Antigen-binding molecules with improved cytoplasmic penetration activity

Abstract

The present invention provides an antigen-binding molecule (e.g., an antibody) having improved cytoplasmic entry ability, the molecule comprising an amino acid substitution in at least one selected from the group consisting of CDRL1, CDRL2, and CDRL3 in the light chain variable region, or an antigen-binding fragment thereof (antibody fragment); as well as a pharmaceutical composition comprising the antigen-binding molecule; a method for specifically delivering the antigen-binding molecule into the cytoplasm of a target cell; a method for removing, suppressing, or activating a cytoplasmic antigen specifically in a target cell using the antigen-binding molecule; and a pharmaceutical composition for diagnosing, preventing, or treating a disease in a subject, comprising the antigen-binding molecule.

Description

[Technical field]

[0001] The present disclosure relates to an antigen-binding molecule (e.g., an antibody) having improved cytoplasmic entry ability, and a cytoplasmic entry antigen-binding molecule (e.g., an antibody) comprising an amino acid mutation in at least one selected from the group consisting of CDRL1, CDRL2, and CDRL3 in the light chain variable region, or an antigen-binding fragment thereof (antibody fragment); as well as a pharmaceutical composition comprising the antigen-binding molecule; a method for specifically delivering the antigen-binding molecule into the cytoplasm of a target cell; a method for removing, suppressing, or activating a cytoplasmic antigen in a target cell-specific manner by using the antigen-binding molecule; and a pharmaceutical composition for diagnosing, preventing, or treating a disease in a subject, comprising the antigen-binding molecule. [Background technology]

[0002] Antibodies are proteins that bind specifically to antigens with high affinity. It is known that various molecules, from low molecular weight compounds to proteins, can act as antigens. Since the development of techniques for producing monoclonal antibodies, antibody engineering techniques have advanced, making it easier to obtain antibodies that recognize specific molecules. Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Antibodies not only bind to antigens, and have agonistic and antagonistic effects, but also induce cytotoxic activity (also called effector function) by effector cells, such as ADCC (Antibody Dependent Cytotoxicity), ADCP (Antibody Dependent Cell phagocytosis), and CDC (Complement Dependent Cytotoxicity). Utilizing such antibody functions, pharmaceuticals for cancer, immune diseases, chronic diseases, infectious diseases, and the like have been developed (Nat Rev Drug Discov. 2018 Mar;17(3):197-223. (Non-Patent Document 1)).

[0003] On the other hand, full-length IgG molecules have a large molecular weight of about 150 kDa and generally do not exhibit cell permeability, so the target antigens of antibody drugs are limited to antigens on the cell membrane or extracellular antigens. Therefore, intrabodies that express antibodies or antibody fragments within cells, antibody-CPP complexes fused with cell penetrating peptides (CPPs), and protein transfection methods have been developed and reported as techniques for acting antibodies on intracellular antigens (MAbs. 2011 Jan-Feb;3(1):3-16. (Non-Patent Document 2)).

[0004] It is also known that some natural full-length antibodies exhibit intracellular translocation ability. For example, it has been reported that anti-DNA autoantibodies identified from patients with systemic lupus erythematosus (SLE) and MRL-mpj / lpr lupus model mice exhibit intracellular translocation ability (Sci Rep. 2015 Jul 9; 5: 12022. (Non-Patent Document 3), Mol Immunol. 2015 Oct; 67 (2 Pt B): 377-87. (Non-Patent Document 4)). Some anti-DNA antibodies have not only cytoplasmic translocation ability, but also DNA hydrolysis activity and DNA repair inhibitory activity, and exhibit cytotoxicity activity (WO2012135831 (Patent Document 1), Sci Rep. 2014 Aug 5; 4: 5958 (Non-Patent Document 5)). In addition, an antibody has been reported in which an antibody isolated from an SLE model mouse, which has the ability to escape from endosomes to the cytoplasm, was humanized and then combined with a heavy chain variable region capable of binding to activated Ras (WO2016013871A1 (Patent Document 2)). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2012 / 135831 [Patent Document 2] WO2016 / 013871A1 [Non-patent literature]

[0006] [Non-Patent Document 1] Nat Rev Drug Discov. 2018 Mar;17(3):197-223. [Non-Patent Document 2] MAbs. 2011 Jan-Feb;3(1):3-16. [Non-Patent Document 3] Sci Rep. 2015 Jul 9;5:12022. [Non-Patent Document 4] Mol Immunol. 2015 Oct;67(2 Pt B):377-87. [Non-Patent Document 5] Sci Rep. 2014 Aug 5;4:5958 Summary of the Invention [Problem to be solved by the invention]

[0007] Most antibody therapeutics developed to date only target surface-exposed or secreted molecules, and therefore cannot target cytoplasmic molecules, and therefore there is a need to develop a method for delivering antibodies through cell membranes. In a non-limiting aspect, the object of the present invention is to provide an antigen-binding molecule (e.g., an antibody) that is efficiently and specifically delivered to the cytoplasm of a target cell, a pharmaceutical composition comprising the antigen-binding molecule, a method for using the antigen-binding molecule, a method for producing the antigen-binding molecule, and a method for specifically delivering an antigen-binding molecule into the cytoplasm of a target cell. [Means for solving the problem]

[0008] The present invention is derived from the above-mentioned need, and provides an antigen-binding molecule (e.g., an antibody) having improved cytoplasmic permeability, and confirms that the antigen-binding molecule or antibody has improved permeability to the cytoplasm. In a non-limiting embodiment, the present invention relates to an antigen-binding molecule (e.g., an antibody) or a fragment thereof having enhanced cytoplasmic penetration ability or activity, comprising CDRL1, CDRL2, and CDRL3 of the amino acid sequence shown in SEQ ID NO: 4, and comprising a light chain variable region containing one or more amino acid substitutions in at least one selected from CDRL1, CDRL2, and CDRL3, preferably CDRL1 and CDRL3.

[0009] In one non-limiting embodiment, the inventors have identified antigen-binding molecules (antibodies) that have improved or enhanced cytoplasmic penetration ability or activity while additionally having superior properties in terms of (1) antibody expression levels, (2) reduced extracellular matrix (ECM) binding profile (improving half-life or pharmacokinetics in vivo), and / or (3) better physicochemical properties as indicated by lower aggregation tendency.

[0010] In one aspect, the present disclosure provides: [1] A cytoplasmic penetration antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDRL1, CDRL2, and CDRL3 of the amino acid sequence set forth in SEQ ID NO:4, wherein the light chain variable region comprises one or more amino acid substitutions in at least one selected from CDRL1, CDRL2, and CDRL3. [2] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in [1], wherein the light chain variable region contains one or more amino acid substitutions at positions 24 to 34 and 89 to 97 according to the Kabat numbering in the amino acid sequence shown in SEQ ID NO:4. [3] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in [2], wherein the light chain variable region contains one or more amino acid substitutions at positions 24 to 32 and 93 to 97 of the Kabat numbering in the amino acid sequence shown in SEQ ID NO:4. [4] The cytoplasmic penetration antibody or antigen-binding fragment thereof of [3], wherein the one or more amino acid substitutions are selected from the group consisting of the following (positions according to Kabat numbering): In the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid K at position 24 with A, D, E, F, G, H, I, L, N, Q, R, S, T, V, or Y; (b) substitution of amino acid S at position 25 with A, D, E, F, I, M, N, T, V, or Y; (c) substitution of amino acid S at position 26 with A, D, E, F, G, H, I, K, M, N, P, R, T, or V; (d) substitution of amino acid Q at position 27 with A, D, E, F, H, I, L, M, S, or Y; (e) substitution of amino acid S at position 27a with D; (f) substitution of amino acid L at position 27b with I, P, Q, T, or V; (g) substitution of amino acid F at position 27c with A, D, E, I, M, N, S, W, or Y; (h) substitution of amino acid N at position 27d with A, D, E, F, H, N, or Q; (i) substitution of amino acid S at position 27e with A, D, E, I, L, M, N, P, Q, T, V, or Y; (j) substitution of amino acid R at position 27f with A, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y; (k) substitution of amino acid T at position 28 with E, H, I, Q, S, V, W, or Y; (l) substitution of amino acid R at position 29 with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; (m) substitution of amino acid K at position 30 with A, D, E, F, H, I, L, M, N, P, Q, S, T, V, W, or Y; (n) substitution of amino acid N at position 31 with D, M, S, or T; (o) substitution of amino acid Y at position 32 with D, E, G, H, or Q; (p) substitution of amino acid Q at position 89 with H, I, T, or Y; (q) substitution of amino acid Y at position 91 with E, F, or N; (r) substitution of amino acid Y at position 93 with I, L, M, N, P, Q, S, T, V, or W; (s) substitution of amino acid H at position 94 with A, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, or Y; (t) substitution of amino acid M at position 95 with A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; (u) substitution of amino acid Y at position 96 with A, D, E, F, G, H, K, M, N, P, Q, S, T, or V; and (v) Substitution of amino acid T at position 97 with A, D, E, G, H, K, P, Q, R, S, or V. [4A] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in [4], wherein each of CDRL1 and CDRL3 in the light chain variable region contains two or more amino acid substitutions selected from the group defined in [4](a) to (v). [5] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in any one of [1] to [4A], wherein the amino acid substitution enhances the cytoplasmic penetration activity of the antibody or fragment thereof compared to a reference antibody or fragment thereof that does not contain the substitution and comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:4. [5A] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in any one of [1] to [5], which exhibits at least 1%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500%, or 1000% enhanced cytoplasmic penetration activity compared to a reference antibody or fragment thereof comprising a light chain variable region that does not contain the substitution and has the amino acid sequence shown in SEQ ID NO:4, preferably when determined using the Split Nluc assay described in Reference Example 1. [5B] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in any one of [1] to [5A], wherein the cytoplasmic penetration activity is mediated by penetration of the antibody or its fragment into the cell membrane and / or endosomal escape after penetration of the antibody or its fragment into the cell membrane. [5C] The cytoplasmic penetration antibody or antigen-binding fragment thereof according to any one of [1] to [5B], which exhibits one or more characteristics compared to a reference antibody, and the one or more characteristics are selected from the group consisting of: (a) increased antibody expression levels compared to a reference antibody; (b) a reduced binding activity to extracellular matrix (ECM) compared to a reference antibody, preferably determined by the method described in Reference Example 2; and (c) improved physicochemical properties compared to a reference antibody, preferably as indicated by a higher monomer peak percentage as determined by size exclusion chromatography (SEC) analysis. [6] The cytoplasmic penetration antibody or antigen-binding fragment thereof according to any one of [1] to [5C], wherein the light chain variable region contains one, two, three or more amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence shown in SEQ ID NO: 4, (a) Substitution of amino acid K at position 24 with N; (b) substitution of amino acid Q at position 27 with E; (c) substitution of amino acid S at position 27a with D; (d) substitution of amino acid N at position 27d with D or E; (e) substitution of amino acid S at position 27e with D or E; (f) substitution of amino acid R at position 27f with D, H, K, L, S, or V; (g) substitution of amino acid T at position 28 with D; (h) substitution of amino acid R at position 29 with E, G, M, or S; (i) substitution of amino acid K at position 30 with L or Q; (j) substitution of amino acid Y at position 32 with E; (k) substitution of amino acid Q at position 89 with H, I, T, or Y; (l) substitution of amino acid Y at position 91 with E, F, or N; (m) substitution of amino acid Y at position 93 with E; (n) substitution of amino acid H at position 94 with E, P, or Q; (o) substitution of amino acid M at position 95 with D, N, or P; and (p) Substitution of amino acid Y at position 96 with E or P. [6A] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in [6], wherein each of CDRL1 and CDRL3 in the light chain variable region contains two or more amino acid substitutions selected from the group defined in [6](a) to (p). [7] The cytoplasmic penetration antibody or antigen-binding fragment thereof of [6], wherein the light chain variable region comprises a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: (a) S27eD / R27fK; (b) S27eD / R27fH; (c) N27dE / S27eD; (d) S27eD / T28D; (e) S27eD / R29E; (f) S27eD / R29S; (g) K24N / S27eD; (h) N27dD / R27fK; (i) N27dE / R27fK; (j) R27fK / T28D; (k) R27fK / R29E; (l) R27fK / R29S; (m) K24N / R27fK; (n) K24N / N27dD; (o) N27dD / T28D; (p)N27dD / R29E; (q) N27dD / R29S; (r) S27eD / Q89H; (s) N27dD / Q89H; (t) R27fK / Q89H; (u) S27eD / Y91E; (v) N27dD / Y91E; (w) R27fK / Y91E; (x) Q89H / H94P; (y) Y91E / H94P; (z) H94P / M95N; (aa) H94P / M95D; (bb) H94P / M95P; (cc) H94E / Y96P; (dd) H94E / M95N; (ee) H94E / M95D; (ff) H94E / M95P; (gg) N27dE / R27fK / Q89H / H94E / M95P; (hh) N27dE / R27fK / Q89Y / H94E / M95P; (ii) N27dE / R27fK / Y91E / H94E / M95P; (jj) N27dE / R27fK / Y91F / H94E / M95P; (kk) N27dE / R27fS / Q89H / H94E / M95P; (ll) N27dE / R27fS / Q89Y / H94E / M95P; (mm) N27dE / R27fS / Y91E / H94E / M95P; (nn) N27dE / R27fS / Y91F / H94E / M95P; (oo) N27dE / R27fS / Q89H / H94Q / M95P; (pp) N27dE / R27fS / Q89Y / H94Q / M95P; (qq) N27dE / R27fS / Y91E / H94Q / M95P; (rr) N27dE / R27fS / Y91F / H94Q / M95P; (ss) N27dE / R27fV / Q89H / H94E / M95P; (tt) N27dE / R27fV / Q89Y / H94E / M95P; (uu) N27dE / R27fV / Y91E / H94E / M95P; (vv) N27dE / R27fV / Y91F / H94E / M95P; (ww) N27dE / R29S / Q89H / H94E / M95P; (xx) N27dE / R29S / Q89Y / H94E / M95P; (yy) N27dE / K30L / Q89H / H94E / M95P; (zz) N27dE / K30L / Q89Y / H94E / M95P; (aaa) S27eD / R27fK / Y91E / H94E / M95P; (bbb) S27eD / R27fK / Y91F / H94E / M95P; (ccc) S27eD / R27fK / Y91E / H94Q / M95P; (ddd) S27eD / R27fK / Y91F / H94Q / M95P; (eee) S27eD / R27fK / Y91E / H94Q; (fff) S27eD / R27fK / Y91F / H94Q; (ggg) S27eE / R27fK / Y91E / H94E / M95P; (hhh) S27eE / R27fK / Y91F / H94E / M95P; (iii) S27eE / R27fK / Y91E / H94Q / M95P; (yyy) S27eE / R27fK / Y91F / H94Q / M95P; (kkk) R27fS / R29S / Q89H / H94E / M95P; (lll) R27fS / R29S / Q89Y / H94E / M95P; (mmm) R27fS / R29S / Y91E / H94E / M95P; (nnn) R27fS / R29S / Y91F / H94E / M95P; (ooo) R27fS / R29S / Q89H / H94Q / M95P; (ppp) R27fS / R29S / Q89Y / H94Q / M95P; (qqq) R27fS / R29S / Y91E / H94Q / M95P; (rrr) R27fS / R29S / Y91F / H94Q / M95P; (sss) N27dE / R27fS / R29S / Q89Y / H94Q / M95P;および (ttt) N27dE / R27fS / R29S / Y91F / H94Q / M95P. [8] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in any one of [1] to [7], wherein the light chain variable region contains a human framework region (FR). [8A] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in [8], wherein the light chain variable region contains amino acid I at position 2 and / or amino acid Q at position 3 according to the Kabat numbering system. [8B] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in [8], wherein the light chain variable region comprises a human Vκ1 FR1 sequence. [8C] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in [8], wherein the light chain variable region comprises a human Vκ3 FR3 sequence. [8D] The cytoplasmic penetration antibody or antigen-binding fragment thereof described in [8], wherein the light chain variable region comprises human Vκ1 FR1, human Vκ1 FR2, and human Vκ3 FR3 sequences. [8E] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in [8], wherein the light chain variable region includes one, two, three, or four of the FR domains (FR1, FR2, FR3, and FR4) included in SEQ ID NO: 123. [8F] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in any one of [2] to [8E], wherein the light chain variable region further contains one, two, three or more amino acid substitutions in CDRL2. [9] The cytoplasmic penetration antibody or antigen-binding fragment thereof according to any one of [1] to [8F], wherein the light chain variable region further comprises one, two, three or more amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In CDRL2 of the amino acid sequence shown in SEQ ID NO: 4 (a) substitution of amino acid W at position 50 with A, G, I, T, or V; (b) substitution of amino acid S at position 52 with F or I; (c) substitution of amino acid T at position 53 with N or Y; and (d) Substitution of amino acid R at position 54 with K or V.

