approximately 10 response units (RU). Following antigen injection, 1M ethanolamine was 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) surfactant were injected at a flow rate of approximately 25 μl / min at 25 °C. Binding rate (kon) and dissociation rate (koff) were calculated by simultaneously fitting sensor maps of binding and dissociation using a simple one-to-one Langmuir binding model (BIACORE evaluation software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio of koff / kon. Furthermore, the apparent dissociation constant (Kd) could also be determined using equilibrium analysis. These methods are described in the BIACORE manual. For example, refer to Chen et al., J.Mol.Biol.293:865-881 (1999) or Methods Enzymol.2000;323:325-40. Furthermore, in the surface plasmon resonance assay, the fixed protein mass or the mass of protein used for the reaction, the temperature, and the solution composition can be changed by those skilled in the art. According to the above surface plasmon resonance assay, the ion velocity exceeds 10... 6 M -1 s -1In such cases, the ion velocity can be determined using a fluorescence extinction technique, measured in a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or a ThermoSpectronic SLM-AMINCO (registered trademark) series 8000 spectrophotometer using a stirred cuvette), by measuring the increase or decrease of the fluorescence intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) of the 20 nM antigen-binding domain in PBS pH 7.2 at 25 °C in the presence of gradually increasing concentrations of antigen.

[0302] Furthermore, the antigen-binding activity of the antigen-binding domain can also be determined using known intermolecular interaction assays such as electrochemiluminescence.

[0303] 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 principles of ELISA or FACS (fluorescence activated cell sorting) with cells expressing the antigen as the antigen.

[0304] In ELISA, the binding activity of an antigen-binding domain against a specific antigen on cells expressing that antigen is quantitatively assessed by comparing the signal levels generated by the enzyme reaction. Specifically, the antigen-binding domain is added to an ELISA plate containing cells immobilized with the antigen, and an enzyme-labeled antibody that recognizes the domain is used to detect the binding of the domain to the cells. Alternatively, in FACS, a series of dilutions of the antigen-binding domain are prepared, and the binding titer of the antibody against the antigen-expressing cells is determined to compare the binding activity of the antigen-binding domain on those cells.

[0305] The binding of the antigen-binding domain to an antigen expressed on the surface of a cell suspended in a buffer solution or similar medium can be detected by flow cytometry. Devices known for flow cytometry include, for example, the following.

[0306] FACSCanto™ II

[0307] FACSAria™

[0308] FACSArray™

[0309] FACSVantage™ SE

[0310] FACSCalibur™ (both are trade names of BD Biosciences)

[0311] EPICS ALTRA HyPerSort

[0312] Cytomics FC 500

[0313] EPICS XL-MCL ADC EPICS XL ADC

[0314] Cell Lab Quanta / Cell Lab Quanta SC (both are product names of Beckman Coulter).

[0315] 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 exemplified. First, staining with a FITC-labeled secondary antibody that recognizes and reacts with cells expressing a certain antigen is performed. 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. Next, fluorescence intensity and cell number are measured using FACSCalibur (BD). The amount of antigen-binding domain binding to the cell is reflected by 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 binding activity of the antigen-binding domain, represented by the amount of binding of the antigen-binding domain, can be determined.

[0316] When an antigen-binding domain shares an epitope with another antigen-binding domain, this can be confirmed by the competition between the two domains for the same epitope. Competition between antigen-binding domains can be detected using methods such as cross-blocking assays. For example, competitive ELISA is a preferred method for cross-blocking assays.

[0317] Specifically, in the 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 to 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 test antigen-binding domain to the well coated with the antigen protein.

[0318] The amount of the antigen-binding domain of a test antigen, mediated by a specific antigen protein, binding to a pore, can be readily determined by pre-labeling the antigen-binding domain. For example, biotin-labeled antigen-binding domains can be determined using an avidin-peroxidase conjugate and a suitable substrate. Cross-blocking assays using enzymes such as peroxidase are specifically called competitive ELISA assays. The antigen-binding domain can be labeled with other detectable or measurable labeling substances. Specifically, radioactive or fluorescent labels are known.

[0319] 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 tested 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.

[0320] In identifying the structure of an epitope bound by an antigen-binding domain against a particular antigen, the common epitope between the test antigen-binding domain and the control antigen-binding domain can be assessed by comparing their binding activity to peptides or polypeptides with amino acid-modified peptides that constitute the epitope.

[0321] As a method for determining such binding activity, for example in the ELISA format described above, 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 quantitatively eluting the antigen-binding domain in the elution buffer after passing the test antigen-binding domain and the control antigen-binding domain through a column containing the variant peptide. Methods for adsorbing the variant peptide onto the column as a fusion peptide, such as GST, are known.

[0322] 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 using the following method. First, prepare cells expressing a certain antigen and cells expressing a variant of the antigen with an epitope introduced into them. Add the test antigen-binding domain and the control antigen-binding domain to a cell suspension suspended in a suitable buffer such as PBS. Next, add a FITC-labeled antibody that recognizes both the test antigen-binding domain and the control antigen-binding domain to the cell suspension washed with a suitable buffer. The fluorescence intensity and cell number of cells stained with the labeled antibody are determined using FACSCalibur (BD). The concentrations of the test antigen-binding domain and the control antigen-binding domain are 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 is used. The binding amount of the labeled antibody to the cells is reflected by the fluorescence intensity obtained through analysis using CELL QUEST Software (BD), i.e., the geometric mean value. That is, by obtaining this geometric mean, the binding activity of the test antigen binding domain and the control antigen binding domain, represented by the amount of labeled antibody bound, can be determined.

[0323] Furthermore, in addition to ELISA or FACS, radiolabeled antigen binding assay (RIA), BIACORE (registered trademark) surface plasmon resonance assay, electrochemiluminescence assay, etc., can also be used to confirm the competition of antigen-binding domains for epitopes that are the same as other antigen-binding domains.

[0324] The geometric mean value reflecting the binding amount of the test antigen-binding domain to the mutant antigen-expressing cells obtained from the analysis (the molecular ΔGeo-Mean value of the mutant antigen) is compared with the ΔGeo-Mean value reflecting the binding amount of the test antigen-binding domain to the antigen-expressing cells. In this case, the concentration of the test antigen-binding domain used in calculating the ΔGeo-Mean value for the mutant antigen-expressing cells and the antigen-expressing cells is preferably prepared to be the same or substantially the same concentration. A pre-identified antigen-binding domain that recognizes an epitope in an antigen is used as a control antigen-binding domain.

[0325] The ΔGeo-Mean comparison value of the test antigen-binding domain to cells expressing a variant antigen is considered "substantially non-binding to cells expressing a variant antigen" if it is less than at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value of the test antigen-binding domain to cells expressing a variant antigen. The formula for calculating the ΔGeo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BDBiosciences). By comparing the comparison values, if they are substantially equivalent, the epitopes of the test antigen-binding domain and the control antigen-binding domain can be assessed as identical.

[0326] 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 this 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 this 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, hydrophobic interactions, etc.

[0327] 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 activity of the antigen-binding domain of the antigen-binding molecule is inhibited by the inhibition domain in the portion removed by cleavage of the linker. Methods for confirming that the antigen-binding activity of the antigen-binding domain is inhibited by the inhibition domain include FACS (fluorescence activated cell sorting), ELISA (enzyme-linked immunosorbent assay), ECL (electrogenated chemiluminescence), SPR (Surface Plasmon Resonance) (Biacore), BLI (Bio-Layer Interferometry) (Octet), etc. 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 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.

[0328] 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 linker cleavage. That is, the antigen-binding activity measured using antigen-binding molecules after linker 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 the antigen-binding activity measured using antigen-binding molecules before linker cleavage. In more specific embodiments, when the antigen-binding activity of the antigen-binding molecules before linker cleavage is measured using one of the methods described above, no binding of the antigen-binding domain to the antigen is found.

[0329] In some embodiments of the present invention, since the linker of the antigen-binding molecule is cleaved by a protease, the comparison of antigen-binding activity can be performed by comparing the antigen-binding activity of the antigen-binding molecule before and after protease treatment. That is, the antigen-binding activity measured using the protease-treated antigen-binding molecule 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 the untreated antigen-binding molecule. In more specific embodiments, when the antigen-binding activity of the untreated antigen-binding molecule is measured using one of the methods selected from the above methods, no binding of the antigen-binding domain to the antigen is found.

[0330] In this invention, antigen-binding molecules before cleavage have a longer half-life in the blood compared to antigen-binding molecules after cleavage. To further extend the half-life of the antigen-binding molecules, in some embodiments of this invention, the antigen-binding molecules before cleavage are designed to have a longer half-life in the blood. Examples of embodiments for extending the half-life in the blood include, but are not limited to, having a large molecular weight, FcRn binding activity, albumin binding activity, or PEGylation of the antigen-binding molecules before cleavage.

[0331] In this invention, the comparison of half-life is preferably made by comparing 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.).

[0332] As one implementation method for extending the blood half-life of antigen-binding molecules, an example is to endow the antigen-binding molecule with FcRn binding property before the linker is cleaved. To achieve FcRn binding property, there are methods that typically incorporate an FcRn binding region into the antigen-binding molecule before the linker is cleaved. An FcRn binding region 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.

[0333] After being taken into cells via the FcRn salvage pathway, which includes an FcRn binding region, IgG molecules are returned to the plasma. For example, the relatively long plasma retention (slow disappearance) of IgG molecules is due to the known function of FcRn as a salvage receptor for IgG. IgG molecules taken into endosomes via endocytosis bind to FcRn expressed in the endosome under acidic conditions. IgG molecules that cannot bind to FcRn enter lysosomes and are broken down there, while IgG molecules bound to FcRn migrate to the cell surface, dissociate from FcRn under neutral conditions in the plasma, and return to the plasma.

[0334] The FcRn binding region is preferably a region that directly binds to FcRn. As a preferred example of an FcRn binding region, the Fc region of an antibody can be cited. However, regions that can bind to peptides with FcRn-binding ability, 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 a peptide with FcRn-binding ability.

[0335] The binding activity of the FcRn binding region of the present invention for FcRn, particularly for human FcRn, as described in the preceding section on binding activity, can be determined by methods known to those skilled in the art, with conditions suitably determined by those skilled in the art. The binding activity for human FcRn can be evaluated using methods such as KD (dissociation constant), apparent KD (apparent dissociation constant), dissociation rate kd (dissociation rate), or apparent kd (apparent dissociation). These can be determined by methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard plot, flow cytometry, etc., can be used.

[0336] The conditions for determining the binding activity of the FcRn binding region to FcRn can be appropriately 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. Alternatively, 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 those using Biacore (GE Healthcare).

[0337] The pH used for the assay conditions 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 for the assay conditions 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, any temperature between 20°C and 35°C, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, can also be used to determine the binding affinity of the FcRn binding region to FcRn. The temperature of 25°C is a non-limiting example of the present invention.

[0338] As an example of an FcRn binding region, the Fc region of an IgG antibody can be cited, but it is not limited to this. 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.

[0339] Furthermore, as long as the Fc region of a natural IgG antibody possesses FcRn binding ability, a variant Fc region with one or more amino acid substitutions can also be used. For example, a variant Fc region containing positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, and 31 of the EU number from the Fc region of an IgG antibody can be used. A variant Fc region in which at least one amino acid at positions 1, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is replaced by another amino acid.

[0340] More specifically, EU numbers selected from the Fc region of IgG antibodies can be used:

[0341] The Gly amino acid at position 237 is replaced with a Met amino acid;

[0342] The Pro at position 238 is replaced with an amino acid substitution for Ala;

[0343] The 239th Ser position is replaced with an amino acid replacement of Lys;

[0344] The Lys position at position 248 is replaced with an Ile amino acid replacement.

[0345] The 250th Thr is replaced with an amino acid substitution of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr.

[0346] The 252nd Met position is replaced with an amino acid substitution of Phe, Trp, or Tyr;

[0347] The 254th Ser position is replaced with an amino acid replacement of Thr;

[0348] The Arg at position 255 is replaced with an amino acid substitution of Glu;

[0349] The Thr at position 256 is replaced with an amino acid substitution of Asp, Glu, or Gln;

[0350] The Pro at position 257 is replaced with an amino acid substitution of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;

[0351] The Glu at position 258 is replaced with an amino acid substitution for His;

[0352] The Asp at position 265 is replaced with an amino acid substitution for Ala;

[0353] The 270th Asp is replaced with a Phe amino acid;

[0354] The 286th Asn position is replaced with an amino acid substitution of Ala or Glu;

[0355] The Thr substitution at position 289 is replaced by an amino acid substitution for His;

[0356] The 297th Asn position is replaced with an amino acid substitution for Ala;

[0357] The 298th Ser position is replaced with an amino acid substitution of Gly;

[0358] The Val substitution at position 303 is replaced with an amino acid substitution of Ala;

[0359] The Val substitution at position 305 is replaced with an amino acid substitution of Ala;

[0360] 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.

[0361] The Val substitution at position 308 is replaced by an amino acid substitution of Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;

[0362] The 309th position is replaced by an amino acid substitution of Ala, Asp, Glu, Pro, or Arg;

[0363] The Gln at position 311 is replaced with an amino acid substitution of Ala, His, or Ile;

[0364] The Asp at position 312 is replaced with an amino acid substitution of Ala or His;

[0365] The Leu position at position 314 is replaced with an amino acid substitution of Lys or Arg.

[0366] The 315th Asn position is replaced with an amino acid substitution of Ala or His;

[0367] The Lys position at position 317 is replaced with an amino acid substitution for Ala.

[0368] The 325th Asn position is replaced with an amino acid substitution of Gly;

[0369] The 332nd Ile is replaced with an amino acid substitution for Val;

[0370] The Lys substitution at position 334 is replaced by an amino acid substitution for Leu;

[0371] The 360th Lys position is replaced by an amino acid substitution for His.

[0372] The Asp at position 376 is replaced with an amino acid substitution for Ala;

[0373] The Glu at position 380 is replaced with an amino acid substitution for Ala;

[0374] The Glu at position 382 is replaced with an amino acid substitution of Ala;

[0375] The 384th position is replaced with an amino acid substitution of Ala, either Asn or Ser.