[10] The cytoplasmic penetration antibody or antigen-binding fragment thereof of [9], wherein the light chain variable region comprises a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence of SEQ ID NO:4 (a) L2I / V3Q / S27eD / R27fK / W50A / Y91F / H94Q; (b) L2I / V3Q / S27eD / R27fK / W50G / Y91F / H94Q; (c) L2I / V3Q / S27eD / R27fK / W50I / Y91F / H94Q; (d) L2I / V3Q / S27eD / R27fK / W50T / Y91F / H94Q; (e) L2I / V3Q / S27eD / R27fK / W50V / Y91F / H94Q; (f) L2I / V3Q / S27eD / R27fK / S52F / Y91F / H94Q; (g) L2I / V3Q / S27eD / R27fK / S52I / Y91F / H94Q; (h) L2I / V3Q / S27eD / R27fK / T53N / Y91F / H94Q; (i) L2I / V3Q / S27eD / R27fK / T53Y / Y91F / H94Q; (j) L2I / V3Q / S27eD / R27fK / R54K / Y91F / H94Q; (k) L2I / V3Q / S27eD / R27fK / W50I / S52F / Y91F / H94Q; (l) L2I / V3Q / S27eD / R27fK / W50I / T53N / Y91F / H94Q; and (m) L2I / V3Q / S27eD / R27fK / W50I / R54V / Y91F / H94Q. [11-1] A cytoplasmic penetrating antibody or an antigen-binding fragment thereof described in any one of [1] to

[10] , which comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 150 and 161 to 173. [11-2] A cytoplasmic penetration antibody or an antigen-binding fragment thereof described in [11-1], which comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 123 to 150. [11-3] A cytoplasmic penetration antibody or an antigen-binding fragment thereof described in [11-1], which comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161 to 173. [11A] A cytoplasmic penetration antibody or an antigen-binding fragment thereof described in any one of [1] to [11-3], which is in the form of Fab or scFv. [11B] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in any one of [1] to [11A], further comprising a heavy chain variable region, optionally comprising the amino acid sequence of SEQ ID NO: 158. [11C] A cytoplasmic penetrating antibody or an antigen-binding fragment thereof described in any one of [1] to [11B], which is a multifunctional antibody. [11D] A cytoplasmic penetration antibody or antigen-binding fragment thereof described in [11C], which comprises a cell surface antigen-binding domain, a cytoplasmic antigen-binding domain, and a cytoplasmic penetration domain, wherein the cytoplasmic penetration domain comprises a light chain variable region described in any one of [1] to [11A].

[12] A nucleic acid encoding a cytoplasmic penetration antibody or an antigen-binding fragment thereof described in any one of [1] to [11D].

[13] A vector comprising the nucleic acid described in

[12] .

[14] A host cell comprising the nucleic acid described in

[12] or the vector described in

[13] .

[15] A method for producing a cytoplasmic penetrating antibody or antigen-binding fragment thereof described in any one of [1] to [11D], comprising the steps of: a) culturing a host cell described in

[14] under conditions suitable for expression of the antibody or fragment thereof; and b) recovering the antibody or fragment thereof.

[16] A composition or pharmaceutical composition for delivering a biologically active molecule into the cytoplasm, comprising a cytoplasmic penetrating antibody or an antigen-binding fragment thereof described in any one of [1] to [11D].

[17] The composition or pharmaceutical composition described in

[16] , wherein the biologically active molecule is at least one selected from the group consisting of a peptide, a protein, a toxin, an antibody, an antibody fragment, RNA, DNA, a small molecule drug, a nanoparticle, and a liposome; and the biologically active molecule is conjugated or fused to the cytoplasm-penetrating antibody or antigen-binding fragment.

[18] An immunoconjugate of a cytoplasmic penetrating antibody or an antigen-binding fragment thereof described in any one of [1] to [11D] and a biologically active molecule.

[19] The immunoconjugate according to

[18] , wherein the physiologically active molecule is selected from the group consisting of a peptide, a protein, a toxin, an antibody, an antibody fragment, RNA, DNA, a small molecule drug, a nanoparticle, and a liposome.

[20] A method for delivering a cytoplasmic penetrating antibody or antigen-binding fragment thereof described in any one of [1] to [11D], or an immunoconjugate described in

[18] or

[19] , through a cell membrane and placing it in the cytoplasm.

[21] A method for targeting, inhibiting or activating a cytoplasmic antigen by using a cytoplasmic penetrating antibody or antigen-binding fragment thereof described in any one of [1] to [11D], wherein the antibody or fragment thereof specifically binds to the cytoplasmic antigen. [21A] A method for imaging a cytoplasmic antigen in a sample cell, comprising contacting the sample cell with a cytoplasmic penetrating antibody or antigen-binding fragment thereof described in any one of [1] to [11D], wherein the antibody or fragment thereof is labeled and specifically binds to the cytoplasmic antigen.

[22] A method for producing a cytoplasmic penetrating antibody or antigen-binding fragment thereof, comprising: (a) providing a parent cytoplasmic penetration antigen binding molecule comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO:4; (b) introducing one or more amino acid substitutions in at least one selected from CDRL1 and CDRL3 in the light chain variable region of the parent cytoplasmic entry antigen binding molecule; and (c) identifying and / or selecting a cytoplasmic entry antibody or antigen-binding fragment thereof comprising said one or more amino acid substitutions, said antibody or antigen-binding fragment exhibiting enhanced cytoplasmic entry ability compared to said parent cytoplasmic entry antigen-binding molecule. [22A] The method of

[22] , further comprising identifying and / or selecting a cytoplasmic penetration antibody or antigen-binding fragment thereof that exhibits one or more properties compared to a parent antibody, wherein the one or more properties are selected from the group consisting of: (a) Increased antibody expression levels; (b) reduced binding activity to extracellular matrix (ECM), preferably as determined by the method described in Reference Example 2; and (c) improved physicochemical properties, preferably as indicated by a higher monomer peak percentage, as determined by size exclusion chromatography (SEC) analysis.

[23] The method according to any one of

[22] to [22A], further comprising: (d) obtaining an expression vector comprising a gene encoding the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced by the method of

[22] and a suitable promoter operably linked thereto; (e) introducing the vector into a host cell and culturing the host cell to produce the cytoplasmic penetrating antibody or antigen-binding fragment thereof; and (f) recovering said cytoplasmic penetrating antibody or antigen-binding fragment thereof from the host cell culture.

[24] The method according to any one of

[22] to

[23] , wherein the one or more amino acid substitutions are located at positions 24 to 34 and positions 89 to 97 according to the Kabat numbering in the amino acid sequence shown in SEQ ID NO:4.

[25] The method according to

[24] , wherein the one or more amino acid substitutions are located at positions 24 to 32 and 93 to 97 according to the Kabat numbering in the amino acid sequence shown in SEQ ID NO:4.

[26] The method according to any one of

[22] to

[25] , wherein the one or more amino acid substitutions are selected from the group consisting of the following (positions according to Kabat numbering): In the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid K at position 24 with A, D, E, F, G, H, I, L, N, Q, R, S, T, V, or Y; (b) substitution of amino acid S at position 25 with A, D, E, F, I, M, N, T, V, or Y; (c) substitution of amino acid S at position 26 with A, D, E, F, G, H, I, K, M, N, P, R, T, or V; (d) substitution of amino acid Q at position 27 with A, D, E, F, H, I, L, M, S, or Y; (e) substitution of amino acid S at position 27a with D; (f) substitution of amino acid L at position 27b with I, P, Q, T, or V; (g) substitution of amino acid F at position 27c with A, D, E, I, M, N, S, W, or Y; (h) substitution of amino acid N at position 27d with A, D, E, F, H, N, or Q; (i) substitution of amino acid S at position 27e with A, D, E, I, L, M, N, P, Q, T, V, or Y; (j) substitution of amino acid R at position 27f with A, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y; (k) substitution of amino acid T at position 28 with E, H, I, Q, S, V, W, or Y; (l) substitution of amino acid R at position 29 with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; (m) substitution of amino acid K at position 30 with A, D, E, F, H, I, L, M, N, P, Q, S, T, V, W, or Y; (n) substitution of amino acid N at position 31 with D, M, S, or T; (o) substitution of amino acid Y at position 32 with D, E, G, H, or Q; (p) substitution of amino acid Q at position 89 with H, I, T, or Y; (q) substitution of amino acid Y at position 91 with E, F, or N; (r) substitution of amino acid Y at position 93 with I, L, M, N, P, Q, S, T, V, or W; (s) substitution of amino acid H at position 94 with A, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, or Y; (t) substitution of amino acid M at position 95 with A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; (u) substitution of amino acid Y at position 96 with A, D, E, F, G, H, K, M, N, P, Q, S, T, or V; and (v) Substitution of amino acid T at position 97 with A, D, E, G, H, K, P, Q, R, S, or V. [26A] The method according to

[26] , wherein each of CDRL1 and CDRL3 in the light chain variable region contains two or more amino acid substitutions selected from the group defined in

[26] (a) to (v).

[27] The method described in any one of

[22] to

[26] , wherein the one or more amino acid substitutions enhance the cytoplasmic penetration activity of the antibody or fragment thereof compared to a reference antibody or fragment thereof that does not contain the substitutions and comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:4.

[28] The method according to any one of

[22] to

[27] , wherein the one or more amino acid substitutions are selected from the group consisting of the following (positions according to Kabat numbering): In the amino acid sequence shown in SEQ ID NO: 4, (a) Substitution of amino acid K at position 24 with N; (b) substitution of amino acid Q at position 27 with E; (c) substitution of amino acid S at position 27a with D; (d) substitution of amino acid N at position 27d with D or E; (e) substitution of amino acid S at position 27e with D or E; (f) substitution of amino acid R at position 27f with D, H, K, L, S, or V; (g) substitution of amino acid T at position 28 with D; (h) substitution of amino acid R at position 29 with E, G, M, or S; (i) substitution of amino acid K at position 30 with L or Q; (j) substitution of amino acid Y at position 32 with E; (k) substitution of amino acid Q at position 89 with H, I, T, or Y; (l) substitution of amino acid Y at position 91 with E, F, or N; (m) substitution of amino acid Y at position 93 with E; (n) substitution of amino acid H at position 94 with E, P, or Q; (o) substitution of amino acid M at position 95 with D, N, or P; and (p) Substitution of amino acid Y at position 96 with E or P. [28A] The method according to

[28] , wherein each of CDRL1 and CDRL3 in the light chain variable region contains two or more amino acid substitutions selected from the group defined in

[28] (a) to (p).

[29] The method according to any one of

[22] to

[28] , wherein the one or more amino acid substitutions include a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: (a) S27eD / R27fK; (b) S27eD / R27fH; (c) N27dE / S27eD; (d) S27eD / T28D; (e) S27eD / R29E; (f) S27eD / R29S; (g) K24N / S27eD; (h) N27dD / R27fK; (i) N27dE / R27fK; (j) R27fK / T28D; (k) R27fK / R29E; (l) R27fK / R29S; (m) K24N / R27fK; (n) K24N / N27dD; (o) N27dD / T28D; (p)N27dD / R29E; (q) N27dD / R29S; (r) S27eD / Q89H (s) N27dD / Q89H; (t) R27fK / Q89H; (u) S27eD / Y91E; (v) N27dD / Y91E; (w) R27fK / Y91E; (x) Q89H / H94P; (y) Y91E / H94P; (z) H94P / M95N; (aa) H94P / M95D; (bb) H94P / M95P; (cc) H94E / Y96P; (dd) H94E / M95N; (ee) H94E / M95D; (ff) H94E / M95P; (gg) N27dE / R27fK / Q89H / H94E / M95P; (hh) N27dE / R27fK / Q89Y / H94E / M95P; (ii) N27dE / R27fK / Y91E / H94E / M95P; (jj) N27dE / R27fK / Y91F / H94E / M95P; (kk) N27dE / R27fS / Q89H / H94E / M95P; (ll) N27dE / R27fS / Q89Y / H94E / M95P; (mm) N27dE / R27fS / Y91E / H94E / M95P; (nn) N27dE / R27fS / Y91F / H94E / M95P; (oo) N27dE / R27fS / Q89H / H94Q / M95P; (pp) N27dE / R27fS / Q89Y / H94Q / M95P; (qq) N27dE / R27fS / Y91E / H94Q / M95P; (rr) N27dE / R27fS / Y91F / H94Q / M95P; (ss) N27dE / R27fV / Q89H / H94E / M95P; (tt) N27dE / R27fV / Q89Y / H94E / M95P; (uu) N27dE / R27fV / Y91E / H94E / M95P; (vv) N27dE / R27fV / Y91F / H94E / M95P; (ww) N27dE / R29S / Q89H / H94E / M95P; (xx) N27dE / R29S / Q89Y / H94E / M95P; (yy) N27dE / K30L / Q89H / H94E / M95P; (zz) N27dE / K30L / Q89Y / H94E / M95P; (aaa) S27eD / R27fK / Y91E / H94E / M95P; (bbb) S27eD / R27fK / Y91F / H94E / M95P; (ccc) S27eD / R27fK / Y91E / H94Q / M95P; (ddd) S27eD / R27fK / Y91F / H94Q / M95P; (eee) S27eD / R27fK / Y91E / H94Q; (fff) S27eD / R27fK / Y91F / H94Q; (ggg) S27eE / R27fK / Y91E / H94E / M95P; (hhh) S27eE / R27fK / Y91F / H94E / M95P; (iii) S27eE / R27fK / Y91E / H94Q / M95P; (jjj) S27eE / R27fK / Y91F / H94Q / M95P; (kkk) R27fS / R29S / Q89H / H94E / M95P (lll) R27fS / R29S / Q89Y / H94E / M95P; (mmm) R27fS / R29S / Y91E / H94E / M95P; (nnn) R27fS / R29S / Y91F / H94E / M95P; (ooo) R27fS / R29S / Q89H / H94Q / M95P; (ppp) R27fS / R29S / Q89Y / H94Q / M95P; (qqq) R27fS / R29S / Y91E / H94Q / M95P; (rrr) R27fS / R29S / Y91F / H94Q / M95P; (sss) N27dE / R27fS / R29S / Q89Y / H94Q / M95P; and (ttt) N27dE / R27fS / R29S / Y91F / H94Q / M95P.

[30] The method according to any one of

[22] to

[29] , further comprising replacing the framework region (FR) of the light chain variable region with a human framework region (FR). [30A] The method according to

[30] , wherein the light chain variable region comprises amino acid I at position 2 and / or amino acid Q at position 3 according to the Kabat numbering system. [30B] The method according to

[30] , wherein the light chain variable region comprises a human Vκ1 FR1 sequence. [30C] The method according to

[30] , wherein the light chain variable region comprises a human Vκ3 FR3 sequence. [30D] The method according to

[30] , wherein the light chain variable region comprises human Vκ1 FR1, human Vκ1 FR2, and human Vκ3 FR3 sequences. [30E] The method according to

[30] , wherein the light chain variable region comprises one, two, three, or four of the FR domains (FR1, FR2, FR3, and FR4) contained in SEQ ID NO: 123. [30F] The method described in any one of

[22] to

[30] , further comprising introducing one, two, three or more amino acid substitutions into CDRL2 of the light chain variable region.

[31] The method according to any one of

[22] to [30F], further comprising introducing one, two, three or more amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In CDRL2 of the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid W at position 50 with A, G, I, T, or V; (b) substitution of amino acid S at position 52 with F or I; (c) substitution of amino acid T at position 53 with N or Y; and (d) Substitution of amino acid R at position 54 with K or V.

[32] The method according to any one of

[22] to

[31] , wherein the amino acid substitutions introduced into the light chain variable region include a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence shown in SEQ ID NO: 4, (a) L2I / V3Q / S27eD / R27fK / W50A / Y91F / H94Q; (b) L2I / V3Q / S27eD / R27fK / W50G / Y91F / H94Q; (c) L2I / V3Q / S27eD / R27fK / W50I / Y91F / H94Q; (d) L2I / V3Q / S27eD / R27fK / W50T / Y91F / H94Q; (e) L2I / V3Q / S27eD / R27fK / W50V / Y91F / H94Q; (f) L2I / V3Q / S27eD / R27fK / S52F / Y91F / H94Q; (g) L2I / V3Q / S27eD / R27fK / S52I / Y91F / H94Q; (h) L2I / V3Q / S27eD / R27fK / T53N / Y91F / H94Q; (i) L2I / V3Q / S27eD / R27fK / T53Y / Y91F / H94Q; (j) L2I / V3Q / S27eD / R27fK / R54K / Y91F / H94Q; (k) L2I / V3Q / S27eD / R27fK / W50I / S52F / Y91F / H94Q; (l) L2I / V3Q / S27eD / R27fK / W50I / T53N / Y91F / H94Q; and (m) L2I / V3Q / S27eD / R27fK / W50I / R54V / Y91F / H94Q. [33-1] The method described in any one of

[22] to

[32] , wherein the cytoplasmic penetrating antibody or its antigen-binding fragment comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 150 and 161 to 173. [33-2] The method described in [33-1], wherein the cytoplasmic penetrating antibody or its antigen-binding fragment comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 123 to 150. [33-3] The method described in [33-1], wherein the cytoplasmic penetrating antibody or its antigen-binding fragment comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161 to 173.

[34] The method described in any one of

[22] to [33-3], wherein the cytoplasmic penetrating antibody or its antigen-binding fragment is in the form of Fab or scFv.

[35] The method described in any one of

[22] to [33-3], wherein the cytoplasmic penetrating antibody or its antigen-binding fragment further comprises a heavy chain variable region, optionally comprising the amino acid sequence of SEQ ID NO: 158.

[0011] In another non-limiting embodiment, the present invention relates to a multifunctional antigen-binding molecule (antibody or antigen-binding fragment thereof) that comprises a cytoplasmic entry domain, a cell surface antigen-binding domain, and / or a cytoplasmic antigen-binding domain in a combination that can be delivered into the cytoplasm in a target cell-specific manner. In a non-limiting embodiment, each domain is derived from an antibody. In a non-limiting embodiment, these antigen-binding molecules are expected to bind to cell surface antigens, thereby internalizing the target cell specifically and translocating to the cytoplasm.

[0012] In particular, the present disclosure encompasses embodiments of bifunctional or multifunctional antigen binding molecules, e.g., as described below.

[36] An antigen-binding molecule comprising a cell surface antigen-binding domain, a cytoplasmic antigen-binding domain, and a cytoplasmic penetration domain, wherein the cytoplasmic penetration domain comprises a light chain variable region according to any one of [1] to [11-3]. [36A] The antigen-binding molecule according to

[36] , which does not bind to an antigen expressed on the cell surface that is different from the cell surface antigen. [36B] The antigen-binding molecule according to

[36] , wherein the cytoplasmic antigen-binding domain and the cytoplasmic penetration domain do not bind to an antigen that is different from the cell surface antigen and is expressed on the cell surface. [36C] The antigen-binding molecule of

[36] or [36B], wherein the cytoplasmic antigen-binding domain and the cytoplasmic penetration domain do not bind to an antigen expressed on the cell surface. [36D] The antigen-binding molecule according to any one of

[36] to [36C], which is a multifunctional antibody or a multifunctional antibody derivative.

[37] An antigen-binding molecule comprising a first and a second Fab region, (a) a first Fab region that specifically binds to a cell surface antigen; and (b) the second Fab region (i) comprises a pair of a heavy chain variable region (VH) that specifically binds to a cytoplasmic antigen and a light chain variable region (VL) according to any one of [1] to [11-3]; Antigen-binding molecules.

[38] An antigen-binding molecule comprising a first and a second Fab region, and a single chain unit, (a) a first Fab region that specifically binds to a cell surface antigen; (b) the second Fab region has the ability to enter the cytoplasm; (c) the single chain unit specifically binds to a cytoplasmic antigen; and The second Fab region comprises a light chain variable region (VL) according to any one of [1] to [11-3]. Antigen-binding molecules.

[39] An antigen-binding molecule comprising a first and a second Fab region, and a single chain unit, (a) a first Fab region that specifically binds to a cytoplasmic antigen; (b) the second Fab region has the ability to enter the cytoplasm; (c) the single chain unit specifically binds to a cell surface antigen; and The second Fab region comprises a light chain variable region (VL) according to any one of [1] to [11-3]. Antigen-binding molecules.

[40] An antigen-binding molecule comprising a first and a second Fab region, and a single chain unit, (a) the first and second Fab regions each include (i) a pair of a heavy chain variable region (VH) that specifically binds to a cell surface antigen and a light chain variable region (VL) according to any one of [1] to [11-3]; and (b) the single chain unit specifically binds to a cytoplasmic antigen; Antigen-binding molecules.

[41] An antigen-binding molecule comprising a first, second, third, and fourth Fab region and an Fc region, (a) one Fab region selected from the first, second, third, and fourth Fab regions specifically binds to a cell surface antigen; (b) the three Fab regions other than (a) each comprise a pair of (i) a heavy chain variable region that specifically binds to a cytoplasmic antigen and a light chain variable region according to any one of [1] to [11-3]; (c) the Fc region comprises a first Fc subunit and a second Fc subunit; (d) the C-terminus of the heavy chain of a first Fab region is fused to the N-terminus of a first Fc subunit, the C-terminus of the heavy chain of a second Fab region is fused to the N-terminus of a second Fc subunit, the C-terminus of the heavy chain of a third Fab region is fused to the N-terminus of the heavy chain of the first Fab region, and the C-terminus of the heavy chain of a fourth Fab region is fused to the N-terminus of the heavy chain of the second Fab region. Antigen-binding molecules.

[42] An antigen-binding molecule comprising the light chain variable region and an Fc region according to any one of [1] to [11-3], wherein the Fc region comprises one or more amino acid modifications for promoting multimerization of the antigen-binding molecule, and the antigen-binding molecule has an increased ability to enter the cytoplasm as compared to an antigen-binding molecule comprising a parent Fc region not comprising the one or more amino acid modifications.

[43] An antigen-binding molecule comprising a first and second Fab region and an Fc region, (a) a first Fab region that specifically binds to a cell surface antigen; (b) the second Fab region comprises (i) a pair of a heavy chain variable region (VH) that specifically binds to a cytoplasmic antigen and a light chain variable region (VL) according to any one of [1] to

[11] ; and (c) the Fc region comprises one or more amino acid modifications to promote multimerization of the antigen-binding molecule; Antigen-binding molecules.

[44] The antigen-binding molecule according to any one of

[37] to

[43] , wherein the Fab region comprising the light chain variable region according to any one of [1] to [11-3] does not bind to an antigen expressed on a cell surface that is different from the cell surface antigen.

[45] The antigen-binding molecule according to any one of

[37] to

[44] , wherein the cell surface antigen is an antigen specifically expressed in a target cell, and the antigen-binding molecule is specifically delivered into the cytoplasm of the target cell.

[46] A pharmaceutical composition comprising the antigen-binding molecule according to any one of

[37] to

[45] and a pharma- ceutical acceptable carrier.

[47] A method for specifically delivering the antigen-binding molecule according to any one of

[37] to

[45] into the cytoplasm of a target cell, wherein the cell surface antigen is an antigen specifically expressed in the target cell.

[48] ​​A method for removing, suppressing, or activating a cytoplasmic antigen in a target cell-specific manner by using the antigen-binding molecule according to any one of

[37] to

[45] , wherein the cell surface antigen is an antigen that is specifically expressed in the target cell.

[49] A pharmaceutical composition for diagnosing, preventing, or treating a disease in a target, wherein diseased cells express a cell surface antigen and a cytoplasmic antigen to which the antigen-binding molecule of any one of

[37] to

[45] specifically binds, and the pharmaceutical composition comprises the antigen-binding molecule of any one of

[37] to

[45] and a pharmaceutical acceptable carrier. [Brief description of the drawings]

[0013] [Figure 1]Size exclusion chromatography (SEC) chromatograms of representative hT4VL.FRv04 mutants are shown. High monomer peak percentage and sharp symmetric peak shape (narrow peak width) indicate good physicochemical properties. All mutants with the mutations listed in Table 6 showed better monomer peak percentage and sharper peak shape, as shown in Figure 1. [Diagram 2] The results of imaging analysis of the cytoplasmic transport of bifunctional antibodies in ASGPR-expressing HeLa cell line (HeLa / ASGPR / BirA cells) are shown. The selected antibodies in bifunctional molecule format with anti-ASGPR antibody (AGA0078) showed higher Alexa488 fluorescence intensity than the antibodies in bifunctional molecule format with anti-KLH antibody (IC17=control). This indicates that the selected bifunctional antibodies can be delivered into HeLa / ASGPR / BirA cells in an ASGPR-dependent manner, supporting the receptor-dependent cytoplasmic transport observed by Split-Nanoluc assay (ASGPR-expressing cell-specific cytoplasmic transport mediated by ASGPR Ab). [Diagram 3] 1 shows the results of a Split Nluc assay for cytoplasmic transport of a bifunctional antibody comprising an hT4VL mutant and an anti-IL6R heavy chain in HepG2 cells that endogenously express IL6R on the cell surface. [Figure 4] 4 is a schematic diagram showing the concept of the antigen-binding molecule of the present disclosure, in which an antigen-binding molecule in the form of a bifunctional antibody is shown as an example to demonstrate the concept that the antigen-binding molecule of the present disclosure is delivered specifically to target cells. [Diagram 5] FIG. 1 is a schematic diagram showing the molecular form of a multivalent antibody as an example of the molecular form of an antigen-binding molecule of the present disclosure. [Figure 6] 1 is a schematic diagram showing an exemplary molecular shape of an antigen-binding molecule of the present disclosure, which comprises a first and a second Fab region. [Figure 7] 1 is a schematic diagram showing an exemplary molecular form of an antigen-binding molecule of the present disclosure, the antigen-binding molecule comprising a first and a second Fab region, and a single-chain unit. [Figure 8] 1 is a schematic diagram showing an exemplary molecular form of an antigen-binding molecule of the present disclosure, which comprises a first and a second single domain antibody variable region, and a single chain unit. [Figure 9] 1 is a schematic diagram showing an exemplary molecular shape of an antigen-binding molecule of the present disclosure, which has the molecular shape of DVD-Ig (registered trademark). [Figure 10] 1 is a schematic diagram showing an exemplary molecular shape of an antigen-binding molecule of the present disclosure, which comprises a first, second and third Fab region. [Figure 11] 1 is a schematic diagram showing an exemplary molecular shape of an antigen-binding molecule of the present disclosure, which comprises a first, second, third and fourth Fab region. [Figure 12] A comparison of the PK of the optimized antibody variant, 3D8H / hT4VL.FRv40563, and the parental 3D8 antibody (3D8H / hT4VL), which contains the same wild-type human IgG1 constant region, is shown. [Figure 13] PK of a bifunctional antibody containing a cytoplasmic entry domain (FRv40563) and a cell surface antigen binding domain (anti-ASGPR antibody, AGA0078) is shown. FRv40563n / / AGA0078p showed faster clearance compared to its control antibody FRv40563n / / IC17p, suggesting that the bifunctional antibody FRv40563n / / AGA0078p can be delivered to the liver in an ASGPR-dependent manner. In addition, FRv40563n / / AGA0078p showed faster clearance than IC17n / / AGA0078p. This could support ASGPR-dependent cytoplasmic transport in vivo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] I. Definition An "acceptor human framework" for the purposes of this specification is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0015] The term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, an antigen-binding fragment of an antibody, or an antibody derivative.

[0016] "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (KD) or "amount of analyte bound per unit amount of ligand". Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0017] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0018] "Binding activity" refers to the strength of the total non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and the binding partner of the molecule (e.g., an antigen). Here, "binding activity" is not strictly limited to 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). For example, when members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of these binding forces. The binding activity of a molecule X to its partner Y can generally be expressed by the dissociation constant (KD) or "amount of analyte bound per unit amount of ligand". Binding activity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0019] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multifunctional antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0020] As used herein, an "agonist" antigen-binding molecule or an "agonist" antibody is an antibody that significantly enhances the biological activity of the antigen to which it binds.