[0376] The Gly amino acid at position 385 is replaced with either Asp or His.

[0377] The Gln at position 386 is replaced with an amino acid substitution of Pro;

[0378] The 387th Pro position is replaced with an amino acid substitution of Glu;

[0379] The 389th Asn position is replaced with an amino acid substitution of Ala or Ser;

[0380] The Ser at position 424 is replaced with an amino acid substitution for Ala;

[0381] 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.

[0382] The His substitution at position 433 is replaced by an amino acid substitution for Lys;

[0383] The 434th Asn position is replaced with an amino acid substitution of Ala, Phe, His, Ser, Trp, or Tyr; and

[0384] The 436th position is replaced by an amino acid substitution of His, either Tyr or Phe.

[0385] The variant Fc region with at least one amino acid substitution.

[0386] On the other hand, EU numbers selected from the Fc region of IgG antibodies can be used:

[0387] Met of the 237th amino acid;

[0388] Ala, the amino acid at position 238;

[0389] Lys of the 239th amino acid;

[0390] Ile of amino acid at position 248;

[0391] The amino acid at position 250 is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr;

[0392] The amino acid at position 252 is Phe, Trp, or Tyr;

[0393] Thr of the 254th amino acid;

[0394] Glu, the amino acid at position 255;

[0395] The amino acid at position 256 is Asp, Glu, or Gln;

[0396] The amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;

[0397] His of the 258th amino acid;

[0398] Ala, the amino acid at position 265;

[0399] Phe, the amino acid at position 270;

[0400] The amino acid at position 286 is either Ala or Glu;

[0401] His of the 289th amino acid;

[0402] Ala, the amino acid at position 297;

[0403] Gly of the 298th amino acid;

[0404] Ala, the amino acid at position 303;

[0405] Ala, the amino acid at position 305;

[0406] 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.

[0407] The amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;

[0408] The amino acid at position 309 is Ala, Asp, Glu, Pro, or Arg;

[0409] The amino acid at position 311 is Ala, His, or Ile;

[0410] The amino acid at position 312 is either Ala or His;

[0411] The 314th amino acid is either Lys or Arg;

[0412] The amino acid at position 315 is either Ala or His;

[0413] Ala, the amino acid at position 317;

[0414] Gly of the 325th amino acid;

[0415] Val of the 332nd amino acid;

[0416] Leu, the amino acid at position 334;

[0417] His of the 360th amino acid;

[0418] Ala, the amino acid at position 376;

[0419] Ala, the amino acid at position 380;

[0420] Ala, the amino acid at position 382;

[0421] Ala, the amino acid at position 384;

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

[0423] Pro of amino acid at position 386;

[0424] Glu, the 387th amino acid;

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

[0426] Ala, the amino acid at position 424;

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

[0430] His of the 436th amino acid;

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

[0432] The antigen-binding molecule may also have FcRn binding in the antigen-binding domain. An embodiment in which the blood half-life of the antigen-binding molecule prior to cleavage is longer than the blood half-life of the antigen-binding domain is also possible. This can be achieved if the antigen-binding domain does not have FcRn binding, or if the antigen-binding domain does have FcRn binding, but if it has a weaker FcRn binding than the antigen-binding molecule prior to cleavage.

[0433] In addition, as an implementation method to prolong the half-life in the blood, there is a method to bind antigen-binding molecules to albumin before the cleavage of the linker. 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). Therefore, it has been reported that the protein bound to albumin increases in size and becomes possible to bind indirectly to FcRn, thus increasing the half-life in the blood (Antibodies 2015, 4(3), 141-156).

[0434] Furthermore, as an implementation scheme to prolong the half-life of proteins in the blood, there is a method for PEGylating antigen-binding molecules before the linker is cleaved. It is believed that by PEGylating proteins, the protein volume increases, and at the same time, the half-life of proteins in the blood is prolonged by inhibiting the degradation of proteins by proteases in the blood (J Pharm Sci. 2008 Oct; 97(10): 4167-83).

[0435] In some embodiments of the present invention, the antigen-binding molecule prior to linker cleavage includes an antibody Fc region. As one specific embodiment, the antigen-binding molecule prior to linker cleavage includes the CH2 and CH3 domains of a human IgG antibody. As another specific embodiment, the antigen-binding molecule prior to linker cleavage includes 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) residue at the C-terminus of the Fc region may or may not be present.

[0436] In some embodiments of the invention, the antigen-binding molecule prior to cleavage comprises an antibody constant region. In a preferred embodiment, the antigen-binding molecule prior to cleavage comprises an IgG antibody constant region. In a preferred embodiment, the antigen-binding molecule prior to cleavage comprises a human IgG antibody constant region.

[0437] In some other embodiments of the invention, the antigen-binding molecule prior to cleavage comprises a region having a structure substantially similar to the constant region of the antibody heavy chain and a region having a structure substantially similar to the antibody light chain, which is bound to the region by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0438] The antigen-binding molecule has a linker that is activated by a protease at a rate of approximately 0.001–1500 × 10⁻⁶. 4 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 1500×10 4 M -1 S -1 Speed-specific cutting.

[0439] In this specification, the term "protease" refers to an enzyme such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, typically 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:

[0440] (1) The protease expressed at a higher level in the target tissue than in the normal tissue.

[0441] (2) Proteases that exhibit higher activity in target tissues than in normal tissues.

[0442] (3) The protease expressed at a higher level in target cells than in normal cells.

[0443] (4) Proteases that have higher activity in target cells than in normal cells.

[0444] In a more specific implementation, cancer-specific proteases or inflammatory tissue-specific proteases may be used.

[0445] The term "target tissue" in this specification refers to tissue containing at least one target cell. In some embodiments of the invention, the target tissue is cancerous tissue. In some embodiments of the invention, the target tissue is inflamed tissue.

[0446] The term "cancer tissue" refers to tissue containing at least one cancer cell. Therefore, cancer tissue can be, for example, any cell type involved in the formation of a tumor mass containing cancer cells and blood vessels. In this specification, a tumor is referred to as a foci of tumor tissue. The term "tumor" is generally used to refer to either benign or malignant growths.

[0447] In this specification, "inflammatory tissue" may be exemplified as follows:

[0448] Joints of rheumatoid arthritis or osteoarthritis

[0449] Lungs (alveoli) in bronchial asthma or COPD

[0450] Inflammatory bowel disease, Crohn's disease, or ulcerative colitis affects the digestive organs.

[0451] Fibrotic tissue in fibrotic diseases of the liver, kidneys, and lungs

[0452] Tissues that cause rejection in organ transplantation

[0453] Blood vessels and heart (myocardium) affected by arteriosclerosis or heart failure.

[0454] Visceral fat in metabolic syndrome

[0455] Skin tissue of atopic dermatitis or other dermatitis

[0456] Spinal nerves involved in herniated discs or chronic low back pain.

[0457] 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, proteases considered to be associated with inflammation have been disclosed in 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., and Biochim Biophys Acta. 2012 Jan; 1824(1): 133-45.

[0458] In addition to proteases specifically expressed in target tissues, there are also proteases specifically activated in target tissues. For example, there are cases where proteases are expressed in an inactive form and later become activated. In many tissues, there are substances that inhibit activated proteases, and 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 specifically activated after escaping inhibition. Activated proteases can be determined using methods that identify antibodies that recognize activated proteases (PNAS 2013 Jan 2; 110(1): 93-98), or by using a method that fluorescently labels the peptide that recognizes the protease, which becomes luminescent before cleavage (quenching) but glows after cleavage (Nat Rev Drug Discov. 2010 Sep; 9(9): 690-701. doi: 10.1038 / nrd3053.).

[0459] From one perspective, the term "target tissue-specific protease" can refer to any of the following:

[0460] (i) Proteases expressed at higher levels in target tissues than in normal tissues

[0461] (ii) Proteases that exhibit higher activity in target tissues than in normal tissues.

[0462] (iii) Proteases expressed at higher levels in target cells than in normal cells.

[0463] (iv) Proteases that have higher activity in target cells than in normal cells.

[0464] Without limiting the interpretation of these, specific examples of proteases include, for instance, 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), metalloproteinases (including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13 and MMP18-23 and MMP26-28) metalloproteinases (MMP1-28), Adisintegrin and metalloproteinase (ADAM), and metalloproteinases with Adisintegrin or platelet-reactive protein motifs (ADAM proteases with thrombospondin motif).ADAMTS), transmembrane peptidases (meprinα, meprinβ), CD10 (CALLA), prostate-specific antigen (PSA), asparagine endopeptidase (legumain), TMPRSS3, TMPRSS4, neutrophil elastase (HNE), β-secretase (BACE), fibroblast activating protein α (FAP), granzyme B, guanidino-benzoatase (GB), heparin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, ostuain2, 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, calpapsin 2, glutamate carboxypeptidase 2, AMSH-like protease, AMSH, γ-secretase, anti-fibrinolytic enzyme A (APCE), Decysin 1, N-acetylated α-linked acidic dipeptidase-like 1 (NAALADL1), furin, etc.

[0465] From another perspective, target tissue-specific proteases can refer to proteases specific to cancerous tissues or proteases specific to inflamed tissues.

[0466] Examples of proteases that are specifically expressed in cancer tissues include those disclosed in international publications such as WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846.

[0467] The higher the specificity of the expression of the cancer-specific protease in the cancerous tissue of the treatment target, the fewer side effects are obtained. Preferably, the concentration of the cancer-specific protease in cancerous tissue is at least 5 times higher than that in normal tissue; more preferably, at least 10 times higher; even more preferably, at least 100 times higher; particularly preferably, at least 500 times higher; and most preferably, at least 1000 times higher. Furthermore, the activity of the cancer-specific protease in cancerous tissue is preferably at least 2 times higher than that in normal tissue; more preferably, at least 3 times, 4 times, 5 times, or 10 times higher; even more preferably, at least 100 times higher; particularly preferably, at least 500 times higher; and most preferably, at least 1000 times higher.

[0468] Furthermore, cancer tissue-specific proteases can bind to the cell membrane of cancer cells or be secreted extracellularly without binding to the cell membrane. In cases where cancer tissue-specific proteases do not bind to the cell membrane of cancer cells, to ensure that the cytotoxicity induced by immune cells is specific to cancer cells, the cancer tissue-specific proteases are preferably located inside or near the cancer tissue. In this specification, "near cancer tissue" refers to the area within which the cancer tissue-specific protease's cleavage sequence is cleaved and its antigen-binding domain exerts antigen-binding activity. However, it is preferable to be located within a range that minimizes harm to normal cells.

[0469] From another perspective, cancer tissue-specific proteases are any of the following:

[0470] (i) Proteases expressed at higher levels in cancerous tissues than in normal tissues.

[0471] (ii) Proteases that exhibit higher activity in cancerous tissues than in normal tissues.

[0472] (iii) Proteases expressed at higher levels in cancer cells than in normal cells.

[0473] (iv) Proteases that have higher activity in cancer cells than in normal cells.

[0474] Cancer tissue-specific proteases can be a single protease or a combination of two or more proteases. The number of cancer tissue-specific proteases can be appropriately determined by those skilled in the art, taking into account the type of cancer being treated.

[0475] From the above perspective, among the proteases exemplified above, the preferred proteases as cancer tissue-specific proteases are serine proteases and metalloproteinases, more preferably mastriptase (including MT-SP1), urokinase (uPA), and metalloproteinases, and even more preferably MT-SP1, uPA, MMP2, and MMP9.

[0476] The higher the specificity of the expression of the inflammatory tissue-specific protease in the inflamed tissue of the treatment subject, the fewer side effects are obtained. Preferably, the concentration of the inflammatory tissue-specific protease in the inflamed tissue 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, most preferably more than 500 times higher, and most preferably more than 1000 times higher. Furthermore, the activity of the inflammatory tissue-specific protease in the inflamed tissue is preferably more than 2 times higher than that in normal tissue, more preferably more than 3 times, 4 times, 5 times, or 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.

[0477] In addition, tissue-specific proteases can bind to the cell membrane of inflammatory cells or be secreted extracellularly without binding to the cell membrane. When tissue-specific proteases do not bind to the cell membrane of inflammatory cells, they are preferably located inside or near the inflamed tissue to ensure that the cytotoxicity induced by immune cells is specific to the inflammatory cells. In this specification, "near the inflamed tissue" refers to the area within which the inflammatory tissue-specific protease's cleavage sequence is cleaved and its antigen-binding domain exerts its antigen-binding activity. However, it is preferable to be located within a range that minimizes harm to normal cells.

[0478] From another perspective, the inflammatory tissue-specific protease is any of the following:

[0479] (i) Proteases expressed at higher levels in inflamed tissues than in normal tissues.

[0480] (ii) Proteases that exhibit higher activity in inflamed tissues than in normal tissues.

[0481] (iii) Proteases expressed at higher levels in inflammatory cells than in normal cells.

[0482] (iv) Proteases that have higher activity in inflammatory cells than in normal cells.

[0483] Inflammatory tissue-specific proteases can be a single protease or a combination of two or more proteases. The types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into account the symptoms of the patient being treated.

[0484] From the above perspective, among the proteases exemplified above, metalloproteinases are preferred as proteases specific to inflamed tissues, and more preferably ADAMTS5, MMP2, MMP7, MMP9, and MMP13 among the metalloproteinases.

[0485] The protease cleavage sequence is the 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.

[0486] 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.

[0487] Specific protease cleavage sequences can be exemplified by target sequences specifically hydrolyzed by proteases expressed in cancerous tissues, inflammatory tissues, etc., as disclosed in International Publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, as illustrated above. Artificially modified sequences, such as those with appropriate amino acid alterations, can also be used. Furthermore, protease cleavage sequences identified using methods known to those skilled in the art, as described in Nature Biotechnology 19, 661-667 (2001), can also be used.

[0488] Furthermore, naturally occurring protease cleavage sequences can also be used. For example, as TGFβ changes to its latent form through protease cleavage, protease-cleaved sequences in proteins whose molecular form is altered by protease cleavage can also be used.