[0021] As used herein, a "blocking" antigen-binding molecule or antibody, or an "antagonist" antigen-binding molecule or antibody, is an antibody that significantly inhibits (either partially or completely) the biological activity of the antigen to which it binds.

[0022] The terms "antibody fragment" and "antigen-binding fragment" refer to molecules other than a complete antibody, including a portion of the complete antibody. Examples of antibody fragments include, but are not limited to, Fv (VH and VL), Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multifunctional and multispecific antibodies formed from antibody fragments.

[0023] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0024] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0025] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0026] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re,153 Sm, 212 Bi, 32 P, 212 radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents, as disclosed below.

[0027] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0028] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to the amount, at dosages and for periods of time necessary, effective to achieve the desired therapeutic or prophylactic result.

[0029] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0030] In the present specification, amino acid modification or substitution in the Fc region or constant region can be represented by a combination of the EU numbering system and amino acid. For example, S424N represents a substitution of serine (Ser) at EU numbering position 424 with asparagine (Asn). Also, EU424N represents a substitution of any amino acid at position 424 with asparagine (Asn).

[0031] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Fc receptors are proteins on the surface of immune cells, such as natural killer cells, macrophages, neutrophils, and mast cells. Fc receptors bind to the Fc (fragment crystallizable) region of antibodies attached to infected cells or invading pathogens, stimulating phagocytes or cytotoxic cells to destroy the microorganism or infected cell by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibitory receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also encompassed by the term "FcR" herein.

[0032] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) as well as immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0033] The in vivo binding to human FcRn and plasma half-life of human FcRn high affinity binding polypeptide can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptide with mutated Fc region is administered. WO2000 / 42072 (Presta) describes antibody mutants with increased or decreased binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0034] The term "Fc region-containing antibody" as used herein refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Thus, a composition comprising an antibody with an Fc region according to the present disclosure can include an antibody with G446-K447, an antibody with G446 but without K447, an antibody with G446-K447 completely removed, or a mixture of the above three types of antibodies.

[0035] As used herein, the terms "subunit of an Fc domain" and "Fc subunit" refer to one of the two polypeptides that form a dimeric Fc region, i.e., a polypeptide that contains the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association. For example, a subunit of an IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

[0036] A "modification that promotes the association of the first and second subunits of the Fc domain" is a peptide backbone manipulation or post-translational modification of the Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunits that form homodimers with the same polypeptide. As used herein, a modification that promotes association particularly includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) whose association is desired, which are complementary to each other so as to promote the association of the two Fc domain subunits. For example, an association-promoting modification may alter the structure or charge of one or both of the Fc domains to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may be non-identical in the sense that the further components (e.g., antigen-binding moieties) fused to each of the subunits are not identical. In some embodiments, an association-promoting modification comprises an amino acid mutation, particularly an amino acid substitution, in the Fc domain. In a particular embodiment, the association modification comprises separate amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0037] In one embodiment, the modification is a so-called knob-into-hole modification, which includes a knob modification in one of the two subunits of the Fc domain and a hole modification in the other of the two subunits of the Fc domain. The knob-into-hole technique is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of identical or smaller size to the protrusion are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg alanine or threonine).

[0038] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0039] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0040] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Also included herein are mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected.

[0041] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0042] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0043] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0044] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen contact residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring 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)); (b) the CDRs occurring at amino acid residues 24-34 (CDRL1), 50-56 (CDRL2), 89-97 (CDRL3), 31-35b (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring 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 (d) combinations of (a), (b), and / or (c), comprising 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). Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0045] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0046] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0047] An "isolated" antibody is one that has been separated from the components of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0048] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0049] "Isolated nucleic acid encoding a cytoplasmic entry antigen-binding molecule" refers to one or more nucleic acid molecules encoding a cytoplasmic entry antigen-binding molecule, including nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell. When the TR cytoplasmic entry antigen-binding molecule is an antibody, "isolated nucleic acid encoding a cytoplasmic entry antigen-binding molecule" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0050] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants including naturally occurring variants or variants that arise during the manufacture of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0051] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0052] "Native antibodies" refer to immunoglobulin molecules with various structures that occur in nature. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons that are composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

[0053] The term "cytoplasmic-penetrating antigen-binding molecule" or "cytoplasmic-penetrating antibody or antigen-binding fragment thereof" refers to an antigen-binding molecule or a cytoplasmic-penetrating antibody or antigen-binding fragment thereof that can enter the cytoplasm of a living cell. The method of entry into the cytoplasm is not particularly limited, and may be via endosomal escape after being taken up via the endocytosis process, or may be any other method. An example other than endosomal escape is when an antigen-binding molecule directly penetrates the cell membrane. The same antigen-binding molecule may enter the cytoplasm by both endosomal escape and direct penetration of the cell membrane. Antigen-binding molecules that can enter the cytoplasm by a method other than endosomal escape have been reported (e.g., WO2013102659). In one embodiment, the cytoplasmic-penetrating antigen-binding molecule is an antibody or an antibody derivative. In another embodiment, the cytoplasmic-penetrating antigen-binding molecule comprises a cytoplasmic entry domain.

[0054] The terms "cytoplasmic entry domain" and "region having cytoplasmic entry ability / cytoplasmic entry activity" are used interchangeably and refer to a domain that can enter the cytoplasm of a living cell. Without wishing to be bound by theory, the method of entry into the cytoplasm is not particularly limited, and may be taken up via the endocytosis process followed by endosome escape, or may be another method. The method of endosomal escape is not particularly limited, and for example, the cytoplasmic entry domain interacts with the endosomal membrane in an endosome at an acidic pH, forming a hole on the endosomal membrane, thereby achieving translocation to the cytoplasm. An example other than endosomal escape is when an antigen-binding molecule directly penetrates the cell membrane. The same antigen-binding molecule may enter the cytoplasm by both methods of endosomal escape and direct penetration of the cell membrane. In one embodiment, the cytoplasmic entry domain has one or both of endosomal escape ability and cell membrane penetration ability. In one embodiment, the cytoplasmic entry domain does not have endocytosis ability. In another embodiment, the cytoplasmic entry domain has endocytosis ability. The cytoplasmic entry domain may be of any type as long as it has the ability to enter the cytoplasm, but in a preferred embodiment, it is an antibody or a fragment thereof. When the cytoplasmic entry domain is an antibody or a fragment thereof, the cytoplasmic entry domain includes a combination of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), or a heavy chain antibody variable region (VHH), an antibody heavy chain variable region (VH), an antibody light chain variable region (VL), and a single domain antibody variable region such as the variable region of an immunoglobulin new antigen receptor (IgNAR) (VNAR). An example of an antibody or a fragment thereof having the ability to enter the cytoplasm is Cytotransmab (Nat Commun. 2017 May 10;8:15090.), which includes a light chain variable region exhibiting the ability to enter the cytoplasm.Different examples of cytoplasmic entry domains include "scFv (single chain Fv)", "single chain antibody", "Fv", "scFv2 (single chain Fv 2)", "Fab" or "F(ab')2". The cytoplasmic entry domain may be used in combination with a peptide moiety or a nucleic acid moiety having cell entry activity. Examples of peptide moieties having cell entry activity include cell penetrating peptides (CPPs), protein transduction peptides (PTDs), and the like (see, for example, WO2017156630). Examples of nucleic acid moieties having cell entry activity include oligonucleic acids having phosphorothioate backbones (see, for example, WO2015031837). Whether or not the domain of interest is a cytoplasmic entry domain (whether or not the domain of interest has the ability to enter the cytoplasm) can be confirmed by various methods known to those skilled in the art. For example, the domain of interest can be fused with a molecule (e.g., an antigen-binding molecule) that does not have the ability to enter the cytoplasm, and the fusion molecule can be evaluated by contacting the fusion molecule with a cell and then observing whether the fusion molecule is detected in the cytoplasm of the cell. Various methods for detecting the presence of a molecule of interest in the cytoplasm are known, for example, an assay using the split NanoLuc® system and imaging analysis using a fluorescent microscope, which are described in Reference Examples 1 and 3 below, respectively. The BirA assay and the assay using the split GFP system can also be used to detect the presence of a molecule of interest in the cytoplasm. In one embodiment, the cytoplasmic entry domain may interact with a cell surface antigen and / or a cytoplasmic antigen.

[0055] The "cytoplasmic transport" ability of an antigen-binding molecule means the ability of the antigen-binding molecule to be transported to the cytoplasm, and the terms "enter", "transport", or "traffick" are used interchangeably. The terms "cytoplasmic entry ability / activity", "cytoplasmic transport ability / activity", "cytoplasmic permeability", and "permeability to the cytoplasm" of an antigen-binding molecule are used interchangeably herein and refer to the ability of the antigen-binding molecule itself to move from an endosome to the cytoplasm or the ability of the antigen-binding molecule to be transported to the cytoplasm.

[0056] "Reduced extracellular matrix (ECM) binding" or "lower ECM binding" of an antigen-binding molecule (antibody) means that the binding of the antigen-binding molecule to ECM is reduced compared to a reference antibody. ECM (extracellular matrix) is an extracellular component and is present at various sites in vivo. Therefore, it is known that antibodies that strongly bind to ECM have poorer kinetics (shorter half-life) in blood (WO2012093704 A1). Therefore, antigen-binding molecules containing amino acid substitutions that enhance cytoplasmic invasion activity but do not increase ECM binding are preferably selected as antigen-binding molecule variants that appear in the antibody library.

[0057] The binding of each antibody to ECM (extracellular matrix) can be evaluated by the following procedure with reference to WO2012093704 A1: ECM Phenol red free (BD Matrigel #356237) was diluted to 2 mg / mL in TBS and 5 μL was added to the center of each well of an ECL assay plate (L15XB-3, MSD KK, high bind) cooled on ice. The coated plate was then sealed and stored overnight at 4°C. The assay was then performed the next day at room temperature. 150 μL of ECL blocking buffer (PBS supplemented with 0.5% BSA and 0.05% Tween 20) was added to each well of the coated plate and the plate was left at room temperature for at least 2 hours. The blocking buffer was then removed. Each antibody sample was diluted to 9 μg / mL in ACES-T and further diluted to 3 μg / mL in ECLDB (PBS supplemented with 0.1% BSA and 0.01% Tween 20). 50 μL of the diluted antibody solution was added to each well and incubated for 1 hour at 30° C. with shaking at 600 rpm. The samples were then removed by gently tapping the inverted plate. Next, 50 μL of glutaraldehyde was added to each well to fix the samples and incubated for 10 minutes at room temperature. The wells were then washed three times with PBS-T (PBS supplemented with 0.05% Tween 20). The secondary antibody was diluted to 1 μg / mL in ECLDB and 50 μL of the secondary antibody solution was added to each well of the plate. The plate was sealed and incubated for 1 hour at 30° C. with shaking at 600 rpm while shielded from light. The secondary antibody solution was then removed. The plates were washed again three times with PBS-T, after which READ buffer (MSD KK) was added at 150 μL / well, followed by immediate detection of luminescent signals using a Sector Imager 2400 (MSD KK).

[0058] The term "lower aggregation tendency" of an antigen-binding molecule means that the mutual self-association of the antigen-binding molecules, i.e., the interaction between monomers, is reduced compared to that of the reference antigen-binding molecule. The aggregation profile was evaluated by size-exclusion chromatography (SEC) analysis using UPLC (Acquity H-Class bio, Waters) for each mutant. 50 mM phosphate / 300 mM NaCl (pH 7.0) was used as the running buffer, and a BEH200 column (Waters, #186005225) was used as the analytical column. Chromatograms were recorded by 220 nM UV absorption. Data were processed using Empower3 software (Waters) to calculate the monomer peak percentage and the full width at half maximum of the monomer peak. A high monomer peak percentage and a sharp symmetrical peak shape (narrow peak width) are considered as good physicochemical properties.

[0059] By "fused" it is meant that the components (eg, Fab region and Fc domain subunits) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0060] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0061] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0062] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one may say a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in said program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0063] The term "pharmaceutical formulation" refers to a preparation in a form such that the biological activity of the active ingredients contained therein can be exerted, and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0064] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0065] As used herein, "treatment" (and its grammatical derivatives, such as "treat", "treating", etc.) refers to a clinical intervention intended to modify the natural course of the individual being treated, and may be performed for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or alleviation of the disease state, and remission or improved prognosis. In some embodiments, the antigen binding molecules of the present disclosure are used to delay the onset of disease or slow the progression of disease.

[0066] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a complementary library of VL or VH domains, respectively, with a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0067] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. A vector can effect expression of a nucleic acid to which it is operatively linked. Such vectors are also referred to herein as "expression vectors."

[0068] As used herein, the expressions "substantially decreased," "substantially increased," or "substantially different" refer to a difference between two numerical values ​​(usually one for a molecule and one for a reference / comparator molecule) that is sufficiently large that one of skill in the art would consider the difference between the two numerical values ​​to be statistically significant in terms of the biological characteristic measured by the numerical values ​​(e.g., KD value).

[0069] As used herein, the terms "substantially similar," "substantially unchanged," or "substantially the same" refer to a similarity between two numerical values ​​(e.g., between one relating to an antigen-binding molecule of the present disclosure and one relating to a reference / comparator antigen-binding molecule) that is sufficiently high that one of skill in the art would consider the difference between the two numerical values ​​to have little or no biological and / or statistical significance in terms of the biological characteristic measured by the numerical value (e.g., KD value).

[0070] The expression "substantially undetectable" as used herein means below the detection limit of standard detection techniques known to those skilled in the art (e.g., Western blotting, capillary immunoassay, imaging by fluorescence microscope, etc.), and means that it is not detected at all, or even if it is detected, it is detected at a background level or noise level. Specifically, for example, when the measured value of the antigen-binding molecule of the present disclosure is substantially similar or substantially the same as the measured value of the negative control (e.g., no statistical significance is observed), it is said that the antigen-binding molecule of the present disclosure is substantially undetectable. "Not substantially delivered" means that the delivery is not substantially detected by a standard detection technique for detecting it. For example, with respect to delivery into the cytoplasm of a cell, if the abundance of the antigen-binding molecule or its complex in the cytoplasm of a cell contacted with the antigen-binding molecule of the present disclosure or its complex is substantially similar or substantially the same (e.g., no statistical significance is observed) as the abundance in the cytoplasm of a cell not contacted, the antigen-binding molecule is said to be not substantially delivered into the cytoplasm of the cell. Similarly, "not substantially removed, suppressed, or activated" means that the removal, suppression, or activation is not substantially detected by a standard detection technique for detecting them. For example, with respect to the removal, suppression, or activation of a cytoplasmic antigen, if the measured value when using an antigen-binding molecule or a complex thereof of the present disclosure is substantially similar or substantially the same as the measured value of a control (e.g., no statistical significance is observed), it is said that the cytoplasmic antigen is not substantially removed, suppressed, or activated.