[0489] Examples of protease cleavage sequences 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 Nos. US2016 / 0289324 and US2016 / 0311903, PNAS(2000)97:7754-7759, and Biochemical. The sequences shown in Journal (2010) 426: 219-228 and Beilstein J Nanotechnol. (2016) 7: 364-373 are not limited to these.

[0490] The protease cleavage sequence, as described above, is preferably an amino acid sequence obtained by specific hydrolysis by a suitable target tissue-specific protease. The amino acid sequence obtained by specific hydrolysis by the target tissue-specific protease preferably includes the following amino acid sequence.

[0491] LSGRSDNH (can be cut via MT-SP1, uPA)

[0492] PLALAG (can be cut using MMP2 and MMP9)

[0493] VPLSLTMG (can be cut with MMP7)

[0494] The following sequences can also be used as protease cleavage sequences.

[0495] TSTSGRSANPRG (can be cut via MT-SP1, uPA)

[0496] ISSGLLSGRSDNH (can be cut via MT-SP1, uPA)

[0497] AVGLLAPPGGLSGRSDNH (can be cut via MT-SP1, uPA)

[0498] GAGVPMSMRGGAG (can be cut with MMP1)

[0499] GAGIPVSLRSGAG ​​(can be cut by MMP2)

[0500] GPLGIAGQ (can be cut by MMP2)

[0501] GGPLGMLSQS (can be cut by MMP2)

[0502] PLGLWA (can be cut by MMP2)

[0503] GAGRPFSMIMGAG (can be cut by MMP3)

[0504] GAGVPLSLTMGAG (can be cut with MMP7)

[0505] GAGVPLSLYSGAG (can be cut with MMP9)

[0506] AANLRN (can be cut by MMP11)

[0507] AQAYVK (can be cut using MMP11)

[0508] AANYMR (can be cut with MMP11)

[0509] AAALTR (can be cut via MMP11)

[0510] AQNLMR (can be cut via MMP11)

[0511] AANYTK (can be cut with MMP11)

[0512] GAGPQGLAGQRGIVAG (can be cut with MMP13)

[0513] PRFKIIGG (can be cleaved by prourokinase)

[0514] PRFRIIGG (can be cleaved by prourokinase)

[0515] GAGSGRSAG (can be cut via uPA)

[0516] SGRSA (can be cut via uPA)

[0517] GSGRSA (can be cut via uPA)

[0518] SGKSA (can be cut via uPA)

[0519] SGRSS (can be cut via uPA)

[0520] SGRRA (can be cut via uPA)

[0521] SGRNA (can be cleaved by uPA)

[0522] SGRKA (can be cut via uPA)

[0523] QRGRSA (can be cut via tPA)

[0524] GAGSLLKSRMVPNFNAG (can be cleaved by cathepsin B)

[0525] TQGAAA (can be cleaved by cathepsin B)

[0526] GAAAAA (can be cleaved by cathepsin B)

[0527] GAGAAG (can be cleaved by cathepsin B)

[0528] AAAAAG (can be cleaved by cathepsin B)

[0529] LCGAAI (can be cleaved by cathepsin B)

[0530] FAQALG (can be cleaved by cathepsin B)

[0531] LLQANP (can be cleaved by cathepsin B)

[0532] LAAANP (can be cleaved by cathepsin B)

[0533] LYGAQF (can be cleaved by cathepsin B)

[0534] LSQAQG (can be cleaved by cathepsin B)

[0535] ASAASG (can be cleaved by cathepsin B)

[0536] FLGASL (can be cleaved by cathepsin B)

[0537] AYGATG (can be cleaved by cathepsin B)

[0538] LAQATG (can be cleaved by cathepsin B)

[0539] GAGSGVVIATVIVITAG (can be cleaved by cathepsin L)

[0540] APMAEGGG (can be cleaved by transmembrane peptidase α and transmembrane peptidase β)

[0541] EAQGDKII (can be cleaved by transmembrane peptidase α and transmembrane peptidase β)

[0542] LAFSDAGP (can be cleaved by transmembrane peptidase α and transmembrane peptidase β)

[0543] YVADAPK (can be cleaved by transmembrane peptidase α and transmembrane peptidase β)

[0544] RRRRR (can be cleaved by furin)

[0545] RRRRRR (can be cleaved by furin)

[0546] GQSSRHRRAL (can be cleaved by furin)

[0547] SSRHRRALD (can be cleaved by TGFβ)

[0548] RKSSIIIRMRDVVL (can be cleaved by plasminogen)

[0549] SSSFDKGKYKKGDDA (can be cleaved by streptokinase)

[0550] SSSFDKGKYKRGDDA (can be cleaved by streptokinase)

[0551] IEGR (can be cleaved by coagulation factor Xa)

[0552] IDGR (can be cleaved by coagulation factor Xa)

[0553] GGSIDGR (can be cleaved by coagulation factor Xa)

[0554] GPQGIAGQ (can be cleaved by collagenase)

[0555] GPQGLLGA (can be cleaved by collagenase)

[0556] GIAGQ (can be cleaved by collagenase)

[0557] GPLGIAG (can be cleaved by collagenase)

[0558] GPEGLRVG (can be cleaved by collagenase)

[0559] YGAGLGVV (can be cleaved by collagenase)

[0560] AGLGVVER (can be cleaved by collagenase)

[0561] AGLGISST (can be cleaved by collagenase)

[0562] EPQALAMS (can be cleaved by collagenase)

[0563] QALAMSAI (can be cleaved by collagenase)

[0564] AAYHLVSQ (can be cleaved by collagenase)

[0565] MDAFLESS (can be cleaved by collagenase)

[0566] ESLPVVAV (can be cleaved by collagenase)

[0567] SAPAVESE (can be cleaved by collagenase)

[0568] DVAQFVLT (can be cleaved by collagenase)

[0569] VAQFVLTE (can be cleaved by collagenase)

[0570] AQFVLTEG (can be cleaved by collagenase)

[0571] PVQPIGPQ (can be cleaved by collagenase)

[0572] LVPRGS (can be cut by thrombin).

[0573] In some embodiments, the protease cleavage sequence is cleaved at least by a cysteine ​​protease. In some embodiments, the protease cleavage sequence is cleaved at least by a metalloproteinase. In some embodiments, the protease cleavage sequence is cleaved at least by a Matriptase. In some embodiments, the protease cleavage sequence is cleaved at least by MT-SP1. In some embodiments, the protease cleavage sequence is cleaved at least by uPA. In some embodiments, the protease cleavage sequence is cleaved at least by both Matriptase and uPA. In some embodiments, the protease cleavage sequence is cleaved at least by both MT-SP1 and uPA.

[0574] 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.

[0575] In one embodiment, the protease cleavage sequence is selected from the group consisting of GPQGIAGQ, GPQGLLGA, GIAGQ, GPLGIAG, GPEGLRVG, YGAGLGVV, AGLLGVVER, AGLGISST, EPQALAMS, QALAMSAI, AAYHLVSQ, MDAFLESS, ESLPVVAV, SAPAVESE, DVAQFVLT, VAQFVLTE, AQFVLTEG, and PVQPIGPQ, which are cleavable by collagenase.

[0576] The sequences shown in sequence numbers 1 to 725 can also be used as protease cleavage sequences.

[0577] The following sequences can also be used as protease cleavage sequences:

[0578] X1-X2-X3-X4-X5-X6-X7-X8

[0579] Where 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, 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.

[0580] The following sequences can also be used as protease cleavage sequences:

[0581] X1-X2-X3-X4-X5-X6-X7-X8

[0582] Wherein, X1 to X8 each represent one amino acid, X1 being an amino acid selected from A, E, F, G, H, K, M, N, P, Q, 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 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, R, S, T, V, W, and Y; X4 is R; and X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, 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.

[0583] The following sequences can also be used as protease cleavage sequences:

[0584] X1-X2-X3-X4-X5-X6-X7-X8

[0585] 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, F, L, M, P, Q, 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 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, ... Amino acids 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.

[0586] The following sequences can also be used as protease cleavage sequences:

[0587] X1-X2-X3-X4-X5-X6-X7-X8

[0588] Where 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, ... The amino acids are 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.

[0589] The following sequences can also be used as protease cleavage sequences:

[0590] X1-X2-X3-X4-X5-X6-X7-X8

[0591] Where 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, ... 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.

[0592] The following sequences can also be used as protease cleavage sequences:

[0593] X1-X2-X3-X4-X5-X6-X7-X8

[0594] 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 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, 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; X5 is R; X6 is R; X7 is R; X8 ... X6 is an amino acid selected from E, F, K, 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.

[0595] The following sequences can also be used as protease cleavage sequences:

[0596] X1-X2-X3-X4-X5-X6-X7-X8

[0597] Where 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 ...5 is R; X6 is R; X7 is R; X8 is R; X8 is R; X9 is R; X1 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; X1 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 R; X9 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; X1 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, The amino acids are 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, 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.

[0598] The following sequences can also be used as protease cleavage sequences:

[0599] X1-X2-X3-X4-X5-X6-X7-X8

[0600] Where 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 ...5 is R; X6 is R; X7 is R; X8 is R; X8 is R; X9 is R; X1 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; X1 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 R; X9 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; X1 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Amino acids 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.

[0601] The following sequences can also be used as protease cleavage sequences:

[0602] X1-X2-X3-X4-X5-X6-X7-X8

[0603] In this context, 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; and X8 is an amino acid selected from H, V, and Y.

[0604] The following sequences can also be used as protease cleavage sequences:

[0605] X1-X2-X3-X4-X5-X6-X7-X8

[0606] 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; and X8 is an amino acid selected from H, P, V and Y.

[0607] The following sequences can also be used as protease cleavage sequences:

[0608] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0609] Wherein, X1 to X9 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; and X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, 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.

[0610] The following sequences can also be used as protease cleavage sequences:

[0611] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0612] Wherein, X1 to X9 each represent one amino acid, X1 being an amino acid selected from A, E, F, G, H, K, M, N, P, Q, 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 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P 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.

[0613] The following sequences can also be used as protease cleavage sequences:

[0614] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0615] Wherein, X1 to X9 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, F, L, M, P, Q, 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 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, ... 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.

[0616] The following sequences can also be used as protease cleavage sequences:

[0617] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0618] Wherein, X1 to X9 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, 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, ... X6 is an amino acid selected from P, Q, R, 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, 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.

[0619] The following sequences can also be used as protease cleavage sequences:

[0620] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0621] Wherein, X1 to X9 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, G, H, I, K, L, ... Amino acids M, N, Q, R, 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.

[0622] The following sequences can also be used as protease cleavage sequences:

[0623] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0624] Wherein, X1 to X9 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, and Y. X6 is an amino acid selected from E, F, 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; and X9 is an amino acid selected from R and G.

[0625] The following sequences can also be used as protease cleavage sequences:

[0626] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0627] Where 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, ... The amino acids are 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, 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; and X9 is an amino acid selected from R and G.

[0628] The following sequences can also be used as protease cleavage sequences:

[0629] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0630] Wherein, X1 to X9 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; and 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 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.

[0631] The following sequences can also be used as protease cleavage sequences:

[0632] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0633] 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.

[0634] The following sequences can also be used as protease cleavage sequences:

[0635] X1-X2-X3-X4-X5-X6-X7-X8-X9

[0636] In this context, 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; and X9 is an amino acid selected from R and G.

[0637] In addition to using the protease cleavage sequences mentioned above, new protease cleavage sequences can also be obtained through screening. For example, by analyzing the crystal structure of known protease cleavage sequences and altering the interaction between the cleavage sequence and the enzyme's active residues or recognition residues, new protease cleavage sequences can be explored. Furthermore, by adding amino acid variations to known protease cleavage sequences and confirming their interaction with the protease, new protease cleavage sequences can be explored. As another example, using in vitro display methods such as phage display and ribosome display to display peptide libraries, or using peptide arrays immobilized on chips or beads, the protease cleavage sequences can be explored by confirming their interaction with the protease. The interaction between the protease cleavage sequence and the protease can be confirmed in vitro or in vivo.

[0638] The protease cleavage sequence disclosed herein can be cleaved by a protease at a rate of approximately 0.001 to 1500 × 10⁻⁶. 4 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 1500×10 4 M -1 S -1 Speed-specific modification (cutting).

[0639] Confirming the method of cleavage by protease

[0640] As a method for evaluating the protease substrates or protease cleavage sequences described in this specification, the method described in Mol Cell Proteomics. 2014 June; 13(6): 1585-97. doi: 10.1074 / mcp.M113.033308.Epub 4 April 2014 is an example.

[0641] The type or concentration of protease used in the evaluation, the treatment temperature or 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 carried out at 37°C for 1 hour.

[0642] Alternatively, serum (including human serum and mouse serum) can be used instead of solutions containing proteases to process the peptide array, using the fluorescence values ​​measured from the chip.

[0643] The type or concentration of serum used in the evaluation, the treatment temperature or treatment time can be appropriately selected. For example, human serum diluted to 80% concentration can be used as the treatment solution and treated at 37°C overnight.

[0644] As a method for qualitatively confirming whether a peptide contains a protease-cleaving sequence, it can be confirmed by performing SDS-PAGE (polyacrylamide gel electrophoresis) on a solution containing the peptide containing the protease-cleaving sequence and determining the molecular weight of the fragment. Alternatively, it can be confirmed by comparing the molecular weights of untreated and protease-treated peptides.

[0645] 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 flanking the protease cleavage sequence of a polypeptide remain connected even in the absence of a protease cleavage sequence and / or the absence of reduction of the cysteine-cysteine ​​disulfide bonds in the protease cleavage sequence.

[0646] Furthermore, by quantifying the amount of cleavage fragments after protease treatment separated by electrophoresis methods such as SDS-PAGE, the protease cleavage sequence can be evaluated, as well as the cleavage rate of molecules with introduced protease cleavage sequences. As a non-limiting method for evaluating the cleavage rate of molecules with introduced protease cleavage sequences, the following method can be cited. For example, when evaluating the cleavage rate of antibody variants with introduced protease cleavage sequences 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), huPA 40 nM or hMT-SP1 3 nM, antibody variant 100 μg / mL PBS is reacted at 37°C for 1 hour, followed by capillary electrophoretic immunoassay. Capillary electrophoresis immunoassay can be performed using Wes (Protein Simple), but is not limited to it. Alternatively, after separation by SDS-PAGE or other methods, detection can be performed by Western blotting, but is not limited to these methods. Anti-human λ chain HRP-labeled antibodies (abcam; ab9007) can be used to detect light chains before and after cleavage, but any antibody can be used to detect the cleaved fragment. The peak areas obtained after protease treatment are output using Wes-specific software (Compass for SW; ProteinSimple). The cleavage rate (%) of the antibody variant can be calculated using the formula ((cleaved light chain peak area) × 100 / (cleaved 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.

[0647] For example, a linker containing any of the protease cleavage sequences exemplified by sequence numbers 1-725 is useful as a protease substrate that is hydrolyzed by a protease. That is, in this invention, a linker that serves as 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 enable the antigen-binding molecule to be selectively cleaved 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 determined by conducting a comprehensive analysis of the sensitivity of each protease substrate to various proteases beforehand. Based on the obtained protease tolerance profile, a protease cleavage sequence with the necessary sensitivity and tolerance can be found.

[0648] 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 characteristics can be selected based on information comparing the tolerance of various protease substrates.

[0649] 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.

[0650] (Protein cleavage sequence)

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

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

[0653] (First flexible linker) - (Protein cleavage sequence) - (Second flexible linker)

[0654] 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 protease 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, especially Gly, Ser, Asp, Asn, Ala, and even more particularly Gly and Ser, especially Gly, etc.) to obtain the desired protease accessibility.

[0655] Flexible linkers suitable for use at both ends of a protease cleavage sequence typically improve the protease's accessibility to the cleavage sequence and increase the protease's cleavage efficiency. Suitable flexible linkers can be readily selected, ranging from 1 amino acid (Gly, etc.) to 20 amino acids, 2 to 15 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and from 3 to 12 amino acids in length. In some embodiments of the present invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0656] As an example of a flexible joint, glycine polymer (G) can be cited. n Glycine-serine polymers (e.g., containing (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 joints known in the art, but not limited thereto.

[0657] Among them, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and can easily function as neutral linkers between components.

[0658] Examples of flexible joints formed from glycine-serine polymers include, for example...

[0659] Ser

[0660] Gly·Ser(GS)

[0661] Ser·Gly (SG)

[0662] Gly·Gly·Ser (GGS)

[0663] Gly·Ser·Gly(GSG)

[0664] Ser·Gly·Gly(SGG)

[0665] Gly·Ser·Ser(GSS)

[0666] Ser·Ser·Gly(SSG)

[0667] Ser·Gly·Ser(SGS)

[0668] Gly·Gly·Gly·Ser(GGGS)

[0669] Gly·Gly·Ser·Gly(GGSG)

[0670] Gly·Ser·Gly·Gly(GSGG)

[0671] Ser·Gly·Gly·Gly(SGGG)

[0672] Gly·Ser·Ser·Gly(GSSG)

[0673] Gly·Gly·Gly·Gly·Ser(GGGGS)

[0674] Gly·Gly·Gly·Ser·Gly(GGGSG)

[0675] Gly·Gly·Ser·Gly·Gly(GGSGG)

[0676] Gly·Ser·Gly·Gly·Gly(GSGGG)

[0677] Gly·Ser·Gly·Gly·Ser(GSGGS)

[0678] Ser·Gly·Gly·Gly·Gly(SGGGG)

[0679] Gly·Ser·Ser·Gly·Gly(GSSGG)

[0680] Gly·Ser·Gly·Ser·Gly(GSGSG)

[0681] Ser·Gly·Gly·Ser·Gly(SGGSG)

[0682] Gly·Ser·Ser·Ser·Gly(GSSSG)

[0683] Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS)

[0684] Ser·Gly·Gly·Gly·Gly·Gly(SGGGGG)

[0685] Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS)

[0686] Ser·Gly·Gly·Gly·Gly·Gly·Gly(SGGGGGG)

[0687] (Gly·Gly·Gly·Gly·Ser(GGGGS)) n

[0688] (Ser·Gly·Gly·Gly·Gly(SGGGG)) n And so on, but not limited to these.

[0689] In this specification, "association" can be interpreted as, for example, the interaction of two or more polypeptide regions. Generally, associative bonds are formed between the target polypeptide regions by hydrophobic bonds, hydrogen bonds, ionic bonds, etc. As a common example of association, it is known that in antibodies representing natural antibodies, the paired structure is maintained by non-covalent bonds between the heavy chain variable region (VH) and the light chain variable region (VL).

[0690] In this specification, "interface" generally refers to the association surface during association (interaction). The amino acid residues forming the interface are usually one or more amino acid residues contained in the polypeptide region that provides the association, and preferably, they refer to amino acid residues that are close to each other and participate in the interaction during association. This interaction specifically includes non-covalent bonds such as hydrogen bonds, electrostatic interactions, and salt bridges formed between amino acid residues that are close to each other during association.

[0691] In this specification, "amino acid residues forming the interface," if detailed, refers to the amino acid residues contained in the polypeptide region constituting the interface. The polypeptide region constituting the interface, by way of example, refers to the polypeptide region that performs selective binding within or between molecules of antibodies, ligands, receptors, substrates, etc. Examples of amino acid residues forming the interface include, but are not limited to, amino acid residues that are close to each other during association. 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 association.

[0692] 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 VL association include amino acid residues that form the interface between VHH and VL. Examples of amino acid residues in VHH that are associated with VL association include, for example, amino acid residues at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125), but are not limited thereto. By promoting the association between VHH and VL, the activity of VHH is inhibited. Similarly, examples of amino acid residues in VL that are associated with VHH association include amino acid residues that form the interface between VHH and VL.

[0693] To promote association between VHH and VL, the amino acid residues in VHH that are associated with VL association can be altered. Examples of such amino acid substitutions include, but are not limited to, F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, and / or S47W. Furthermore, VHH can also be used with amino acid residues from the original 37V, 44G, 45L, and / or 47W, without altering any of the residues in the VHH.

[0694] Furthermore, as long as the goal of promoting association between VHH and VL can be achieved, the amino acids in VHH can be changed without changing them, but the amino acid residues in VL that are associated with VHH association can be changed. Furthermore, amino acid changes can also be introduced into both VHH and VL.

[0695] To alter the amino acid sequence of a polypeptide, site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR, among other known methods, can be appropriately employed. Alternatively, amino acid alteration by replacing amino acids with those other than natural amino acids can also be achieved using several known methods (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 (amber codon), one of the stop codons, contains tRNA that binds to non-natural amino acids.

[0696] In other embodiments of the invention, using VHH as the antigen-binding domain and VH or VHH as the repressive domain allows the antigen-binding domain and the repressive domain to associate. To promote the association between the antigen-binding domain VHH and the repressive domain VH or VHH, amino acid residues in the VHH of the antigen-binding domain that are associated with the association of the repressive domain VH or VHH can be identified and modified. Additionally, amino acid residues in the repressive domain VH or VHH that are associated with the association of the antigen-binding domain VHH can be identified and modified.

[0697] In addition, when using single-domain antibodies other than VHH as antigen-binding domains, association-related amino acid residues in the antigen-binding or inhibitory domains can be identified and modified.

[0698] In a particular embodiment, the protease cleavage sequence is located within the antibody constant region of the antigen-binding molecule. In this case, it is preferable that the protease cleavage sequence is located within the antibody constant region, thereby freeing the antigen-binding domain 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 amino acid position 140 (EU number), preferably on the antigen-binding domain side of amino acid position 122 (EU number). In other specific embodiments, the protease cleavage sequence is located within the antibody light chain constant region contained in the antigen-binding molecule, more specifically, on the antigen-binding domain side of amino acid position 130 (Kabat number), preferably on the antigen-binding domain side of amino acid position 113 (Kabat number).

[0699] In a specific implementation, the protease-cleavable linker is located near the boundary between the variable and constant regions, or near the boundary between CH1 and CH2 within the constant region. The boundary between the variable and constant regions refers to the area before and after the junction of VH and CH1, or before and after the junction of VL and CL, where the secondary structure of the antigen-binding domain is not significantly affected, including the elbow hinge region (positions 109 to 140). The boundary between CH1 and CH2 refers to the area before and after the junction of CH1 and CH2, where the secondary structure of the antigen-binding domain is not significantly affected, including the upper hinge region (positions 215 to 220) and the lower hinge region (positions 221 to 230).

[0700] 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 boundary between the antigen-binding domain and the antibody constant region can refer to the boundary between the antigen-binding domain and the antibody heavy chain constant region, or 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 antibody heavy chain constant region, the boundary between the antigen-binding domain and the antibody constant region 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 made of VH or where VHH is connected 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 boundary between the antigen-binding domain and the constant region of the antibody refers to the area before and after the site connecting VHH and CH2, which does not significantly affect the secondary structure of the antigen-binding domain, including the lowerhinge region, beginning at position 96 (Kabat number) of the single-domain antibody, preferably beginning at position 104 (Kabat number) of the single-domain antibody.

[0701] 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 constant region of the antibody heavy chain, preferably on the variable region side of amino acid position 122 (EU number) in the constant region of the antibody heavy chain. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid position 118 (EU number) to amino acid position 140 (EU number) in the constant region of the antibody heavy chain. In other more specific embodiments, the cleavage site / protease cleavage sequence is located on the variable region side of amino acid position 130 (Kabat number) in the constant region of the antibody light chain, preferably on the variable region side of amino acid position 113 (Kabat number) or amino acid position 112 (Kabat number) in the constant region of the antibody light chain. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid position 108 (Kabat number) to amino acid position 131 (Kabat number) in the constant region of the antibody light chain.

[0702] 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 amino acids from position 96 (Kabat number) of the antibody VL and position 130 (EU number) of the antibody light chain constant region, preferably between amino acids from position 104 (Kabat number) of the antibody VL and position 113 (EU number) of the antibody light chain constant region, or between amino acids from position 105 (Kabat number) of the antibody VL and position 112 (EU number) of the antibody light chain constant region. In the case where the antibody VL and the antibody heavy chain constant region are linked, the boundary between the antibody VL and the antibody heavy chain constant region can refer to the area between the 96th amino acid (Kabat number) of the antibody VL and the 140th amino acid (EU number) of the antibody heavy chain constant region, preferably between the 104th amino acid (Kabat number) of the antibody VL and the 122nd amino acid (EU number) of the antibody heavy chain constant region, or between the 105th amino acid (Kabat number) of the antibody VL and the 122nd amino acid (EU number) of the antibody heavy chain constant region.

[0703] In one embodiment, the cleavage site / protease cleavage sequence is introduced near the CH2 / CH3 interface of the antibody heavy chain constant region. Here, the region near the CH2 / CH3 interface is from position 335 (EU number) to position 345 (EU number).

[0704] Multiple cleavage sites / protease cleavage sequences can be provided in the ligand-binding molecule, for example, they can be located at multiple positions selected from within the antibody constant region, within antibody VH, within antibody VL, near the boundary between antibody VH and the antibody constant region, and near the boundary between antibody VL and the antibody constant region. Furthermore, anyone skilled in the art who has access to this invention can replace antibody VH and antibody VL, etc., and change the molecular morphology containing antibody VH, antibody VL, and the antibody constant region, without departing from the scope of this invention.

[0705] As used in this specification, the term "IgG antibody-like molecule" is used to define a portion of a molecule having a structure substantially similar to that of an IgG antibody, including constant domains or constant regions, and a portion of a molecule having a structure substantially similar to that of an IgG antibody, including variable domains or variable regions, and having a stereostructure substantially similar to that of an IgG antibody. However, "IgG antibody-like molecule" in this specification is not limited to those molecules that maintain a structure similar to that of an IgG antibody while exerting antigen-binding activity.

[0706] When the antigen-binding molecule is an IgG antibody-like molecule, an embodiment in which antigen-binding domains are respectively provided in the two variable regions corresponding to the IgG antibody is an embodiment that will be understood by 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 will be understood by those skilled in the art and is clearly defined without departing from the scope of the invention.

[0707] In this specification, the term "specificity" refers to the property that a molecule of a specifically binding molecule does not substantially bind to molecules other than those of its target molecules. It is also used when an antigen-binding domain is specific to an epitope contained in a particular antigen. Additionally, it is used when an antigen-binding domain is specific to a particular epitope among multiple epitopes contained in an antigen. Here, "substantially non-binding" can be confirmed by the methods described in the binding activity section. The binding activity of a specifically binding molecule to molecules other than those of its target molecules refers to a binding activity exhibiting 80% or less of the binding activity to those target molecules, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less.

[0708] As used in this specification, the term "treatment" (and its grammatical derivatives, such as "to treat," "to treat," etc.) refers to a clinical intervention intended to alter the natural course of the treated individual, which may 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.

[0709] The pharmaceutical compositions of this invention generally refer to agents used for the treatment or prevention of diseases, or for examination and diagnosis. In this invention, when the pharmaceutical composition is used in combination with other ingredients, the pharmaceutical composition may be administered simultaneously, separately, or continuously with the other ingredients. The pharmaceutical compositions of this invention may also contain other ingredients as components.

[0710] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be administered non-orally as an injectable form of a sterile solution or suspension of water or other pharmaceutically permissible liquids. For instance, 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 the unit dosage 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.

[0711] 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 and isotonic solutions containing glucose or other excipients (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable co-solvents such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbitol 80(TM), HCO-50, etc.) can be used in conjunction.

[0712] As an oily liquid, examples include sesame oil and soybean oil, and it can also be formulated with benzyl benzoate and / or benzyl alcohol as a cosolvent. Additionally, 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 typically filled into appropriate ampoules.

[0713] The pharmaceutical compositions of the present invention are preferably administered by non-oral administration. Administration methods include, for example, injectable, nasal, pulmonary, and transdermal formulations. Systemic or local administration is also possible, for example, via intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0714] 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, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, a dosage of, for example, 0.001 to 100,000 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 those skilled in the art can consider these conditions to determine an appropriate dosage and method of administration.