[0071] II. Compositions and Methods In one aspect, the present disclosure is based in part on the discovery of an antigen-binding molecule (e.g., an antibody) comprising a light chain variable region having improved cytoplasmic penetration ability or activity compared to an existing cytoplasmic penetration antibody light chain variable region (VL of h3D8 antibody: hT4VL disclosed in WO2016 / 013870). The present disclosure is further based in part on the discovery that an antigen-binding molecule (antibody) having an improved cytoplasmic penetration antibody further exhibits improved physicochemical properties as indicated by improved expression levels, reduced ECM (extracellular matrix) binding, and / or lower aggregation tendency. In certain embodiments, the cytoplasmic entry antigen-binding molecule (antibody) or fragment thereof provided herein comprises CDRL1, CDRL2, and CDRL3 of hT4VL.FRv4 (SEQ ID NO: 4), which differs from hT4VL only by having human VK3 (VK3) FR3 instead of human VK1 FR3, and comprises a light chain variable region comprising one or more amino acid substitutions in at least one selected from CDRL1, CDRL2, and CDRL3, preferably CDRL1 and CDRL3. In another aspect, the multifunctional antigen-binding molecule provided herein comprises a combination of a cytoplasmic entry antibody or a fragment of the antibody, an antibody or a fragment of the antibody that binds to a cell surface antigen, and / or an antibody or a fragment of the antibody that binds to a cytoplasmic antigen. The multifunctional antigen-binding molecule is delivered into the cytoplasm in a target cell-specific manner. In certain embodiments, the multifunctional antigen-binding molecule provided herein comprises a cell surface antigen-binding domain, a cytoplasmic antigen-binding domain, and a cytoplasmic entry domain comprising the light chain variable region described above. The antigen-binding molecules of the present disclosure are useful, for example, for the diagnosis, prevention, or treatment of diseases caused by cytoplasmic antigens.

[0072] A-1. Exemplary Cytoplasmic Entry Antigen Binding Molecules (Antibodies) In one embodiment, the antigen-binding molecule of the present disclosure is an antigen-binding molecule with improved (enhanced) cytoplasmic penetration ability (activity). In one embodiment, the cytoplasmic penetration antigen-binding molecule of the present disclosure is delivered (translocated) to the cytoplasm more efficiently than existing cell-penetrating antibodies. In one embodiment, when a certain amount of the cytoplasmic penetration antigen-binding molecule of the present disclosure is administered to a subject or contacted with a cell, a greater amount of the antigen-binding molecule is delivered to the cytoplasm of the target cell than the same amount of existing cytoplasmic penetration antibodies. In one embodiment, the antigen-binding molecule of the present disclosure further exhibits improved physicochemical properties, as indicated by improved expression levels, reduced ECM (extracellular matrix) binding, and / or lower aggregation tendency, compared to existing cell-penetrating antibodies.

[0073] In a preferred embodiment, the cytoplasmic entry antigen binding molecule of the present disclosure comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 of the amino acid sequence set forth in SEQ ID NO: 4 (i.e., CDRL1, CDRL2, and CDRL3 contained in the amino acid sequence set forth in SEQ ID NO: 4), wherein the light chain variable region comprises one or more amino acid substitutions in at least one selected from CDRL1, CDRL2, and CDRL3. In a further preferred embodiment, the light chain variable region comprises one or more amino acid substitutions in at least one selected from CDRL1 and CDRL3.

[0074] In a preferred embodiment, the light chain variable region comprises one or more amino acid substitutions at positions 24 to 34 and positions 89 to 97 according to the Kabat numbering in the amino acid sequence set forth in SEQ ID NO: 4. More preferably, the one or more amino acid substitutions are located at positions 24 to 32 and positions 93 to 97 according to the Kabat numbering in the amino acid sequence set forth in SEQ ID NO: 4.

[0075] In a preferred embodiment, the light chain variable region of the antigen binding molecule of the present disclosure comprises one or more amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid K at position 24 with A, D, E, F, G, H, I, L, N, Q, R, S, T, V, or Y; (b) substitution of amino acid S at position 25 with A, D, E, F, I, M, N, T, V, or Y; (c) substitution of amino acid S at position 26 with A, D, E, F, G, H, I, K, M, N, P, R, T, or V; (d) substitution of amino acid Q at position 27 with A, D, E, F, H, I, L, M, S, or Y; (e) substitution of amino acid S at position 27a with D; (f) substitution of amino acid L at position 27b with I, P, Q, T, or V; (g) substitution of amino acid F at position 27c with A, D, E, I, M, N, S, W, or Y; (h) substitution of amino acid N at position 27d with A, D, E, F, H, N, or Q; (i) substitution of amino acid S at position 27e with A, D, E, I, L, M, N, P, Q, T, V, or Y; (j) substitution of amino acid R at position 27f with A, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y; (k) substitution of amino acid T at position 28 with E, H, I, Q, S, V, W, or Y; (l) substitution of amino acid R at position 29 with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; (m) substitution of amino acid K at position 30 with A, D, E, F, H, I, L, M, N, P, Q, S, T, V, W, or Y; (n) substitution of amino acid N at position 31 with D, M, S, or T; (o) substitution of amino acid Y at position 32 with D, E, G, H, or Q; (p) substitution of amino acid Q at position 89 with H, I, T, or Y; (q) substitution of amino acid Y at position 91 with E, F, or N; (r) substitution of amino acid Y at position 93 with I, L, M, N, P, Q, S, T, V, or W; (s) substitution of amino acid H at position 94 with A, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, or Y; (t) substitution of amino acid M at position 95 with A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; (u) substitution of amino acid Y at position 96 with A, D, E, F, G, H, K, M, N, P, Q, S, T, or V; and (v) Substitution of amino acid T at position 97 with A, D, E, G, H, K, P, Q, R, S, or V.

[0076] In a preferred embodiment, the one or more amino acid substitutions in the light chain variable region of the present disclosure enhance the cytoplasmic entry activity of the antibody or fragment thereof compared to a reference antibody or fragment thereof that does not contain the substitution and that comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 4. In a further preferred embodiment, the antibody or fragment thereof of the present disclosure exhibits at least 1%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500% or 1000% enhanced cytoplasmic entry activity compared to a reference antibody or fragment thereof that does not contain the substitution and that comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 4, preferably as determined using the Split Nluc assay described in Reference Example 1. In one embodiment, the cytoplasmic entry activity is mediated by entry of the antibody or fragment thereof into the cell membrane and / or endosomal escape.

[0077] In a preferred embodiment, the light chain variable region of the disclosure contains one, two, three or more amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence shown in SEQ ID NO: 4, (a) Substitution of amino acid K at position 24 with N; (b) substitution of amino acid Q at position 27 with E; (c) substitution of amino acid S at position 27a with D; (d) substitution of amino acid N at position 27d with D or E; (e) substitution of amino acid S at position 27e with D or E; (f) substitution of amino acid R at position 27f with D, H, K, L, S, or V; (g) substitution of amino acid T at position 28 with D; (h) substitution of amino acid R at position 29 with E, G, M, or S; (i) substitution of amino acid K at position 30 with L or Q; (j) substitution of amino acid Y at position 32 with E; (k) substitution of amino acid Q at position 89 with H, I, T, or Y; (l) substitution of amino acid Y at position 91 with E, F, or N; (m) substitution of amino acid Y at position 93 with E; (n) substitution of amino acid H at position 94 with E, P, or Q; (o) substitution of amino acid M at position 95 with D, N, or P; and (p) Substitution of amino acid Y at position 96 with E or P.

[0078] In a preferred embodiment, the light chain variable region of the disclosure comprises a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: (a) S27eD / R27fK; (b) S27eD / R27fH; (c) N27dE / S27eD; (d) S27eD / T28D; (e) S27eD / R29E; (f) S27eD / R29S; (g) K24N / S27eD; (h) N27dD / R27fK; (i) N27dE / R27fK; (j) R27fK / T28D; (k) R27fK / R29E; (l) R27fK / R29S; (m) K24N / R27fK; (n) K24N / N27dD; (o) N27dD / T28D; (p) N27dD / R29E; (q) N27dD / R29S; (r) S27eD / Q89H; (s) N27dD / Q89H; (t) R27fK / Q89H; (u) S27eD / Y91E; (v) N27dD / Y91E; (w) R27fK / Y91E; (x) Q89H / H94P; (y) Y91E / H94P; (z) H94P / M95N; (aa) H94P / M95D; (bb) H94P / M95P; (cc) H94E / Y96P; (dd) H94E / M95N; (ee) H94E / M95D; (ff) H94E / M95P; (gg) N27dE / R27fK / Q89H / H94E / M95P; (hh) N27dE / R27fK / Q89Y / H94E / M95P; (ii) N27dE / R27fK / Y91E / H94E / M95P; (jj) N27dE / R27fK / Y91F / H94E / M95P; (kk) N27dE / R27fS / Q89H / H94E / M95P; (ll) N27dE / R27fS / Q89Y / H94E / M95P; (mm) N27dE / R27fS / Y91E / H94E / M95P; (nn) N27dE / R27fS / Y91F / H94E / M95P; (oo) N27dE / R27fS / Q89H / H94Q / M95P; (pp) N27dE / R27fS / Q89Y / H94Q / M95P; (qq) N27dE / R27fS / Y91E / H94Q / M95P; (rr) N27dE / R27fS / Y91F / H94Q / M95P; (ss) N27dE / R27fV / Q89H / H94E / M95P; (tt) N27dE / R27fV / Q89Y / H94E / M95P; (uu) N27dE / R27fV / Y91E / H94E / M95P; (vv) N27dE / R27fV / Y91F / H94E / M95P; (ww) N27dE / R29S / Q89H / H94E / M95P; (xx) N27dE / R29S / Q89Y / H94E / M95P; (yy) N27dE / K30L / Q89H / H94E / M95P; (zz) N27dE / K30L / Q89Y / H94E / M95P; (aaa) S27eD / R27fK / Y91E / H94E / M95P; (bbb) S27eD / R27fK / Y91F / H94E / M95P; (ccc) S27eD / R27fK / Y91E / H94Q / M95P; (ddd) S27eD / R27fK / Y91F / H94Q / M95P; (eee) S27eD / R27fK / Y91E / H94Q; (fff) S27eD / R27fK / Y91F / H94Q; (ggg) S27eE / R27fK / Y91E / H94E / M95P; (hhh) S27eE / R27fK / Y91F / H94E / M95P; (iii) S27eE / R27fK / Y91E / H94Q / M95P; (jjj) S27eE / R27fK / Y91F / H94Q / M95P; (kkk) R27fS / R29S / Q89H / H94E / M95P; (lll) R27fS / R29S / Q89Y / H94E / M95P; (mmm) R27fS / R29S / Y91E / H94E / M95P; (nnn) R27fS / R29S / Y91F / H94E / M95P; (ooo) R27fS / R29S / Q89H / H94Q / M95P; (ppp) R27fS / R29S / Q89Y / H94Q / M95P; (qqq) R27fS / R29S / Y91E / H94Q / M95P; (rrr) R27fS / R29S / Y91F / H94Q / M95P; (sss) N27dE / R27fS / R29S / Q89Y / H94Q / M95P; and (ttt) N27dE / R27fS / R29S / Y91F / H94Q / M95P.

[0079] In a preferred embodiment, the light chain variable region of the present disclosure comprises a human framework region (FR). In a particular embodiment, the light chain variable region comprises amino acid I at position 2, and / or amino acid Q at position 3 according to the Kabat numbering.

[0080] In a preferred embodiment, the light chain variable region of the present disclosure may comprise a human Vκ1, Vκ2, or Vκ3 FR1 sequence. More preferably, the light chain variable region comprises a human Vκ1 FR1 sequence. In a preferred embodiment, the light chain variable region may comprise a human Vκ1, Vκ2, or Vκ3 FR2 sequence. More preferably, the light chain variable region comprises a human Vκ1 FR2 sequence. In a preferred embodiment, the light chain variable region may comprise a human Vκ1, Vκ2, or Vκ3 FR3 sequence. More preferably, the light chain variable region comprises a human Vκ1 FR3 sequence. In a further preferred embodiment, the light chain variable region comprises a human Vκ1 FR1, human Vκ1 FR2, and human Vκ3 FR3 sequence.

[0081] In a preferred embodiment, the light chain variable region of the disclosure comprises one, two, three, or four of the FR domains (FR1, FR2, FR3, and FR4) contained in SEQ ID NO:123.

[0082] In a preferred embodiment, the light chain variable region of the disclosure further comprises one, two, three, or more amino acid substitutions in CDRL2. In a preferred embodiment, the one, two, three, or more amino acid substitutions are located at positions 50-56 of the Kabat numbering in the amino acid sequence set forth in SEQ ID NO:4. In a specific embodiment, the one, two, three, or more amino acid substitutions in CDRL2 are selected from the group consisting of the following (positions according to Kabat numbering): In the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid W at position 50 with A, G, I, T, or V; (b) substitution of amino acid S at position 52 with F or I; (c) substitution of amino acid T at position 53 with N or Y; and (d) Substitution of amino acid R at position 54 with K or V.

[0083] In some embodiments, the light chain variable region of the disclosure comprises a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence of SEQ ID NO: 4, (a) L2I / V3Q / S27eD / R27fK / W50A / Y91F / H94Q; (b) L2I / V3Q / S27eD / R27fK / W50G / Y91F / H94Q; (c) L2I / V3Q / S27eD / R27fK / W50I / Y91F / H94Q; (d) L2I / V3Q / S27eD / R27fK / W50T / Y91F / H94Q; (e) L2I / V3Q / S27eD / R27fK / W50V / Y91F / H94Q; (f) L2I / V3Q / S27eD / R27fK / S52F / Y91F / H94Q; (g) L2I / V3Q / S27eD / R27fK / S52I / Y91F / H94Q; (h) L2I / V3Q / S27eD / R27fK / T53N / Y91F / H94Q; (i) L2I / V3Q / S27eD / R27fK / T53Y / Y91F / H94Q; (j) L2I / V3Q / S27eD / R27fK / R54K / Y91F / H94Q; (k) L2I / V3Q / S27eD / R27fK / W50I / S52F / Y91F / H94Q; (l) L2I / V3Q / S27eD / R27fK / W50I / T53N / Y91F / H94Q; and (m) L2I / V3Q / S27eD / R27fK / W50I / R54V / Y91F / H94Q.

[0084] In a preferred embodiment, the cytoplasmic penetrating antigen-binding molecule of the present disclosure comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 150 and 161 to 173. In one embodiment, the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 123 to 150. In one embodiment, the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161 to 173.

[0085] In a further preferred embodiment, the cytoplasmic entry antigen-binding molecule of the present disclosure can be in the form of scFv (single chain Fv), single chain antibody, Fv, scFv2 (single chain Fv2), Fab, or F(ab')2. In a further preferred embodiment, the cytoplasmic entry antigen-binding molecule of the present disclosure can be in the form of Fab or scFv.

[0086] In a preferred embodiment, the cytoplasmic entry molecule of the present disclosure further comprises a heavy chain variable region. The cytoplasmic entry antibody or antigen-binding fragment thereof of the present disclosure may further comprise a single domain antibody variable region, such as a VHH.