[0715] In this invention, polynucleotides are typically 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, when using *E. coli* as the host, the preferred cloning vector is pBluescript (manufactured by Stratagene), but various commercially available vectors can be used. In the implementation of this invention, vectors are used for the purpose of producing polypeptides (e.g., chimeric receptors, IgG antibodies, bispecific antibodies, antigen-binding molecules, etc.), and expression vectors are particularly useful. As an expression vector, any vector that expresses the polypeptide in vitro, in E. coli, in cultured cells, or in an individual organism is acceptable, without particular limitation. For example, for expression in vitro, pBEST vector (manufactured by Promega) is preferred; for expression in E. coli, pET vector (manufactured by Invitrogen) is preferred; for expression in cultured cells, pME18S-FL3 vector (GenBank accession number AB009864) is preferred; and for expression in an individual 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 performed 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).

[0716] There are no particular limitations on the host cells used; various host cells can be used depending on the purpose. Examples of cells used for expressing polypeptides include bacterial cells (such as Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, and Bacillus subtilis), fungal cells (such as yeast and Aspergillus), insect cells (such as Drosophila S2 and Spodoptera SF9), animal cells (such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293, and 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, edited by Ausubel et al., (1987) Published by John Wiley & Sons, Section 9.1-9.9), lipid transfection (GIBCO-BRL), and microinjection.

[0717] To induce the secretion of peptides expressed in host cells into the endoplasmic reticulum lumen, pericellular lumen, or extracellular environment, appropriate secretion signals can be introduced into the target peptide. These signals can be endogenous or xenogeneic for the target peptide.

[0718] In the above-described manufacturing method, the peptide is recovered. If the peptide of the present invention is secreted into a culture medium, the culture medium is recovered. If the peptide of the present invention is generated within a cell, the cell is first dissolved, and then the peptide is recovered.

[0719] For the recovery and purification of the polypeptides of the present invention 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.

[0720] Furthermore, as antigen-binding domains used in some embodiments of the present invention, examples include single-domain antibodies, which inhibit antigen-binding activity by associating with a specific VL, or with a specific VHH, or with a specific VHH. The present invention also relates to methods for screening such single-domain antibodies.

[0721] 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, novel VL / VH / VHH sequences identified from human antibody libraries can also be used. By combining these sequences to prepare proteins and measuring their binding activity using the methods described above, the VL / VH / VHHs that inhibit the binding activity of single-domain antibodies can be selected.

[0722] 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:

[0723] (a) Steps for obtaining single-domain antibodies with target antigen binding activity;

[0724] (b) The step of associating the single-domain antibody obtained in step (a) with a specific VL;

[0725] (c) The step of confirming that the binding activity of the single-domain antibody associated with the specific VL in step (b) is reduced or lost against the antigen.

[0726] 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.

[0727] In one embodiment of the present invention, a method for screening single-domain antibodies that inhibit antigen-binding activity by associating with a specific VH is provided, comprising the following steps:

[0728] (a) Steps for obtaining single-domain antibodies with target antigen binding activity;

[0729] (b) The step of associating the single-domain antibody obtained in step (a) with a specific VH;

[0730] (c) The step of confirming that the binding activity of the single-domain antibody associated with the specific VH in step (b) is reduced or lost against the antigen.

[0731] 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.

[0732] In one embodiment of the present invention, a method for screening single-domain antibodies that inhibit antigen-binding activity by associating with a specific VHH is provided, comprising the following steps:

[0733] (a) Steps for obtaining single-domain antibodies with target antigen binding activity;

[0734] (b) The step of associating the single-domain antibody obtained in step (a) with a specific VHH;

[0735] (c) The step of confirming that the binding activity of the single-domain antibody associated with the specific VHH in step (b) is reduced or lost against the antigen.

[0736] 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.

[0737] As an example of a method for associating a single-domain antibody with a specific VL / VH / VHH, one can exemplify a method in which a molecule containing both VH and VL is designed, for example, in a complete antibody, Fab, Fab', (Fab)2, or other antibody or antibody fragment, and the sequence of the single-domain antibody is used to replace the sequence of either VH or VL, thereby enabling the expression of a peptide having that sequence.

[0738] 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 single-domain antibodies that inhibit antigen-binding activity by promoting association between the single-domain antibody and 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.

[0739] 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 following steps:

[0740] (a) The step of preparing a variant single-domain antibody that replaces amino acid residues associated with the association of antibody VL in a single-domain antibody, thereby maintaining the binding activity of the single-domain antibody to the target antigen.

[0741] 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 following steps:

[0742] (b) The step of associating the variant single-domain antibody prepared in step (a) with a specific VL;

[0743] (c) The step of confirming that the antigen-binding activity of the variant single-domain antibody associated with the VL is weakened or lost.

[0744] 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.

[0745] 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 following steps:

[0746] (a) The step of preparing a variant single-domain antibody that replaces amino acid residues associated with the association of antibody VH in a single-domain antibody, thereby maintaining the binding activity of the single-domain antibody to the target antigen.

[0747] 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 following steps:

[0748] (b) The step of associating the variant single-domain antibody prepared in step (a) with a specific VH;

[0749] (c) The step of confirming that the antigen-binding activity of the variant single-domain antibody associated with the VH is reduced or lost.

[0750] 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.

[0751] 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 following steps:

[0752] (a) The step of preparing a variant single-domain antibody that replaces amino acid residues associated with the association of antibody VHH in a single-domain antibody, thereby maintaining the binding activity of the single-domain antibody to the target antigen.

[0753] 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 following steps:

[0754] (b) The step of associating the variant single-domain antibody prepared in step (a) with a specific VHH;

[0755] (c) The step of confirming that the antigen-binding activity of the variant single-domain antibody associated with the VHH is reduced or lost.

[0756] 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.

[0757] 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.

[0758] According to one 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 peptides that link the single-domain antibody to a first association support domain.

[0759] As an embodiment of the “library” in this specification, a library of single-domain antibodies that can be effectively obtained by associating with specific VL / VH / VHH antibodies to inhibit or lose antigen-binding activity may be provided.

[0760] In this specification, a "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 a library are not single sequences, but fusion peptides with different sequences from each other.

[0761] In this specification, "sequences differing from each other" in the context of multiple fusion peptides refers to the fact that the sequences of each fusion peptide in the library are different from each other. More preferably, it refers to the different sequences of the single-domain antibody portions of each fusion peptide in the library. That is, the number of sequences that are different from each other in the library reflects the number of independent clones with different sequences in the library, also known as the "library size". A typical phage display library has a size of 10-1. 6 Up to 10 12 By employing known techniques such as ribosome display, the library size can be expanded to 10. 14 However, the actual number of phage particles used in phage display screening is typically 10 to 10,000 times larger than the library size. This excess factor is also called the "library equivalent number," indicating that there can be 10 to 10,000 times more clones with the same amino acid sequence. Therefore, "sequences different from each other" in this invention means that the sequences of the various peptides in the library are different from each other, in addition to the library equivalent number, and more specifically, that there are 10 to 10,000 times more peptides with different sequences. 6 Up to 10 14 10 molecules, preferably 10 7 Up to 10 12 Each molecule.

[0762] Furthermore, the term "multiple" in the present invention, referring to a library primarily composed of multiple fusion peptides, means that, for example, the peptides, polynucleotide molecules, vectors, or viruses of the present invention are typically a collection of two or more types of the substance. For example, if two or more substances differ from each other in terms of specific properties, it indicates that two or more types of the substance exist. As an example, variant amino acids observed at specific amino acid positions in the amino acid sequence can be cited. For example, in the case of two or more peptides of the present invention that are substantially identical, preferably having the same sequence, except for differences in specific variant amino acids at very diverse amino acid positions exposed on the surface, multiple peptides of the present invention exist. In another example, if two or more polynucleotide molecules of the present invention are substantially identical, preferably having the same sequence, except for differences in the bases encoding specific variant amino acids at very diverse amino acid positions exposed on the surface, then multiple polynucleotide molecules of the present invention exist.

[0763] Screening methods for fusion peptides using binding activity as an indicator can also appropriately employ phage panning. 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, phages expressing the fusion peptide on their surface can be obtained. By contacting this phage with the desired antigen and recovering the antigen-bound phage, the DNA encoding the fusion peptide with the target binding activity can be recovered. This process can be repeated as needed to concentrate the fusion peptide with the desired binding activity.

[0764] Besides phage display, other known techniques for obtaining fusion peptides using libraries through panning include cell-free translation systems, techniques for presenting fusion peptides on cell or viral surfaces, and emulsification techniques. For example, cell-free translation systems include: ribosome display, which forms a complex of mRNA and translated protein via ribosomes by removing the stop codon; cDNA display, which uses compounds such as puromycin to covalently bind the gene sequence to the translated protein; mRNA display; and CIS display, which uses proteins that bind to nucleic acids to form a complex of the gene and translated protein. Furthermore, techniques for presenting fusion peptides on cell or viral surfaces include phage display, as well as E. coli display, Gram-positive bacteria display, yeast display, mammalian cell display, and virus display. Emulsification techniques include in vitro virus display, which uses an emulsion containing the gene and translation-related molecules. 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 Mar2; 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).

[0765] 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 the library that inhibit or lose antigen-binding activity by associating with specific VL / VH / VHH is provided.

[0766] "Antigen binding activity below a certain value" can refer to antigen binding activity that is below a certain benchmark when measured using, for example, the method exemplified in this specification. Similarly, "antigen binding activity above a certain value" can refer to antigen binding activity that is above a certain benchmark when measured using, for example, the method exemplified in this specification. Compared to fusion peptides with antigen binding activity below a certain value, fusion peptides with antigen binding activity above a certain value bind to antigens more strongly.

[0767] The following describes some implementation schemes that use 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.

[0768] Fusion peptides containing the target single domain antibody can be screened from a library containing multiple fusion peptides linked to the CH1 domain of an IgG antibody.

[0769] Some embodiments of the present invention provide a library comprising a plurality of fusion peptides linked to a single-domain antibody and the CH1 domain of an IgG antibody, 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.

[0770] In a particular embodiment, a method is provided for screening fusion peptides containing single-domain antibodies that inhibit or lose antigen-binding activity by associating with a specific VL from a library comprising multiple fusion peptides containing a single-domain antibody linked to the CH1 domain of an IgG antibody. Specifically, a method for screening single-domain antibodies is provided, comprising the following steps:

[0771] (a) The step of displaying the fusion peptide of the library in this invention in vitro;

[0772] (b) Steps for preparing specific VL and IgG antibody light chain constant region fusion associated couples;

[0773] (c) Associating the fusion peptide shown in step (a) with the association partner prepared in step (b), 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 described above.

[0774] (d) Selecting a fusion polypeptide selected in step (c) whose single-domain antibody binds to the antigen in a state of not associating with the VL mentioned above, or whose antigen-binding activity is above a certain value.

[0775] The conjugate 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 non-associative state of the single-domain antibody and VL. The location of the protease cleavage sequence in the conjugate is not limited as long as it eliminates the association between the single-domain antibody and VL during cleavage. As an example of the location of the protease cleavage sequence, it can be located near the boundary between the VL and the constant region of the IgG antibody light chain of the conjugate, 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, 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.

[0776] Alternatively, instead of using an association pair containing a protease cleavage sequence, a protease cleavage sequence can be introduced into the fusion peptide in the library. The association between the single-domain antibody and VL is then 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. For example, the protease cleavage sequence could be located near the boundary between the single-domain antibody and the CH1 domain of the IgG antibody in the fusion peptide.

[0777] Furthermore, in step (d) above, the full length of the fusion polypeptide selected in step (c) or the portion containing the single-domain antibody can 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.

[0778] In a particular embodiment, a method is provided for screening fusion peptides containing single-domain antibodies that inhibit or lose antigen-binding activity by associating with specific VH chains, from a library comprising multiple fusion peptides containing a single-domain antibody linked to the 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:

[0779] (a) The step of displaying the fusion peptide of the library in this invention in vitro;

[0780] (b) Steps for preparing specific VH and IgG antibody CH1 domain fusion conjugates;

[0781] (c) Associating the fusion peptide shown in step (a) with the association partner prepared in step (b), 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 described above.

[0782] (d) Selecting a fusion polypeptide containing a single-domain antibody in step (c) that binds to the antigen in a state that does not associate with the VH mentioned above, or a fusion polypeptide with antigen-binding activity above a certain value.

[0783] The conjugate prepared in step (b) above also contains 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 the antigen-binding activity of the single-domain antibody in the non-associative state of the single-domain antibody and VH. The location of the protease cleavage sequence in the conjugate is not limited, as long as it eliminates the association between the single-domain antibody and VH during cleavage. As an example of the location of the protease cleavage sequence, it can be located near the boundary between the VH and the CH1 domain of the IgG antibody in the conjugate, 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.

[0784] Alternatively, instead of using an association pair containing a protease cleavage sequence, a protease cleavage sequence can be introduced into the fusion peptide in the library. The association between the single-domain antibody and VH is then 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 during cleavage and maintains the antigen-binding activity of the single-domain antibody after cleavage. For example, the protease cleavage sequence could be located near the boundary between the single-domain antibody and the constant region of the IgG antibody light chain in the fusion peptide.

[0785] Furthermore, in step (d) above, the full length of the fusion polypeptide selected in step (c) or the portion containing the single-domain antibody can 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 VH.

[0786] The amino acids contained in the amino acid sequence described in this invention may also be subject to post-translational modifications (for example, the modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is a modification known to those skilled in the art). Even such post-translational modifications of amino acids are naturally included in the amino acid sequence described in this invention.

[0787] Methods for preparing antibodies with desired binding activity are well known to those skilled in the art. In this invention, an antigen-binding molecule that is expressed on the surface of a target cell (lesion 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 antibodies that bind to the tumor antigen is illustrated.

[0788] Antibodies that bind to tumor antigens can be obtained using known methods, either polyclonal or monoclonal. These antibodies are preferably prepared as monoclonal antibodies derived from mammals. Monoclonal antibodies derived from mammals include those produced via hybridomas and those produced by genetic engineering methods using host cells transformed with an expression vector containing an antibody gene.

[0789] Hybridomas that produce monoclonal antibodies can be prepared using known techniques, such as those described below. Specifically, a tumor antigen protein is used as the sensitizing antigen, and mammals are immunized according to standard immunization methods. The resulting immune cells are then fused with known parental cells using standard cell fusion methods. The monoclonal antibody-producing cells are then screened using standard screening methods, and hybridomas that produce antibodies against the tumor antigen are selected.

[0790] Specifically, the preparation of monoclonal antibodies is performed as follows. First, a tumor antigen gene is expressed to obtain a tumor antigen protein that can be used as a sensitizing antigen for antibody production. 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. 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. Alternatively, purified native GPC3 protein can also be used as a sensitizing antigen.

[0791] The purified tumor antigen protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of the tumor antigen can also be used as a sensitizing antigen. In this case, the partial peptide can also be obtained by chemical synthesis from the amino acid sequence of a human tumor antigen. Alternatively, it can be obtained by inserting a partial tumor antigen gene into an expression vector for expression. Furthermore, it can also be obtained by decomposing the tumor antigen protein using a proteolytic enzyme, 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.

[0792] Alternatively, fusion proteins formed by fusing a desired portion of a tumor antigen protein with different polypeptides can be used as sensitizing antigens. To manufacture fusion proteins for use as sensitizing antigens, Fc fragments of antibodies or peptide tags are preferably used, for example. A vector expressing the fusion protein is fused with a gene encoding two or more desired polypeptide fragments in a frame-compliant manner, and this fusion gene is inserted into the expression vector as described above. Methods for preparing fusion proteins are 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, methods for obtaining GPC3 as a sensitizing antigen and methods for immunization using it are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0793] The mammals immunized with this sensitizing antigen are not limited to specific animals, but the suitability of the parent cells used for cell fusion is preferably taken into consideration. Rodents, such as mice, rats, hamsters, or rabbits, monkeys, etc., are generally suitable.

[0794] The animals described above are immunized using sensitizing antigens according to known methods. For example, as a routine method, immunization is performed by intraperitoneal or subcutaneous injection of the sensitizing antigen into 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 Freund's complete adjuvant, emulsified, and administered to mammals several times every 4 to 21 days. Furthermore, a suitable carrier can be used for sensitizing antigen immunization. Particularly 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 or keyhole hemocyanin.

[0795] 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 stimulating the expression of the sensitizing antigen within the immunized animal and providing immune stimulation. Compared to conventional immunization methods that administer protein antigens to immunized animals, DNA immunization is expected to offer the following advantages.

[0796] - It can maintain the structure of membrane proteins and provide immune stimulation.

[0797] - It is not necessary to purify the immune antigen.

[0798] To obtain the monoclonal antibody of the present invention via DNA immunization, DNA encoding a tumor antigen protein is first administered to an immunized animal. The DNA encoding the tumor antigen can be synthesized using known methods such as PCR. The obtained DNA is inserted into a suitable expression vector and administered to the immunized animal. Commercially available expression vectors, such as pcDNA3.1, can be suitable. The method of administering the vector to the organism can utilize commonly used methods. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of the immunized animal using a gene gun. Furthermore, the antibody recognizing the tumor antigen can also be prepared using the method described in International Publication WO2003 / 104453.

[0799] After immunizing mammals in this way, and confirming an increase in antibody titers that bind 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.

[0800] The cells fused with the aforementioned immune cells are mammalian myeloma cells. Myeloma cells preferably possess suitable selection markers for screening purposes. Selection markers refer to traits that indicate survival (or non-survival) under specific culture conditions. Selection markers such as hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter referred to as HGPRT deficiency) or thymidine kinase deficiency (hereinafter referred to as TK deficiency) are well known. Cells with HGPRT or TK deficiencies exhibit hypoxanthine-aminopterin-thymidine sensitivity (hereinafter referred to as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA and die in HAT-selective media, but when fused with normal cells, they can continue DNA synthesis using the salvage pathways of normal cells, thus proliferating even in HAT-selective media.

[0801] HGPRT-deficient or TK-deficient cells can be selected in media 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, cells deficient in these enzymes that do not take up these pyrimidine analogs can survive in selective media. In addition, a selection marker called G418 resistance, transmitted via a neomycin resistance gene, provides resistance to 2-deoxystreptamine antibiotics (gentamicin analogs). Various myeloma cell lines suitable for cell fusion are well known.

[0802] Such myeloma cells can be appropriately 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), 269-2 70), 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.

[0803] Basically based on well-known methods, for example The above-mentioned cell fusion of immune cells and myeloma cells was performed using methods similar to those of Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0804] More specifically, for example, cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter. Fusion promoters used include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). To further improve fusion efficiency, auxiliary agents such as dimethyl sulfoxide can be added as needed.

[0805] 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, RPMI 1640 medium or MEM medium suitable for the proliferation of the above-mentioned myeloma cell line can be used; in addition, conventional culture media used for such cell culture can be used; furthermore, serum supplements such as fetal bovine serum (FCS) can be appropriately added.

[0806] Cell fusion involves uniformly mixing specific amounts of the aforementioned immune cells and myeloma cells in the aforementioned culture medium, typically adding a PEG solution (e.g., with an average molecular weight of approximately 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 process of centrifugation and removal of the supernatant is repeated to remove cell fusion agents and other substances detrimental to hybridoma growth.

[0807] The hybridomas thus obtained can be selected by culturing them in a conventional selection medium, such as HAT medium (containing hypoxanthine, aminopterin, and thymidine). Culture in the aforementioned HAT medium continues until sufficient time (typically several days to several weeks) has elapsed to kill cells other than the desired hybridomas (non-fusion cells). Then, selection and monoclonalization of hybridomas producing the desired antibodies are performed using the conventional limiting dilution method.

[0808] The hybridomas thus obtained can be selected using a selective medium containing selection markers specific to the myeloma cells used for cell fusion. For example, cells deficient in HGPRT or TK are selected using HAT medium (containing hypoxanthine, aminopterin, and thymidine). That is, when 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 aforementioned HAT medium continues until sufficient time has passed for the cells other than the desired hybridoma (non-fused cells) to die. Specifically, after several to several weeks of culture, the desired hybridomas can be selected. Then, using the usual limiting dilution method, the hybridomas producing the desired antibodies can be screened and monocloned.

[0809] The screening and monoclonalization of 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.

[0810] To screen hybridomas that produce the monoclonal antibodies of this invention via FACS, cells expressing GPC3 are first prepared. Preferred cells for screening are mammalian cells that are forcibly expressed with the tumor antigens used. By using untransformed mammalian cells as host cells as controls, the binding activity of the antibodies to the tumor antigens 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 cells forcibly expressing GPC3, hybridomas producing monoclonal antibodies against the tumor antigens can be obtained.

[0811] Alternatively, the binding activity of antibodies to immobilized tumor antigen-expressing cells can be assessed based on the principles of ELISA. 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 antibodies binding to the immobilized cells are detected. In cases where the monoclonal antibody is derived from mice, anti-mouse immunoglobulin antibodies can be used to detect the antibodies binding to the cells. Hybridomas selected through such screening that produce the desired antibodies capable of binding to the antigen can be further screened using limiting dilution methods, etc.

[0812] The hybridomas prepared in this way that produce monoclonal antibodies can be passaged in conventional culture media. Furthermore, these hybridomas can be stored long-term in liquid nitrogen.

[0813] The hybridoma can be cultured using standard methods, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be introduced into a suitable mammal to proliferate, and the monoclonal antibody can be obtained from its ascites. The former method is suitable for obtaining high-purity antibodies.

[0814] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be appropriately utilized. The cloned antibody gene is inserted into a suitable vector and introduced into a host cell to express the antibody encoded by that 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). Methods for manufacturing recombinant antibodies, as described below, are also well known.

[0815] For example, cDNA encoding the variable region (V region) of a hybridoma cell that produces an antibody that binds to a tumor antigen can be obtained. For this purpose, total RNA is usually extracted from the hybridoma first. Methods for extracting mRNA from cells include, for example, the following: - guanidine ultracentrifugation (Biochemistry (1979) 18(24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162(1), 156-159).

[0816] The extracted mRNA can be purified using an mRNA purification kit (manufactured by GE Healthcare BioSciences). Alternatively, kits for directly extracting total mRNA from cells are also commercially available, 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 an 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 SMARTRACE cDNA amplification kit (manufactured by Clontech) and the 5'-RACE method using PCR (Prac. 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.

[0817] 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 after selecting colonies such as *E. coli* for introduction, the desired recombinant vector can be prepared from the *E. coli* colonies that form them. Then, it is confirmed whether the recombinant vector possesses the base sequence of the target cDNA using known methods, such as dideoxynucleotide chain termination.

[0818] To obtain genes encoding variable regions, the 5'-RACE method using primers for variable region gene amplification is a simple approach. First, RNA extracted from hybridoma cells is used as a template to synthesize cDNA, resulting in a 5'-RACE cDNA library. Commercially available kits such as the SMART RACE cDNA amplification kit can be appropriately used for the synthesis of 5'-RACE cDNA libraries.

[0819] The obtained 5'-RACE cDNA library was 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 subclass in advance using commercially available kits such as the Iso Strip Mouse Monoclonal Antibody Isotype Kit (Roche Diagnostics).

[0820] Specifically, for example, when aiming to obtain the gene encoding mouse IgG, possible primers can be used to amplify genes encoding heavy chains γ1, γ2a, γ2b, and γ3, and light chains κ and λ. To amplify the IgG variable region gene, primers that anneal to the corresponding constant region near the variable region are generally used for the 3' end primer. On the other hand, primers for the 5' end are used, as provided in the 5'-RACE cDNA library preparation kit.

[0821] The amplified PCR product can be used to reconstruct immunoglobulins composed of combinations of heavy and light chains. The binding activity of the reconstructed immunoglobulins to antigens can be used as an indicator to screen for desired antibodies. For example, when aiming to obtain antibodies against GPC3, antibodies that specifically bind to GPC3 are preferred. The antibodies used in this invention can be screened, for example, as described below:

[0822] (1) The step of contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with an antigen-expressing cell;

[0823] (2) The steps for detecting the binding of antigen-expressing cells to antibodies; and

[0824] (3) The step of selecting antibodies that bind to antigen-expressing cells.

[0825] 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 using methods such as FACS. Stabilized samples of tumor antigen-expressing cells can be appropriately used to assess antibody binding activity.

[0826] Antibody screening methods that use binding activity as an indicator can also appropriately employ phage vector-based panning. In cases where libraries containing antibody genes as heavy and light chain subclasses are obtained from polyclonal antibody expression cell populations, phage vector-based screening is advantageous. Genes encoding the variable regions of the heavy and light chains can be ligated using 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 this phage contacts the desired antigen, the DNA encoding the scFv with the desired binding activity can be recovered by recovering the antigen-bound phage. Repeating this process as needed allows for the concentration of scFvs with the desired binding activity.

[0827] After obtaining cDNA encoding the V region of an antibody that binds to a target tumor antigen, the cDNA is digested by a restriction enzyme that recognizes restriction enzyme sites inserted 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. Further, to ensure that one copy of the digested fragment is inserted into the vector in the correct orientation, the insertion of a restriction enzyme that produces sticky ends is preferred. An antibody expression vector can be obtained by inserting the digested cDNA encoding the V region of the 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. A chimeric antibody expression vector can be constructed by pre-inserting the aforementioned V region gene into an expression vector having the constant region. Specifically, for example, a restriction enzyme recognition sequence for the restriction enzyme that digests the aforementioned V region gene can be appropriately configured at the 5' end of an expression vector containing DNA encoding the desired antibody constant region (C region). The two restriction enzymes digested with the same combination are fused together in a reading frame conformal manner to construct a chimeric antibody expression vector.

[0828] To manufacture monoclonal antibodies, an antibody gene is inserted into an expression vector for expression under the control of an expression control region. This expression control region contains, for example, an enhancer or a promoter. Additionally, a suitable signal sequence can be added to the amino terminus to induce extracellular secretion of the expressed antibody. In the embodiments described later, the signal sequence uses, for example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS, but other suitable signal sequences may be added. The expressed polypeptide is cleaved at the carboxyl terminus of the aforementioned sequence, and the cleaved polypeptide, as a mature polypeptide, can be secreted extracellularly. Then, by transforming suitable host cells with this expression vector, recombinant cells expressing DNA encoding an antibody that binds to a target tumor antigen can be obtained.

[0829] To express antibody genes, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is inserted into different expression vectors. Antibody molecules with both H and L chains can be expressed by 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).

[0830] Various combinations of host cells and expression vectors for preparing antibodies by introducing isolated antibody genes into suitable hosts are known. These expression systems can be used 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, animal cells can be exemplified by the following cells.

[0831] (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, etc.

[0832] (2) Amphibian cells: oocytes of the African clawed frog (Xenopus laevis), etc.

[0833] (3) Insect cells: sf9, sf21, Tn5, etc.

[0834] Alternatively, antibody gene expression systems derived from plant cells of the genus *Nicotiana*, such as *Nicotiana tabacum*, are known. Transformation of plant cells can be appropriately achieved using cells cultured from callus tissue.

[0835] Furthermore, fungal cells can utilize the following cells.

[0836] - Yeasts: *Saccharomyces*, such as *Saccharomyces serevisiae*, and *Pichia*, such as *Pichia pastoris*.

[0837] - Filamentous fungi: Aspergillus species such as Aspergillus niger.

[0838] Furthermore, antibody gene expression systems utilizing prokaryotic cells are also known. For example, in cases using bacterial cells, Escherichia coli (E. coli) and Bacillus subtilis, among other bacterial cells, can be appropriately used. An expression vector containing the target antibody gene is transformed and introduced into these cells. The desired antibody can be obtained from the culture of the transformed cells by culturing them in vitro.