[0087] A-2. Exemplary multifunctional antigen-binding molecules with cytoplasmic entry ability In some embodiments, the cytoplasmic entry antigen-binding molecule of the present disclosure is a multifunctional antigen-binding molecule (antibody). In one embodiment, the multifunctional antigen-binding molecule comprises a cell surface antigen-binding domain, a cytoplasmic antigen-binding domain, and a cytoplasmic entry domain comprising the light chain variable region described above.

[0088] In a preferred embodiment, the multifunctional antigen-binding molecule of the present disclosure does not bind to an antigen expressed on a cell surface that is different from the cell surface antigen. In a preferred embodiment, the cytoplasmic antigen-binding domain and / or the cytoplasmic penetration domain (i) does not bind to an antigen expressed on a cell surface that is different from the cell surface antigen, or (ii) does not bind to any antigen expressed on a cell surface.

[0089] Here, it is known that an existing internalizing antibody (Tmab4 (heavy chain: 3D8, light chain: hT4VL)) is taken up into intracellular endosomes by heparan sulfate proteoglycan (HSPG)-dependent endocytosis on the cell surface, and in the endosomes at an acidic pH, the structure of the light chain changes, interacting with the endosomal membrane and forming a pore on the membrane, thereby achieving translocation to the cytoplasm (J Control Release. 2016 Aug 10;235:165-175). It has also been reported that an antibody with reduced binding to HSPG (Tmab4-03 (heavy chain: 3D8, light chain: hT4VL.03)) maintains pore-forming ability at pH 5.5, but has impaired endocytosis ability (J Control Release. 2016 Aug 10;235:165-175). This means that Tmab4-03 cannot achieve cell-specific intracellular translocation as it is. Furthermore, it is expected that in order to form a hole on the endosomal membrane, it is important that Tmab4 interacts with a molecule on the endosomal membrane to distort the membrane (PNAS, 2011 Oct. 108; 41; 16883-16888). Therefore, in one embodiment, in order to improve the cytoplasmic entry ability, it is desirable that the cytoplasmic entry domain that interacts with the endosomal membrane is multivalent.

[0090] On the other hand, as mentioned above, Tmab4 is endocytosed via HSPGs, which are ubiquitously expressed in epithelial cells, making it difficult to deliver cytoplasmic antigen-binding antibodies specifically to target cells. In other words, when administered systemically, it is expected that the delivery of cytoplasmic antigen-binding antibodies to target tissues will be low. Therefore, in one embodiment, the inventors have considered making the cytoplasmic entry domain monovalent to suppress nonspecific uptake, and further improving target specificity by adding a target cell surface binding domain, while at the same time creating a state in which the interaction between the antibody and the endosomal membrane is multivalent, thereby maintaining or improving the cytoplasmic entry ability.

[0091] Unlike the above-mentioned existing antibodies, in one aspect, the cytoplasmic penetration antigen-binding molecule of the present disclosure comprises a cell surface antigen-binding domain, a cytoplasmic antigen-binding domain, and a cytoplasmic penetration domain. Thus, in one aspect, the cytoplasmic penetration antigen-binding molecule of the present disclosure is expected to be endocytosed specifically in the target cell by binding to a cell surface antigen of the target cell, and then to migrate from the endosome to the cytoplasm. In one aspect, the cytoplasmic penetration antigen-binding molecule of the present disclosure is delivered more selectively into the cytoplasm of the target cell compared to existing cytoplasmic penetration antibodies. In one aspect, when a certain amount of the cytoplasmic penetration antigen-binding molecule of the present disclosure is administered or contacted to a subject, more antigen-binding molecules are delivered into the cytoplasm of the target cell compared to the same amount of existing cytoplasmic penetration antibodies. In one aspect, the cytoplasmic penetration antigen-binding molecule of the present disclosure is delivered specifically into the cytoplasm of the target cell, and is not substantially delivered to cells that do not express the cell surface antigen to which the antigen-binding molecule specifically binds. In one aspect, when the cytoplasmic-penetrating antigen-binding molecule of the present disclosure is used as a pharmaceutical, the cytoplasmic-penetrating antigen-binding molecule exhibits stronger efficacy and / or causes fewer side effects than existing cytoplasmic-penetrating antibodies. Also, in one aspect, a pharmaceutical composition comprising the cytoplasmic-penetrating antigen-binding molecule of the present disclosure can exhibit its efficacy at a smaller dose and / or with a smaller number of administrations than a pharmaceutical composition comprising an existing cytoplasmic-penetrating antibody.

[0092] In a further preferred embodiment, the multifunctional cytoplasmic-penetrating antigen-binding molecule of the present disclosure may be in any of the following molecular forms 1 to 8:

[0093] 1. Cytoplasmic entry antigen-binding molecule comprising a first and a second Fab region (FIG. 4, FIG. 6) In a preferred embodiment of the present disclosure, the cytoplasmic entry antigen binding molecule comprises a first and a second Fab region. In a particular embodiment, (a) the first Fab region specifically binds to a cell surface antigen; and (b) the second Fab region comprises a pair of a heavy chain variable region (VH) that specifically binds to a cytoplasmic antigen and a light chain variable region (VL) that has cytoplasmic penetration ability. In another specific embodiment, (a) the first Fab region comprises a pair of a heavy chain variable region (VH) that specifically binds to a cell surface antigen and a light chain variable region (VL) that has cytoplasmic penetration ability; and (b) the second Fab region comprises a pair of a heavy chain variable region (VH) that binds to a cytoplasmic antigen and a light chain variable region (VL) that has cytoplasmic penetration ability. The cytoplasmic entry antigen binding molecule in these embodiments may further comprise an Fc region, which may comprise a modification that promotes association of the first Fc subunit and the second Fc subunit. The light chain variable region (VL) having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments is as defined in A-1. Exemplary cytoplasmic entry antigen-binding molecules (antibodies).

[0094] 2. Cytoplasmic entry antigen-binding molecule comprising a first and a second Fab region and a single chain unit (Figure 7) In a preferred embodiment of the present disclosure, the cytoplasmic entry antigen binding molecule comprises a first and a second Fab region and a single chain unit. In particular embodiments, (a) the first Fab region specifically binds to a cell surface antigen; (b) the second Fab region has cytoplasmic entry ability; and (c) the single chain unit specifically binds to a cytoplasmic antigen. In another specific embodiment, (a) the first Fab region specifically binds to a cytoplasmic antigen; (b) the second Fab region has cytoplasmic entry ability; and (c) the single chain unit specifically binds to a cell surface antigen. In yet another specific embodiment, (a) the first Fab region specifically binds to a cell surface antigen; (b) the second Fab region specifically binds to a cytoplasmic antigen; and (c) the single chain unit has cytoplasmic entry capability. In yet another specific embodiment, (a) the first and second Fab regions comprise a pair of a heavy chain variable region (VH) that specifically binds to a cell surface antigen and a light chain variable region (VL) that has cytoplasmic penetration ability; and (b) the single chain unit specifically binds to a cytoplasmic antigen. In certain embodiments, the single-chain units are fused to (i) the N-terminus of the heavy chain variable region (VH) of the first Fab region and / or the N-terminus of the heavy chain variable region (VH) of the second Fab region; (ii) the N-terminus of the light chain variable region (VL) of the first Fab region and / or the N-terminus of the light chain variable region (VL) of the second Fab region; or (iii) the C-terminus of the light chain constant region (CL) of the first Fab region and / or the C-terminus of the light chain constant region (CL) of the second Fab region. In these embodiments, the cytoplasmic entry antigen-binding molecule may further comprise an Fc region comprising a first Fc subunit and a second Fc subunit, and the single chain unit may be fused to (i) the C-terminus of the first Fc subunit, or (ii) the C-terminus of the second Fc subunit. The first Fc subunit and the second Fc subunit may be the heavy chain constant region CH2 and CH3 domains of an IgG antibody, or may comprise modifications that promote association of the first Fc subunit and the second Fc subunit. The single chain unit contained in the cytoplasmic entry antigen-binding molecule in these embodiments may be a single domain antibody variable region or a single chain antibody (scFv). Examples of single domain antibody variable regions include, but are not limited to, heavy chain antibody variable regions (VHH), heavy chain variable regions (VH), light chain variable regions (VL) or variable regions of immunoglobulin neoantigen receptors (VNAR). The light chain variable region (VL) having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments is as defined in A-1. Exemplary cytoplasmic entry antigen-binding molecules (antibodies). The Fab region having cytoplasmic entry ability and the single-chain unit having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments also contain a light chain variable region having cytoplasmic entry ability.

[0095] 3. Cytoplasmic entry antigen-binding molecules comprising a first and a second single domain antibody variable region and a single chain unit (Figure 8) In one preferred embodiment of the present disclosure, the cytoplasmic entry antigen binding molecule comprises a first and a second single domain antibody variable region, and a single chain unit. In a particular embodiment, (a) the first single domain antibody variable region specifically binds to a cell surface antigen; (b) the second single domain antibody variable region has cytoplasmic penetration ability; and (c) the single chain unit specifically binds to a cytoplasmic antigen. In a particular embodiment, the single chain unit is fused to the N-terminus of the first single domain antibody variable region and / or to the N-terminus of said second single domain antibody variable region. In these embodiments, the cytoplasmic entry antigen-binding molecule may further comprise an Fc region comprising a first Fc subunit and a second Fc subunit, and the single chain unit may be fused to (i) the C-terminus of the first Fc subunit, or (ii) the C-terminus of the second Fc subunit. The first Fc subunit and the second Fc subunit may be the heavy chain constant region CH2 and CH3 domains of an IgG antibody, or may comprise modifications that promote association of the first Fc subunit and the second Fc subunit. The single chain unit contained in the cytoplasmic entry antigen-binding molecule in these embodiments may be a single domain antibody variable region or a single chain antibody (scFv). Examples of single domain antibody variable regions include, but are not limited to, heavy chain antibody variable regions (VHH), heavy chain variable regions (VH), light chain variable regions (VL) or variable regions of immunoglobulin neoantigen receptors (VNAR). The single domain antibody variable region having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments comprises a light chain variable region (VL) having cytoplasmic entry ability as defined in A-1. Exemplary cytoplasmic entry antigen-binding molecules (antibodies).

[0096] 4. Cytoplasmic entry antigen-binding molecule having the molecular form of DVD-Ig (registered trademark) (Figure 9) In a preferred embodiment of the present disclosure, the cytoplasmic entry antigen binding molecule comprises a first and a second polypeptide chain and has the molecular shape of a DVD-Ig®. In a particular embodiment, (a) the first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable region that specifically binds a cytoplasmic antigen, VD2 is a second heavy chain variable region that specifically binds a cell surface antigen, C is a heavy chain constant region, CH1, X1 is a linker that is not CH1, X2 is an Fc region, and n is 0 or 1; (b) the second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable region that specifically binds to a cytoplasmic antigen, VD2 is a second light chain variable region that specifically binds to a cell surface antigen, C is a light chain constant region, CL, X1 is a linker that is not CL, X2 does not comprise an Fc region, and n is 0 or 1. In another specific embodiment, (a) the first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable region having cytoplasmic entry capability, VD2 is a second heavy chain variable region that specifically binds to a cell surface antigen, C is a heavy chain constant region CH1, X1 is a linker that is not CH1, X2 is an Fc region, and n is 0 or 1; (b) the second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable region having cytoplasmic entry capability, VD2 is a second light chain variable region that specifically binds to a cell surface antigen, C is a light chain constant region CL, X1 is a linker that is not CL, X2 does not comprise an Fc region, and n is 0 or 1. In yet another specific embodiment, (a) the first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable region that specifically binds to a cell surface antigen, VD2 is a second heavy chain variable region that specifically binds to a cytoplasmic antigen, C is a heavy chain constant region CH1, X1 is a linker that is not CH1, X2 is an Fc region, and n is 0 or 1; (b) the second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable region that specifically binds to a cell surface antigen, VD2 is a second light chain variable region having cytoplasmic entry capability, C is a light chain constant region CL, X1 is a linker that is not CL, X2 does not comprise an Fc region, and n is 0 or 1. The light chain variable region having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments is as defined in A-1. Exemplary cytoplasmic entry antigen-binding molecules (antibodies).

[0097] 5. Cytoplasmic entry antigen-binding molecule comprising a first, second and third Fab region (Figure 10) In a preferred embodiment of the present disclosure, the cytoplasmic entry antigen binding molecule comprises a first, a second and a third Fab region. In a particular embodiment, (a) the first Fab region specifically binds to a cell surface antigen; (b) the second and third Fab regions comprise a pair of a heavy chain variable region that specifically binds to a cytoplasmic antigen and a light chain variable region having cytoplasmic penetration ability; (c) the Fc region comprises a first Fc subunit and a second Fc subunit; and (d) the C-terminus of the heavy chain of the first Fab region is fused to the N-terminus of the first Fc subunit, the C-terminus of the heavy chain of the second Fab region is fused to the N-terminus of the second Fc subunit, and the C-terminus of the heavy chain of the third Fab region is fused to the N-terminus of the heavy chain of the second Fab region. In another specific embodiment, (a) the first and third Fab regions comprise a pair of a heavy chain variable region that specifically binds to a cytoplasmic antigen and a light chain variable region having cytoplasmic penetration ability; (b) the second Fab region specifically binds to a cell surface antigen; (c) the Fc region comprises a first Fc subunit and a second Fc subunit; and (d) the C-terminus of the heavy chain of the first Fab region is fused to the N-terminus of the first Fc subunit, the C-terminus of the heavy chain of the second Fab region is fused to the N-terminus of the second Fc subunit, and the C-terminus of the heavy chain of the third Fab region is fused to the N-terminus of the heavy chain of the second Fab region. In yet another specific embodiment, (a) the first and second Fab regions comprise a pair of a heavy chain variable region that specifically binds to a cytoplasmic antigen and a light chain variable region having cytoplasmic penetration ability; (b) the third Fab region specifically binds to a cell surface antigen; (c) the Fc region comprises a first Fc subunit and a second Fc subunit; and (d) the C-terminus of the heavy chain of the first Fab region is fused to the N-terminus of the first Fc subunit, the C-terminus of the heavy chain of the second Fab region is fused to the N-terminus of the second Fc subunit, and the C-terminus of the heavy chain of the third Fab region is fused to the N-terminus of the heavy chain of the second Fab region. In a further specific embodiment, the first and / or second Fab region comprises any one of the following substitutions: (i) a substitution of a Fab light chain variable region (VL) with a Fab heavy chain variable region (VH); (ii) a substitution of a Fab light chain constant region (CL) with a Fab heavy chain constant region (CH1); and (iii) a substitution of a Fab light chain (VL-CL) with a Fab heavy chain (VH-CH1), and the first Fab region and the second Fab region do not comprise an identical substitution. In these embodiments, the first and second Fc subunits may be the heavy chain constant region CH2 and CH3 domains of an IgG antibody, and may contain modifications that facilitate association of the first and second Fc subunits. The light chain variable region (VL) having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments is as defined in A-1. Exemplary cytoplasmic entry antigen-binding molecules (antibodies).