[0839] 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, a fusion gene can be constructed by inserting the antibody gene into the gene encoding a protein that is normally produced in milk, in a frame-compliant manner. Proteins such as sheep β-casein can be used as secreted in milk. A DNA fragment containing the fusion gene with the inserted antibody gene is injected into a sheep embryo, and the injected embryo is introduced into a female sheep. The fusion protein of the desired antibody and milk protein can be obtained from the milk produced by the transgenic sheep (or its offspring) born from the sheep that received the embryo. In addition, 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).

[0840] When administering the antigen-binding molecule described in this specification to humans, a domain derived from an artificially modified recombinant antibody may be appropriately used as the structural domain containing the antibody variable region of the antigen-binding molecule for the purpose of reducing heteroantigenicity against humans. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies can be suitably manufactured using known methods.

[0841] 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) enclosed by 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, binding specificity of one antibody can generally be transferred to other antibodies through CDR transfer.

[0842] Humanized antibodies are also called reshaped human antibodies. Specifically, it is known to transfer the CDR of a non-human animal, such as a mouse antibody, into a human antibody to create human-derived antibodies. General gene recombination methods for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is known as a method for transferring a mouse antibody CDR into a human FR. In overlap extension PCR, the base sequence encoding the mouse antibody CDR to be transferred is added to primers used to synthesize the human antibody FR. Primers are prepared for each of the four FRs. Generally, in the transfer of mouse CDRs into human FRs, 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 preferable to use human FRs composed of amino acid sequences with high identity to the amino acid sequences of the FRs adjacent to the mouse CDR to be transferred.

[0843] Furthermore, the ligation base sequences were designed to be linked in a reading frame-compliant manner. Human FRs were synthesized separately using their respective primers. The results yielded products containing DNA encoding mouse CDRs for each FR. The base sequences encoding mouse CDRs in each product were designed to overlap. Next, using a human antibody gene as a template, the overlapping CDR portions of the synthesized products were annealed to each other, followed by complementary strand synthesis. Through this reaction, human FRs were ligated via mouse CDR sequences.

[0844] Finally, the V region gene, linking 3 CDRs and 4 FRs, is renatured at its 5' and 3' ends, and its full length is amplified using primers with appropriate restriction enzyme recognition sequences. The DNA obtained as described above and the DNA encoding the C region of the human antibody are 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 these recombinant cells to express the DNA encoding the humanized antibody, the humanized antibody is produced in the culture of these cells (European Patent Publication EP239400, International Publication WO1996 / 002576).

[0845] Qualitative or quantitative determination and evaluation of the antigen-binding activity of the humanized antibody prepared as described above allows for the appropriate selection of the FR of the human antibody, where the CDR forms a good antigen-binding site upon CDR linkage. 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 the primers renatured to the FR. FRs synthesized using such primers contain these base sequence variations. The antigen-binding activity of the amino acid-substituted variant antibody 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).

[0846] In addition, transgenic animals with antibody gene libraries containing all 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 antibodies through DNA immunization.

[0847] Furthermore, techniques for obtaining human antibodies through panning human antibody libraries are also known. For example, the V region of a human antibody can be expressed as a single-chain antibody (scFv) on the surface of a phage via phage display. Phages expressing scFvs that bind to antigens can be selected. By analyzing the genes of the selected phages, the DNA sequence encoding the V region of the antigen-binding human antibody can be determined. After determining the DNA sequence of the antigen-binding scFv, 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 that human antibody. Such methods are already known (see international publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

[0848] Domains containing antibody variable regions with T-cell receptor complex binding activity

[0849] In this specification, "a domain comprising an antibody variable region having T-cell receptor complex binding activity" refers to 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 can be the T-cell receptor itself or an aptor molecule that, together with the T-cell receptor, constitutes the T-cell receptor complex. A suitable aptor is CD3.

[0850] Domains containing antibody variable regions with T-cell receptor binding activity

[0851] In this specification, "a domain comprising a variable region of an antibody having T-cell receptor binding activity" refers to 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 this invention can be a variable region or a constant region, preferably an epitope present in the constant region. Examples of sequences of constant regions include, for example, the sequences of the T-cell receptor α chain (RefSeq accession number CAA26636.1), 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).

[0852] Domains containing antibody variable regions with CD3 binding activity

[0853] 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) and a heavy chain variable region (VH) of the anti-CD3 antibody.

[0854] The present invention includes a domain comprising an antibody variable region having CD3 binding activity. If an epitope is present in the γ, δ, or ε chain sequence constituting human CD3, the domain may also be a domain of an antibody variable region binding to any epitope. Preferably, in the present invention, domains comprising the light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody that bind to an epitope present in the extracellular domain of the e chain of the human CD3 complex are suitably used. As such domains, in addition to the light chain variable region (VL) and heavy chain variable region (VH) of the anti-CD3 antibody described in the examples, domains of OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) or various known CD3-binding domains comprising the light chain variable region (VL) and 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, can be suitably used. The domain containing the antibody variable region having CD3 binding activity becomes the anti-CD3 antibody of origin, and as described above, suitable humanized antibodies or human antibodies can be suitably used. The polynucleotide sequences of the structures constituting the γ, δ, or ε chain of CD3 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.

[0855] Specificity

[0856] Specificity refers to a molecule that specifically binds to a particular molecule, while not exhibiting any significant binding to molecules other than those it binds to. Additionally, a domain containing an antibody variable region can be used to specifically bind to a particular epitope among multiple epitopes contained in an antigen. Furthermore, when the epitope bound by the domain containing the antibody variable region is contained in multiple different antigens, an antigen-binding molecule having the domain containing that antibody variable region can bind to multiple antigens containing that epitope.

[0857] Epitope

[0858] An epitope, referring to an antigenic determinant present in an antigen, is a site on the antigen that binds to a domain containing an antibody variable region in an antigen-binding molecule disclosed in this specification. Therefore, an epitope can be defined, for example, based on its structure. Alternatively, an epitope can be defined based on its binding activity to the antigen in an antigen-binding molecule that recognizes it. When the antigen is a peptide or polypeptide, the epitope can also be determined based on the amino acid residues constituting it. Furthermore, when the epitope is a glycan, it can be determined based on its specific glycan structure.

[0859] A linear epitope is an epitope that contains an amino acid primary sequence that is identified. A linear epitope typically contains at least 3, most commonly at least 5, amino acids, such as about 8 to about 10, or 6 to 20, amino acids in the intrinsic sequence.

[0860] Unlike linear epitopes, stereoepitopes are not epitopes whose primary amino acid sequence is the single defining component of the epitope being recognized (e.g., the primary amino acid sequence is not necessarily the epitope recognized by an antibody targeting a specific epitope). Stereoepitopes, compared to linear epitopes, can contain a significantly larger 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, allowing the antibody to recognize the epitope. Methods for determining the stereostructure of epitopes include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-specific spin tagging, and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0861] The following are examples of methods for confirming epitope binding by antigen-binding molecules that bind to tumor antigens.

[0862] For example, it can be confirmed that an antigen-binding molecule that binds to a tumor antigen recognizes a linear epitope present in the tumor antigen molecule. For this 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 via genetic engineering methods using a region in the cDNA of the tumor antigen encoding the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide formed from the amino acid sequence constituting the extracellular domain with a domain having an antibody variable region containing an antibody that is active in binding to the tumor antigen is evaluated. For example, the binding activity of the antigen-binding molecule to 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. Through these experiments, the binding activity of the antigen-binding molecule to the linear peptide can be determined.

[0863] Furthermore, it can be confirmed, as described below, that a test antigen-binding molecule having a domain containing an antibody variable region with binding activity against tumor antigens recognizes a stereospecific epitope. For the above purpose, cells expressing tumor antigens are prepared. For example, when a test antigen-binding molecule having a domain containing an antibody variable region with binding activity against tumor antigens comes into contact with a tumor antigen-expressing cell, it binds strongly to the cell; on the other hand, the antigen-binding molecule substantially does not bind to a linear peptide formed by a fixed amino acid sequence constituting the extracellular domain of the tumor antigen. Here, "substantially does not bind" means a binding activity of 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity against human tumor antigen-expressing cells.

[0864] Methods for determining the binding activity of test antigen-binding molecules containing antigen-binding domains targeting tumor antigens on tumor antigen-expressing cells include, for example, the methods described in the Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, this can be assessed using the principles of ELISA or FACS (fluorescence-activated cell sorting) with GPC3-expressing cells as the antigen.

[0865] In ELISA, the binding activity of a test antigen-binding molecule containing an antigen-binding domain targeting a target tumor antigen to tumor antigen-expressing cells is quantitatively assessed by comparing the signal levels generated after the enzyme reaction. Specifically, the test antigen-binding molecule is added to an ELISA plate immobilized with tumor antigen-expressing cells, and an enzyme-labeled antibody that recognizes the test antigen-binding molecule is used to detect the molecule binding to the cells. Alternatively, in FACS, a series of dilutions of the test antigen-binding molecule is prepared, and the binding titer to tumor antigen-expressing cells is determined to compare the binding activity of the test antigen-binding molecule to these cells.

[0866] The binding of the antigen-binding molecule to an antigen expressed on the surface of a cell suspended in a buffer solution or similar medium can be detected by flow cytometry. Devices known for flow cytometry include, for example, the following.

[0867] FACSCanto™ II

[0868] FACSAria™

[0869] FACSArray™

[0870] FACSVantage™ SE

[0871] FACSCalibur™ (both are trade names of BD Biosciences)

[0872] EPICS ALTRA HyPerSort

[0873] Cytomics FC 500

[0874] EPICS XL-MCL ADC EPICS XL ADC

[0875] Cell Lab Quanta / Cell Lab Quanta SC (both are product names from Beckman Coulter)

[0876] For example, as a suitable method for determining the binding activity of a test antigen-binding molecule against an antigen, the following method can be exemplified. First, staining with a FITC-labeled secondary antibody that identifies and reacts with cells expressing the target tumor antigen is performed. 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 cells is reflected by the fluorescence intensity obtained through analysis using CELLQUEST Software (BD), i.e., the geometric mean. That is, by obtaining this geometric mean, the binding activity of the test antigen-binding molecule, expressed as the amount of the test antigen-binding molecule bound, can be determined.

[0877] When a tested antigen-binding molecule shares an epitope with another antigen-binding molecule, this can be confirmed by the competition between the two molecules for the same epitope. Competition between antigen-binding molecules can be detected using methods such as cross-blocking assays. For example, competitive ELISA is a preferred method for cross-blocking assays.

[0878] Specifically, in the cross-blocking assay, tumor antigen proteins coated on wells of a microtiter plate are pre-incubated in the presence or absence of candidate competing antigen-binding molecules, followed by the addition of a test antigen-binding molecule. The amount of test antigen-binding molecule binding to the tumor antigen protein in the well is indirectly related to the binding ability of the candidate competing antigen-binding molecule 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 well coated with the tumor antigen protein.

[0879] The amount of antigen-binding molecules mediated by tumor antigen proteins and bound to pores can be readily determined by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be determined using an avidin-peroxidase conjugate and a suitable substrate. Cross-blocking assays using enzymes such as peroxidase are specifically called competitive ELISA assays. Antigen-binding molecules can be labeled with other detectable or measurable labeling substances. Specifically, radioactive or fluorescent labels are known.

[0880] Compared to the binding activity obtained in a control test 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 binding to the same epitope as the competing antigen-binding molecule, or is an antigen-binding molecule competing for binding to the same epitope.

[0881] In 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 their binding activity to peptides or polypeptides with amino acid modifications at the peptide constituting the epitope.

[0882] As a method for determining such binding activity, for example in the aforementioned ELISA format, it can be determined by comparing the binding activity of the test antigen-binding molecule and the control antigen-binding molecule for the introduced variant linear peptide. As a method other than ELISA, the binding activity against the variant peptide bound to the column can also be determined by quantitatively eluting antigen-binding molecules from the elution buffer after passing the test antigen-binding molecule and the control antigen-binding molecule through a column bound to the variant peptide. Methods for adsorbing the variant peptide onto the column as a fusion peptide, such as GST, are known.

[0883] In addition, when the identified epitope is a stereoepitaxy, the common epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated using the following method. First, prepare cells expressing tumor antigens and cells expressing epitopes introduced with the variant tumor antigen. Add the test antigen-binding molecule and the control antigen-binding molecule to a cell suspension suspended in a suitable buffer such as PBS. Next, add a FITC-labeled antibody that recognizes the test antigen-binding molecule and the control antigen-binding molecule to the cell suspension washed with a suitable buffer. The fluorescence intensity and cell number of cells stained with the labeled antibody are determined using FACSCalibur (BD). The concentrations of the test antigen-binding molecule and the control antigen-binding molecule are 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 is used. The binding amount of the labeled antibody to the cells is reflected by the fluorescence intensity obtained through analysis using CELL QUEST Software (BD), 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 bound, can be determined.

[0884] The following method can be used to determine whether cells expressing the variant tumor antigen substantially do not bind to it. First, the test antigen-binding molecule and the control antigen-binding molecule bound to cells expressing the variant tumor antigen are stained with a labeled antibody. Next, the fluorescence intensity of the cells is detected. When using FACSCalibur for fluorescence detection as flow cytometry, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The geometric mean of the presence and absence of the antigen-binding molecule is used to calculate the comparison value (ΔGeo-Mean), which indicates the percentage increase in fluorescence intensity due to the binding of the antigen-binding molecule.

[0885] ΔGeo-Mean = Geo-Mean (in the presence of antigen-binding molecules) / Geo-Mean (in the absence of antigen-binding molecules)

[0886] Fv (Variable Region Fragment)

[0887] In this specification, the term "Fv (variable ffagment)" refers to the smallest unit of an antibody's antigen-binding domain, 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 a bacterial signal sequence, the gene could be induced to express and be prepared uniformly and sustainably from periplasmic fractions of *E. coli* (Science (1988) 240 (4855), 1038-1041). In Fv prepared from periplasmic fractions, VH and VL are associated in a form that binds to the antigen.

[0888] In this specification, Fv is, for example, the following antigen-binding molecule: which also suitably includes a pair of Fv, in the case of antigen-binding molecules including:

[0889] (1) A bivalent antigen-binding domain; the bivalent antigen-binding domain is a bivalent scFv, which is 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.

[0890] (2) A domain containing an Fc region, wherein the Fc region is an Fc region in IgG1, IgG2a, IgG3, or IgG4 that does not possess Fcγ receptor-binding activity among the amino acids constituting the Fc region; and

[0891] (3) A CD3-binding domain with at least one valence, wherein the light chain Fv fragment and the heavy chain Fv fragment are associated in a manner that binds to CD3 to form a CD3-binding domain.

[0892] scFv, single-chain antibody, or sc(Fv)2

[0893] 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 for the formation of desired structures that are thought to permit 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 U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, the single-chain antibody may be bispecific and / or humanized.

[0894] scFv is an antigen-binding domain formed by the peptide linker connecting the VH and VL components of Fv (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). This peptide linker maintains the VH and VL in a close proximity state.

[0895] sc(Fv)2 is a single-chain antibody consisting of four variable regions, two VLs and two VHs, linked by linkers such as peptide linkers (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. 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 linkers such as peptide linkers.

[0896] Examples of antibodies that constitute the antigen-binding domain of sc(Fv)2 in this specification include antibodies characterized by two VH and two VL molecules arranged in the order of VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) with the N-terminal side of a single-chain polypeptide as the base point. The order of the two VH and two VL molecules is not particularly limited to the above configuration, and any arrangement is acceptable. For example, configurations with the following order are also possible.

[0897] [VL] connector [VH] connector [VH] connector [VL]

[0898] [VH] connector [VL] connector [VL] connector [VH]

[0899] [VH] connector [VH] connector [VL] connector [VL]

[0900] [VL] connector [VL] connector [VH] connector [VH]

[0901] [VL] connector [VH] connector [VL] connector [VH]

[0902] 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 molecules disclosed in this specification.

[0903] Furthermore, 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. Additionally, by inserting glycan appendage sequences, it is possible to appropriately add them to achieve the desired effect for the glycan chain.

[0904] As the linker for the variable region binding the antibody, any genetically engineered peptide linker or synthetic compound linker (e.g., the linker disclosed in Protein Engineering, 9(3), 299-305, 1996) can be used, but peptide linkers are preferred in this invention. The length of the peptide linker is not particularly limited and can be suitably 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 linkers, all peptide linkers can be of the same length, or peptide linkers of different lengths can be used.

[0905] For example, in the case of peptide linkers, the following sequences can be cited:

[0906] Ser

[0907] Gly·Ser

[0908] Gly·Gly·Ser

[0909] Ser·Gly·Gly

[0910] Gly·Gly·Gly·Ser

[0911] Ser·Gly·Gly·Gly

[0912] Gly·Gly·Gly·Gly·Ser

[0913] Ser·Gly·Gly·Gly·Gly

[0914] Gly·Gly·Gly·Gly·Gly·Ser

[0915] Ser·Gly·Gly·Gly·Gly·Gly

[0916] Gly·Gly·Gly·Gly·Gly·Gly·Ser

[0917] Ser·Gly·Gly·Gly·Gly·Gly·Gly

[0918] (Gly·Gly·Gly·Gly·Ser) n

[0919] (Ser·Gly·Gly·Gly·Gly) n

[0920] [Where 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.

[0921] Synthetic chemical linkers (chemical crosslinking agents) are commonly used crosslinking agents for crosslinking peptides. Examples include: N-hydroxysuccinimide (NHS), disuccinimide octanoate (DSS), bis(sulfosuccinimide) octanoate (BS3), dithiobis(succinimide propionate) (DSP), dithiobis(sulfosuccinimide propionate) (DTSSP), ethylene glycol bis(succinimide succinic acid) (EGS), ethylene glycol bis(sulfosuccinimide succinic acid) (sulfon-EGS), disuccinimide tartrate (DST), disulfosuccinimide tartrate (sulfon-DST), bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl] sulfone (sulfon-BSOCOES), etc. These crosslinking agents are all commercially available.

[0922] In the case of connecting four antibody variable regions, three adapters are usually required. All three can be the same adapter, or different adapters can be used.

[0923] A "Fab" molecule consists of a light chain and a heavy chain with a CH1 region and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0924] "F(ab')2" and "Fab'" refer to antibody fragments prepared by treating immunoglobulins (monoclonal antibodies) with proteolytic enzymes such as pepsin or papain, resulting in digestion before and after the disulfide bond between the two H chains located in the hinge region. For example, IgG can be treated with papain and cleaved upstream of the disulfide bond between the two H chains in the hinge region, preparing two identical antibody fragments: an L chain composed of VL (variable region of the L chain) and CL (constant region of the L chain), and an H chain composed of VH (variable region of the H chain) and CHγ1 (γ1 region in the constant region of the H chain), linked by a disulfide bond at the C-terminus. These two identical antibody fragments are referred to as Fab'.

[0925] "F(ab')2" comprises two light chains and two heavy chains, the two heavy chains containing constant regions of a CH1 domain and a portion of a CH2 domain, such that disulfide bonds are formed between the two heavy chains. The F(ab')2 constituting the antigen-binding molecule disclosed herein can be suitably obtained by partially digesting a full-length monoclonal antibody having the desired antigen-binding domain with a proteolytic enzyme such as pepsin, thereby removing the Fc fragment by adsorption onto a protein A column. As the proteolytic enzyme, any enzyme capable of selectively generating F(ab')2 by digesting a full-length antibody under appropriate pH or other enzyme reaction conditions is acceptable; there are no particular limitations. Examples include pepsin or ficin.

[0926] The Fc domain constituting the antigen-binding molecule disclosed in this specification can be suitably obtained as follows: after partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin, the fragment is adsorbed onto a protein A column or a protein G column, and then eluted with a suitable elution buffer. As for the proteolytic enzyme, any enzyme capable of digesting the antibody such as a monoclonal antibody by appropriately setting the enzyme reaction conditions such as pH is acceptable; there are no particular limitations. For example, pepsin or ficin can be used.

[0927] The antigen-binding molecules described in this specification contain an Fc region with reduced Fcγ receptor-binding activity in the amino acids constituting the Fc region of IgG1, IgG2, IgG3, or IgG4.

[0928] Antibody isotypes are determined by the structure of their constant regions. The constant regions of each isotype of IgG1, IgG2, IgG3, and IgG4 are referred to as Cγ1, Cγ2, Cγ3, and Cγ4, respectively.

[0929] The Fc region refers to the region excluding F(ab')2, which comprises two light chains and two heavy chains. The two heavy chains include a portion of a constant region between the CH1 domain and the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains. The Fc domain constituting the antigen-binding molecule disclosed in this specification can be suitably obtained by partially digesting monoclonal antibodies such as IgG1, IgG2, IgG3, and IgG4 with a proteolytic enzyme such as pepsin, followed by elution of the component adsorbed onto a protein A column. As the proteolytic enzyme, any enzyme capable of selectively generating F(ab')2 by appropriately setting the enzyme reaction conditions such as pH can be used; there are no particular limitations. Examples include pepsin or ficin.

[0930] The Fcγ receptor refers to the receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies. It also represents all members of the protein family encoded by the Fcγ receptor gene. In humans, this family includes: FcγRI (CD64), which contains isoforms FcγRIa, FcγRib, and FcγRIc; FcγRII (CD32), which contains isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRHI (CD16), which contains isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as human-like FcγR or FcγR isoforms or alloforms that have not yet been discovered, but is not limited to these. FcγRs include, but are not limited to, those from humans, mice, rats, rabbits, and monkeys, and can also originate from any organism. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as undiscovered mouse FcγR classes or FcγR allotypes or allotypes, but are not limited to these. Suitable examples of such Fcγ receptors are human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16). The polynucleotide and amino acid sequences of FcγRI are recorded as RefSeq accession numbers NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIA are recorded as RefSeq accession numbers BC020823.1 and 30AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIB are recorded as RefSeq accession numbers BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIA are recorded as RefSeq accession numbers BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are recorded as BC128562.1 and AAI28563.1, respectively.Whether the Fcγ receptor has binding activity with the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies can be confirmed, in addition to the FACS or ELISA methods described above, by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) or by BIACORE method using the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0931] Additionally, "Fc ligand" or "effect ligand" refers to a molecule from any organism, preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. The binding of an Fc ligand to Fc preferably induces one or more effector functions. Fc ligands include, but are not limited to, Fc receptors, FcγR, FcαR, FcεR, FcRn, Clq, C3, mannan-binding lectins, mannose receptors, staphylococcal protein A, staphylococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologues (FcRH) belonging to the Fc receptor family, which are of the same type as FcγR (Davis et al., (2002) Immunological Reviews 190, 123-136). Fc ligands may also include undiscovered molecules that bind to Fc.

[0932] The Fc region exhibits reduced Fcγ receptor-binding activity for any of FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB. This can be confirmed by the FACS or ELISA methods described above, as well as by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) or the BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0933] ALPHA screening is performed using ALPHA technology with two types of beads: donor and acceptor. The molecules bound to the donor bead interact biologically with those bound to the acceptor bead, and a luminescent signal is detected only when the two beads are in close proximity. A photosensitizer within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. This singlet oxygen diffuses towards the donor bead, triggering a chemiluminescent reaction within the nearby acceptor bead, ultimately emitting light. When the molecules bound to the donor and acceptor beads do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, thus not triggering a chemiluminescent reaction.

[0934] For example, a biotin-labeled antigen-binding molecule is bound to a donor bead, and an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to a receptor bead. In the absence of an antigen-binding molecule with a competing mutant Fc region, an antigen-binding molecule with a wild-type Fc region interacts with the Fcγ receptor, generating a signal at 520-620 nm. Antigen-binding molecules with untagged mutant Fc regions and those with wild-type Fc regions compete for interaction with the Fcγ receptor. The relative binding affinity can be determined by the decrease in fluorescence resulting from quantitative competition. Biotinylation of antigen-binding molecules such as antibodies using Sulfo-NHS-biotin is well known. As a method for tagging the Fcγ receptor with GST, suitable methods include fusing a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST within a reading frame, expressing the resulting fusion gene in cells holding a vector for potential expression, and purifying it using a glutathione column. The resulting signal is suitable for analysis using a one-site competition model based on nonlinear regression analysis, employing software such as GRAPCAD PRISM (GraphPad; San Diego).

[0935] If one of the interacting substances (ligand) is fixed onto a gold film on a sensor chip, and light is shone from the back side of the chip, causing total internal reflection at the interface between the gold film and the glass, a portion of the reflected light with reduced intensity (SPR signal) is formed. If the other interacting substance (analyte) is allowed to flow onto the surface of the sensor chip, allowing the ligand to bind to the analyte, the mass of the fixed ligand molecules increases, and the refractive index o...

Claims

1. The use of cells expressing chimeric receptors in the preparation of drugs for administration in combination with antigen-binding molecules, wherein... Antigen-binding molecules contain variable and constant regions of the antibody, as well as a protease-cleavable linker, which enables them to bind to the target antigen after cleavage. Chimeric receptors bind to the peptide ends of antigen-binding molecules through cleavage at the linker. The antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a single-domain antibody. By cleaving the connector, Fab, (Fab)2, VL, VH or VHH, or their antigen-binding fragments, are obtained. Chimeric receptors comprise an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain is capable of binding to antigen-binding molecules cleaved by the adaptor. Through binding to these antigen-binding molecules, the receptor can bind to cells expressing the target antigen. 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. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) is 0.1 or less.

2. The use of antigen-binding molecules in the preparation of drugs for use in combination with cells expressing chimeric receptors, wherein... Antigen-binding molecules contain variable and constant regions of the antibody, as well as a protease-cleavable linker, which enables them to bind to the target antigen after cleavage. Chimeric receptors bind to the peptide ends of antigen-binding molecules through cleavage at the linker. The antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a single-domain antibody. By cleaving the connector, Fab, (Fab)2, VL, VH or VHH, or their antigen-binding fragments, are obtained. Chimeric receptors comprise an extracellular binding domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular binding domain is capable of binding to antigen-binding molecules cleaved by the adaptor. Through binding to these antigen-binding molecules, the receptor can bind to cells expressing the target antigen. 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. D The ratio of values ​​(K) D (After cutting) / K D (Before cutting) is 0.1 or less.

3. The use according to claim 1 or 2, wherein, The antigen-binding molecule is an IgG antibody.

4. The use according to claim 1 or 2, wherein, An antigen-binding molecule whose linker has been cleaved by a protease contains an antigen-binding domain and a portion of the cleaved linker.

5. The use according to claim 1 or 2, 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.

6. The use according to claim 1 or 2, wherein, The antigen-binding molecule after cleavage is the antibody's VL, VH, or VHH, or its antigen-binding fragment.

7. The use according to claim 1 or 2, wherein, The antigen-binding molecule is a single-domain antibody containing a protease-cleavable linker. The antigen-binding molecule after linker cleavage contains the antigen-binding domain of the single-domain antibody and a portion of the linker.

8. The use according to claim 1 or 2, wherein, Protease-cleavable adapters contain protease-cleaving sequences.

9. The use according to claim 1 or 2, wherein, The protease-cleavable adapter contains a peptide having a protease-cleaving sequence of any sequence number 1 to 725.

10. The use according to claim 1 or 2, wherein, The drug is used to treat cancer.

11. Use of the isolated nucleic acid in the preparation of the medicament as defined in any one of claims 1 to 10, wherein, The isolated nucleic acid encodes the antigen-binding molecule or chimeric receptor contained in the drug as defined in any one of claims 1 to 10.

12. Use of the carrier in the preparation of the medicament as defined in any one of claims 1 to 10, wherein, The vector comprises the isolated nucleic acid as described in claim 11.

13. The use according to claim 12, wherein, The vector is operatively linked to at least one regulatory element for the expression of an antigen-binding molecule or a chimeric receptor.

14. Use of cells in the preparation of the medicament as defined in any one of claims 1 to 10, wherein, The cells were obtained by transfection or transduction with the isolated nucleic acid as described in claim 11 or the vector as described in claim 12 or 13.

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