[0098] 6. Cytoplasmic entry antigen-binding molecule comprising a first, second, third and fourth Fab region (Figure 11) In a preferred embodiment of the present disclosure, the cytoplasmic entry antigen binding molecule comprises a first, a second, a third and a fourth Fab region. In a specific embodiment, (a) one Fab region selected from the first, second, third, and fourth Fab regions specifically binds to a cell surface antigen; (b) the three Fab regions other than (a) above comprise a pair of a heavy chain variable region that specifically binds to a cytoplasmic antigen and a light chain variable region having cytoplasmic penetration ability; (d) the Fc region comprises a first Fc subunit and a second Fc subunit; and (e) the C-terminus of the heavy chain of the first Fab region is fused to the N-terminus of the first Fc subunit, the C-terminus of the heavy chain of the second Fab region is fused to the N-terminus of the second Fc subunit, the C-terminus of the heavy chain of the third Fab region is fused to the N-terminus of the heavy chain of the first Fab region, and the C-terminus of the heavy chain of the fourth Fab region is fused to the N-terminus of the heavy chain of the second Fab region. In another specific embodiment, (a) two Fab regions selected from the first, second, third and fourth Fab regions specifically bind to a cell surface antigen; (b) the two Fab regions other than (a) above comprise a pair of a heavy chain variable region that specifically binds to a cytoplasmic antigen and a light chain variable region having cytoplasmic penetration ability; (d) the Fc region comprises a first Fc subunit and a second Fc subunit; and (e) the C-terminus of the heavy chain of the first Fab region is fused to the N-terminus of the first Fc subunit, the C-terminus of the heavy chain of the second Fab region is fused to the N-terminus of the second Fc subunit, the C-terminus of the heavy chain of the third Fab region is fused to the N-terminus of the heavy chain of the first Fab region, and the C-terminus of the heavy chain of the fourth Fab region is fused to the N-terminus of the heavy chain of the second Fab region. In yet another specific embodiment, (a) three Fab regions selected from the first, second, third and fourth Fab regions specifically bind to a cell surface antigen; (b) one Fab region other than (a) above comprises a pair of a heavy chain variable region that specifically binds to a cytoplasmic antigen and a light chain variable region having cytoplasmic penetration ability; (d) the Fc region comprises a first Fc subunit and a second Fc subunit; and (e) the C-terminus of the heavy chain of the first Fab region is fused to the N-terminus of the first Fc subunit, the C-terminus of the heavy chain of the second Fab region is fused to the N-terminus of the second Fc subunit, the C-terminus of the heavy chain of the third Fab region is fused to the N-terminus of the heavy chain of the first Fab region, and the C-terminus of the heavy chain of the fourth Fab region is fused to the N-terminus of the heavy chain of the second Fab region. In a further specific embodiment, the first and / or second Fab region comprises any one of the following substitutions: (i) a substitution of a Fab light chain variable region (VL) with a Fab heavy chain variable region (VH); (ii) a substitution of a Fab light chain constant region (CL) with a Fab heavy chain constant region (CH1); and (iii) a substitution of a Fab light chain (VL-CL) with a Fab heavy chain (VH-CH1), and the first Fab region and the second Fab region do not comprise an identical substitution. In yet another specific embodiment, the third and / or fourth Fab region comprises any one of the following substitutions: (i) a substitution of a Fab light chain variable region (VL) with a Fab heavy chain variable region (VH); (ii) a substitution of a Fab light chain constant region (CL) with a Fab heavy chain constant region (CH1); and (iii) a substitution of a Fab light chain (VL-CL) with a Fab heavy chain (VH-CH1), and the third Fab region and the fourth Fab region do not comprise the same substitution. In these embodiments, the first and second Fc subunits may be the heavy chain constant region CH2 and CH3 domains of an IgG antibody, and may contain modifications that facilitate association of the first and second Fc subunits. The light chain variable region (VL) having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments is as defined in A-1. Exemplary cytoplasmic entry antigen-binding molecules (antibodies).

[0099] 7. Cytoplasmic entry antigen-binding molecules containing modified Fc regions that promote multimerization (Figure 5) In a preferred embodiment of the present disclosure, the cytoplasmic penetration antigen-binding molecule comprises a region having cytoplasmic penetration ability and an Fc region, the Fc region comprising one or more amino acid modifications that promote multimerization of the cytoplasmic penetration antigen-binding molecule, and has increased cytoplasmic penetration ability compared to a cytoplasmic penetration antigen-binding molecule comprising a parent Fc region that does not comprise the one or more amino acid modifications. The cytoplasmic penetration antigen-binding molecule may further comprise a cell surface antigen-binding domain and a cytoplasmic antigen-binding domain. In a specific embodiment, the cytoplasmic entry antigen-binding molecule comprises a first and a second Fab region, and an Fc region, wherein (a) the first Fab region specifically binds to a cell surface antigen; (b) the second Fab region comprises a pair of a heavy chain variable region (VH) that specifically binds to a cytoplasmic antigen and a light chain variable region (VL) that has cytoplasmic entry ability; and (c) the Fc region comprises one or more amino acid modifications that promote multimerization of the cytoplasmic entry antigen-binding molecule. The cytoplasmic entry antigen-binding molecule comprising such a modified Fc region has an increased cytoplasmic entry ability compared to a cytoplasmic entry antigen-binding molecule comprising a parent Fc region that does not comprise the one or more amino acid modifications, and can form a multimeric cytoplasmic entry antigen-binding molecule complex comprising a plurality of the cytoplasmic entry antigen-binding molecules. The multimer may be a dimer, trimer, tetramer, pentamer or hexamer. The amino acid modifications may be a combination of E345R / E430G / S440Y or a combination of T437R / K248E (numbers indicate the positions of the substitutions according to EU numbering). In these embodiments, the first and second Fc subunits may be the heavy chain constant region CH2 and CH3 domains of an IgG antibody, and may contain modifications that facilitate association of the first and second Fc subunits. The light chain variable region (VL) having cytoplasmic entry ability contained in the cytoplasmic entry antigen-binding molecule in these embodiments is as defined in A-1. Exemplary cytoplasmic entry antigen-binding molecules (antibodies).

[0100] 8. Cytoplasmic entry antigen-binding molecules conjugated to heterologous moieties In one aspect, the antigen binding molecule of the present disclosure is a cytoplasmic entry antigen binding molecule conjugated to a heterologous moiety. In one embodiment, the cytoplasmic entry antigen binding molecule of the present disclosure comprises a cell surface antigen binding domain and a cytoplasmic entry domain, but does not comprise a cytoplasmic antigen binding domain. In a preferred embodiment, the cytoplasmic entry antigen binding molecule of the present disclosure does not bind to an antigen that is different from the cell surface antigen and is expressed on the surface of a cell. In a preferred embodiment, the cytoplasmic entry domain and / or the heterologous moiety (i) does not bind to an antigen that is different from the cell surface antigen and is expressed on the surface of a cell, or (ii) does not bind to any antigen expressed on the surface of a cell.

[0101] In one aspect, as described above, the inventors conceived of making the cytoplasmic entry domain monovalent to suppress nonspecific uptake, and further improving target specificity by adding a target cell surface binding domain, while at the same time creating a multivalent state in the interaction between the antibody and the endosomal membrane, thereby maintaining or improving the cytoplasmic entry ability.

[0102] In one aspect, the cytoplasmic penetration antigen-binding molecule of the present disclosure comprises a cell surface antigen-binding domain and a cytoplasmic penetration domain. Thus, in one aspect, the cytoplasmic penetration antigen-binding molecule of the present disclosure is expected to be endocytosed in a target cell-specific manner by binding to a cell surface antigen on a target cell, and then to migrate from the endosome to the cytoplasm, thereby delivering a heterologous moiety into the cytoplasm.

[0103] In one embodiment, the cytoplasmic entry antigen binding molecule of the present disclosure is more selectively delivered into the cytoplasm of a target cell compared to an existing cytoplasmic entry antibody, and therefore the cytoplasmic entry antigen binding molecule of the present disclosure can more selectively deliver a heterologous moiety into the cytoplasm of a target cell. In one embodiment, when a certain amount of the cytoplasmic entry antigen binding molecule of the present disclosure is administered to or contacted with a subject, a larger amount of the heterologous moiety conjugated to the cytoplasmic entry antigen binding molecule is delivered to the cytoplasm of the target cell compared to the same amount of the heterologous moiety conjugated to an existing cytoplasmic entry antibody. In one embodiment, the heterologous moiety conjugated to the cytoplasmic entry antigen binding molecule of the present disclosure is specifically delivered into the cytoplasm of the target cell, but is not substantially delivered to cells that do not express the cell surface antigen to which the antigen binding molecule specifically binds.

[0104] In one aspect, when the cytoplasmic penetration antigen-binding molecule of the present disclosure is used as a pharmaceutical, the heterologous moiety conjugated to the cytoplasmic penetration antigen-binding molecule exerts stronger pharmaceutical effects and / or causes fewer side effects compared to the heterologous moiety conjugated to an existing cytoplasmic penetration antibody. In one aspect, a pharmaceutical composition comprising a heterologous moiety conjugated to a cytoplasmic penetration antigen-binding molecule can exert its pharmaceutical effects at a smaller dosage and / or number of administrations compared to the pharmaceutical composition comprising a heterologous moiety conjugated to an existing cytoplasmic penetration antibody.

[0105] In one embodiment, the cytoplasmic penetration antigen-binding molecule comprises a first and a second Fab region, (a) the first Fab region specifically binds to a cell surface antigen, and (b) the second Fab region has the ability to penetrate the cytoplasm, and the antigen-binding molecule is conjugated to a heterologous moiety. In a preferred embodiment, the heterologous moiety is conjugated to the C-terminus (preferably the biotin-labeled C-terminus) of the L chain of the antigen-binding molecule.

[0106] In one embodiment, the heterologous moiety conjugated to the cytoplasmic penetration antigen binding molecule is a peptide, a nucleic acid, a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or a fragment thereof), or a radioisotope. In one embodiment, the heterologous moiety is a peptide.

[0107] A-3. In further aspects, an antigen-binding molecule according to any of the above embodiments may incorporate any of the following features.

[0108] The terms "antigen-binding domain" and "antigen-binding fragment (of an antibody)" are used interchangeably herein and refer to a portion of an antigen-binding molecule (antibody) that comprises a region that specifically binds to and is complementary to all or a portion of an antigen. If the molecular weight of the antigen is large, the antigen-binding domain can only bind to a specific portion of the antigen. The specific portion is called an epitope. An antigen-binding domain may be provided by one or more variable domains of an antibody. In a preferred embodiment, the antigen-binding domain comprises a combination of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), or all or a portion of a single domain antibody variable region, such as a heavy chain antibody variable region (VHH), an antibody heavy chain variable region (VH), an antibody light chain variable region (VL), and a variable region of an immunoglobulin new antigen receptor (IgNAR) (VNAR). Examples of different antigen-binding domains include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv 2 (scFv2)", "Fab" or "F(ab')2".

[0109] As used herein, the terms "single domain antibody," "single domain antibody variable region," "single domain antibody," and "single domain antibody variable region" are not limited in structure as long as the domain alone can exhibit antigen-binding activity. Conventional antibodies, exemplified by IgG antibodies, exhibit antigen-binding activity when the variable region is formed by pairing VH and VL, whereas single domain antibodies are known to be able to exhibit antigen-binding activity by the domain structure of the single domain antibody alone, without pairing with other domains. Single domain antibodies usually have a relatively low molecular weight and exist in the form of a monomer. Examples of single domain antibodies include, but are not limited to, antigen-binding molecules that congenitally lack light chains, such as camelid VHH and shark VNAR (variable region of immunoglobulin new antigen receptor (IgNAR)), or antibody fragments that contain all or a part of the VH domain or all or a part of the VL domain of an antibody. Examples of single domain antibodies that are antibody fragments that contain all or a part of the VH / VL domains of an antibody include, but are not limited to, single domain antibodies artificially created starting from human antibody VH or human antibody VL, such as those described in U.S. Pat. No. 6,248,516 B1. In some embodiments of the present invention, a single domain antibody has three CDRs (CDR1, CDR2, and CDR3). 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, but are not limited to, camelids and transgenic animals into which a gene capable of producing single domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing single domain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and US Patent Publication US2011 / 0123527 A1. Humanized single domain antibodies can also be obtained by making the framework sequence of a single domain antibody obtained from an animal a human germline sequence or a sequence similar thereto. Humanized single domain antibodies (e.g., humanized VHH) are also an embodiment of the single domain antibody of the present invention. "Humanized single domain antibody" refers to a chimeric single domain antibody comprising amino acid residues from non-human CDR and human FR. In one embodiment, all or substantially all CDRs of a humanized single domain antibody correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. In a humanized antibody, even if some of the residues in the FR do not correspond to those of a human antibody, it is considered as an example in which substantially all of the FRs correspond to those of a human antibody. For example, when humanizing a VHH, which is one embodiment of a single domain antibody, some of the residues in the FR must be residues that do not correspond to those of a human antibody (C Vincke et al., The Journal of Biological Chemistry 284, 3273-3284.). Single domain antibodies can be obtained by ELISA, panning, or the like from a polypeptide library containing single domain antibodies. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive 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 created 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) are among the most famous.

[0110] As used herein, the term "specifically binds" refers to a state in which one of the specifically binding molecules does not show any significant binding to molecules other than the one or more molecules to which it binds. The term is also used when an antigen-binding domain is specific to a specific epitope among multiple epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0111] The "cell surface antigen-binding domain" in the antigen-binding molecule of the present disclosure can bind to an epitope present in an antigen expressed on the cell surface. A "cell surface antigen" refers to an antigen structure expressed by a cell and present on the cell surface so that the antigen-binding domain can access it. In one embodiment, the cytoplasmic penetration antigen-binding molecule of the present disclosure contains a cell surface antigen-binding domain, and is therefore specifically incorporated into a cell (target cell) expressing the cell surface antigen. This enables the specific delivery of the cytoplasmic penetration antigen-binding molecule to the target cell. In one embodiment, the cell surface antigen-binding domain can be provided by one or more antibody variable domains, and may be in any of the forms exemplified as the "antigen-binding domain" described above.

[0112] The "cytoplasmic antigen-binding domain" in the antigen-binding molecule of the present disclosure can bind to an epitope present in an antigen expressed in the cytoplasm. The "cytoplasmic antigen" refers to an antigen structure expressed by a cell and present in the cytoplasm. The cytoplasmic antigen may be a protein, a nucleic acid such as DNA or RNA, or a molecule expressed in an organelle such as a cell nucleus or mitochondria. Since the cytoplasmic antigen-binding molecule of the present disclosure contains a cytoplasmic antigen-binding domain, it is possible to bind to the cytoplasmic antigen in the cytoplasm of a target cell and neutralize, inhibit, activate, or the like the function of the antigen. In one embodiment, the cytoplasmic antigen-binding domain can be provided by one or more variable domains of antibodies and may be in any of the forms exemplified as the "antigen-binding domain" described above. In a further embodiment, the cytoplasmic antigen-binding domain may be an enzyme, shRNA, or the like.

[0113] As used herein, the phrase "an antigen-binding molecule has improved (enhanced) cytoplasmic entry ability (activity)" means that after contacting the antigen-binding molecule with a cell, a) the antigen-binding molecular weight in the cytoplasm of the cell at any time point is increased by at least 1%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500%, or 1000%, or more, compared to a reference antigen-binding molecular weight present in the cytoplasm of the cell at the same time point. In one embodiment, the reference antigen-binding molecule comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 contained in the amino acid sequence shown in SEQ ID NO: 4, without any substitutions in CDRL2, CDRL2, and CDRL3. In one embodiment, the reference antigen-binding molecule comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 4.

[0114] In one embodiment, when the antigen-binding molecular weight present in the cytoplasm at any time point is represented by an HRP luminescence signal, fluorescent signal, or luminescence signal, a) the HRP luminescence signal intensity, fluorescent signal intensity, or luminescence signal intensity at any time point after contacting the antigen-binding molecule with a cell is b) 0.01-fold or more, 0.05-fold or more, 0.08-fold or more, 0.1-fold or more, 0.12-fold or more, 0.15-fold or more, 0.18-fold or more, 0.2-fold or more, 0.25-fold or more, 0.3-fold or more, 0.35-fold or more, 0.4-fold or more, 0.5-fold or more, 0.6-fold or more, 0.7-fold or more, 0.8-fold or more, 0.9-fold or more, 1-fold or more, 2-fold or more, 5-fold or more, or 10-fold or more of the HRP luminescence signal intensity, fluorescent signal intensity, or luminescence signal intensity obtained by a reference antigen-binding molecule at the same time point. In one embodiment, when a method including a cell expressing biotin ligase (BirA) is used, the cytoplasmic-invaded antigen-binding molecular weight present in the cytoplasm at any time point can be represented by HRP luminescence signal. In a different embodiment, when an imaging analysis (such as the method described in Example 4) is used, the cytoplasmic-invaded antigen-binding molecular weight present in the cytoplasm at any time point can be represented by a fluorescent signal. In a different embodiment, when a split protein system (such as the method described in Reference Example 1) is used, the cytoplasmic-invaded antigen-binding molecular weight present in the cytoplasm at any time point can be represented by a luminescence signal. In one embodiment, the reference antigen-binding molecule comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 contained in the amino acid sequence shown in SEQ ID NO: 4, without any substitution in CDRL2, CDRL2, and CDRL3. In one embodiment, the reference antigen-binding molecule comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 4.

[0115] In one embodiment, the cytoplasmic entry antigen-binding molecule is delivered into the cytoplasm of a target cell specifically, meaning that after the cytoplasmic entry antigen-binding molecule is contacted with a target cell and a non-target cell, a) the amount of cytoplasmic entry antigen-binding molecular weight present in the cytoplasm of the non-target cell at any time point is less or substantially reduced compared to b) the amount of cytoplasmic entry antigen-binding molecular weight present in the cytoplasm of the target cell at the same time point. In a preferred embodiment, after the cytoplasmic entry antigen-binding molecule of the present disclosure is contacted with a non-target cell, the cytoplasmic entry antigen-binding molecule is substantially not detected in the cytoplasm of the non-target cell. In a preferred embodiment, the cell is a cell derived from a Hela cell line, a CHO cell line, an MDCK cell, or a HepG2 cell line, which expresses or does not express a cell surface antigen. The amount of the cytoplasmic entry antigen-binding molecule to be contacted with the cell may be determined arbitrarily, but the amount of the cytoplasmic entry antigen-binding molecular weight in the a) and the cytoplasmic entry antigen-binding molecular weight in the b) are the same. The contact of the cytoplasmic entry antigen-binding molecule with the cell is performed by any method, including incubation.

[0116] In one embodiment, the cytoplasmic penetration antigen-binding molecular weight present in the cytoplasm is measured 0 hours, 0.25 hours, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, and / or 16 hours after contacting the cytoplasmic penetration antigen-binding molecule with cells. The cytoplasmic penetration antigen-binding molecular weight present in the cytoplasm may be measured only once or multiple times after contacting the cytoplasmic penetration antigen-binding molecule with cells. By measuring the cytoplasmic penetration antigen-binding molecular weight present in the cytoplasm multiple times, it is possible to observe the change in the cytoplasmic penetration antigen-binding molecular weight present in the cytoplasm over time.

[0117] In a preferred embodiment, after contacting the cytoplasmic-penetrating antigen-binding molecule with a non-target cell and a target cell, a) the amount of cytoplasmic-penetrating antigen-binding molecule present in the cytoplasm of the non-target cell at any given time point is b) 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less of the amount of cytoplasmic-penetrating antigen-binding molecule present in the cytoplasm of the target cell at the same time point.

[0118] In one embodiment, when the cytoplasmic invading antigen-binding molecular weight present in the cytoplasm at any time point is represented by an HRP luminescence signal or a fluorescent signal or a luminescence signal, after the cytoplasmic invading antigen-binding molecule is contacted with the non-target cell and the target cell, a) the HRP luminescence signal intensity or the fluorescent signal intensity or the luminescence signal intensity of the non-target cell at any time point is b) 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, 0.1 times or less, or 0.05 times or less than the HRP luminescence signal intensity or the fluorescent signal intensity or the luminescence signal intensity of the target cell at the same time point. In one embodiment, when a method including a cell expressing biotin ligase (BirA) is used, the amount of the cytoplasmic invading antigen-binding molecule present in the cytoplasm at any time point can be represented by an HRP luminescence signal. In a different embodiment, when imaging analysis (such as the method described in Example 4 and Reference Example 3) is used, the amount of the cytoplasmic invading antigen-binding molecule present in the cytoplasm at any time point can be represented by a fluorescent signal. In a different embodiment, when a split protein system (such as the method described in Reference Example 1) is used, the amount of cytoplasm-invaded antigen-binding molecule present in the cytoplasm at any time point can be represented by a luminescence signal.

[0119] In one embodiment of the present invention, a peptide linker may be used to fuse a cell surface antigen-binding domain and a cytoplasmic penetration domain, a cell surface antigen-binding domain and a cytoplasmic antigen-binding domain, a cytoplasmic penetration domain and a cytoplasmic antigen-binding domain, a cell surface antigen-binding domain and an Fc subunit, a cytoplasmic penetration domain and an Fc subunit, or a cytoplasmic antigen-binding domain and an Fc subunit. In addition, in an antigen-binding molecule having molecular forms 1 to 7 described below, a peptide linker may be used to fuse one Fab region to another Fab region, or to fuse a Fab region to an Fc subunit. For example, an amino acid selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn, Ala, particularly Gly and Ser, particularly Gly, etc. are included.

[0120] A suitable peptide linker can be readily selected by one skilled in the art from among different lengths, such as 1 amino acid (such as Gly) to 21 amino acids, 2 to 15 amino acids, or 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, including 3 to 12 amino acids.

[0121] Examples of peptide linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., including (GS)n, (GSGGS: SEQ ID NO:184)n, and (GGGS: SEQ ID NO:175)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Of these, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and thus tend to function as neutral tethers between components. Examples of flexible linkers made of glycine-serine polymers include, but are not limited to, Ser Gly·Ser(GS) Ser·Gly(SG) Gly Gly Ser (GGS) Gly·Ser·Gly (GSG) Ser Gly Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser Gly Ser (SGS) Gly Gly Gly Ser (GGGS: SEQ ID NO: 175) Gly Gly Ser Gly (GGSG: SEQ ID NO: 176) Gly·Ser·Gly·Gly (GSGG: SEQ ID NO: 177) Ser Gly Gly Gly (SGGG: SEQ ID NO: 178) Gly·Ser·Ser·Gly (GSSG: SEQ ID NO: 179) Gly Gly Gly Gly Ser (GGGGS: SEQ ID NO: 180) Gly Gly Gly Ser Gly (GGGSG: SEQ ID NO: 181) Gly Gly Ser Gly Gly (GGSGG: SEQ ID NO: 182) Gly·Ser·Gly·Gly·Gly (GSGGG: SEQ ID NO: 183) Gly·Ser·Gly·Gly·Ser (GSGGS: SEQ ID NO: 184) Ser Gly Gly Gly Gly (SGGGG: SEQ ID NO: 185) Gly·Ser·Ser·Gly·Gly (GSSGG: SEQ ID NO: 186) Gly·Ser·Gly·Ser·Gly (GSGSG: SEQ ID NO: 187) Ser Gly Gly Ser Gly (SGGSG: SEQ ID NO: 188) Gly·Ser·Ser·Ser·Gly (GSSSG: SEQ ID NO: 189) Gly Gly Gly Gly Gly Ser (GGGGGS: SEQ ID NO: 190) Ser Gly Gly Gly Gly Gly (SGGGGG: SEQ ID NO: 191) Gly Gly Gly Gly Gly Gly Ser (GGGGGGS: SEQ ID NO: 192) Ser Gly Gly Gly Gly Gly Gly (SGGGGGG: SEQ ID NO: 193) (Gly·Gly·Gly·Gly·Ser (GGGGS: SEQ ID NO: 194)) (Ser·Gly·Gly·Gly·Gly (SGGGG: SEQ ID NO: 195))n, where n is an integer of 1 or more. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0122] The amino acids contained in the amino acid sequences described herein may be post-translationally modified (e.g., modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art). Of course, such sequences having post-translationally modified amino acids are also included in the amino acid sequences described herein.

[0123] B. Antibodies In a further aspect of the disclosure, the antigen-binding molecule according to any of the above-mentioned embodiments is an antibody, which in a preferred embodiment is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the antigen-binding molecule is an antibody fragment (antigen-binding fragment), such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, for example, a complete IgG1 antibody, or other antibody class or isotype as defined herein.

[0124] In further aspects, an antibody according to any of the above embodiments may incorporate, either alone or in combination, any of the features described in items 1 to 7 below.

[0125] 1. Antibody avidity and affinity In certain embodiments, the binding activity or affinity of the antibodies provided herein is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 is the dissociation constant (KD) of the ATPase (M).

[0126] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-lives in vivo.

[0127] Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0128] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0129] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0130] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0131] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0132] Humanized antibodies and methods for making them are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) and also see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and further described in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0133] 4. Human antibodies In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0134] 5. Library-derived Antibodies Antibodies of the present disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described in, e.g., McCafferty et al., Nature 348:552-554;Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0135] 6. Multifunctional antibodies In certain embodiments, the antibody provided herein is a multifunctional antibody. A multifunctional antibody is a monoclonal antibody that has different functions at at least two different sites. For example, a multifunctional antibody is a monoclonal antibody that has two functions: (i) binding specificity and (ii) a function different from the binding specificity. An example of a function different from the binding specificity is cytoplasmic invasion ability. In one embodiment, a multifunctional antibody is a bifunctional antibody (having two functions: antigen binding specificity and cytoplasmic invasion ability). In one embodiment, a multifunctional antibody is a multispecific antibody (e.g., a bispecific antibody). A multispecific antibody is a monoclonal antibody that has binding specificity at at least two different sites. A bispecific antibody can be prepared as a full-length antibody or an antibody fragment. In one embodiment, a multifunctional antibody has binding specificity for an antigen and cytoplasmic invasion ability. In one embodiment, a multifunctional antibody has binding specificity for at least two different antigens and cytoplasmic invasion ability.

[0136] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and the knob-in-hole technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1993)). (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991). It will be appreciated that techniques similar to those for making multispecific antibodies described above can be used to make multifunctional antibodies.

[0137] Engineered antibodies with three or more functional antigen binding sites are also included herein, including "octopus antibodies" (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0138] Antibodies or fragments herein also include "dual acting Fabs" or "DAFs" (see, e.g., US Patent Application Publication No. 2008 / 0069820).

[0139] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody, such as the ability to enter the cytoplasm. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0140] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0141] [Table 1]

[0142] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) Residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0143] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Usually, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0144] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve the affinity of the antibody. Such modifications can be made in "hot spots" of HVRs, i.e., residues encoded by codons where mutations occur frequently during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that contact antigens, and the resulting mutated VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0145] In certain embodiments, substitution, insertion, or deletion may be made within one or more HVRs, as long as such modification does not substantially reduce the ability of the antibody to bind to antigen.For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.Such modifications may, for example, be outside the antigen contact residues of HVRs.In certain embodiments of the above mutant VH and VL sequences, each HVR is unmodified or contains only one, two, or three amino acid substitutions.

[0146] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is that described by Cunningham and Wells (1989) Science, 244:1081-1085, called "alanine scanning mutagenesis". In this method, a residue or group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as replacement candidates or excluded from replacement candidates. Mutants can be screened to determine whether they contain the desired properties.

[0147] Amino acid sequence insertions include fusions ranging in length from one residue to polypeptides containing 100 or more residues at the amino and / or carboxyl termini, as well as single or multiple amino acid residue insertions within the sequence. An example of a terminal insertion includes an antibody with a methionyl residue at the N-terminus. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0148] b) Glycosylation Mutants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0149] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, which are usually attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include, for example, various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present disclosure may be made to generate antibody variants with specific improved properties.

[0150] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 represents an asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 can also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0151] Further provided are antibody variants having bisected oligosaccharides, for example, bisected oligosaccharides added to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide added to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0152] c) Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0153] In certain embodiments, antibody variants with some, but not all, effector functions are also contemplated by the present disclosure, which make the antibody a desirable candidate for applications where its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). In addition, FcRn binding and in vivo clearance / half-life determinations may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0154] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0155] Certain antibody variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0156] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residues in EU numbering) of the Fc region).

[0157] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0158] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) have been described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution at Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0159] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0160] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0161] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly 1,3 dioxolane, poly 1,3,6, trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0162] In another embodiment, a conjugate is provided between an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that heat the non-protein moiety to a temperature that is not harmful to normal cells but that kills cells in close proximity to the antibody-non-protein moiety.

[0163] C. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods and constructs, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an antigen-binding molecule described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of an antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, Sp2 / 0 cell)). In one aspect, there is provided a method for producing an antigen-binding molecule of the present disclosure, comprising culturing a host cell comprising a nucleic acid encoding the antigen-binding molecule as described above under conditions suitable for expression of the antigen-binding molecule, and optionally recovering the antigen-binding molecule from the host cell (or host cell culture medium).

[0164] For recombinant production of the antigen-binding molecules of the present disclosure, nucleic acids encoding the antigen-binding molecules (such as those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids may be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an antibody).

[0165] When the antigen-binding molecule of the present disclosure is an antibody, suitable host cells for cloning or expressing the vector encoding the antibody include prokaryotic or eukaryotic cells as described herein. For example, the antibody may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste and may be further purified.

[0166] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0167] Host cells derived from multicellular organisms (invertebrate and vertebrate) are also suitable for the expression of glycosylated antibodies. Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified for conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0168] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0169] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include DHFR cells, -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0170] D. Assays The antigen-binding molecules provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0171] 1. Measurement of binding activity and affinity In one embodiment, the binding activity or affinity of an antibody is measured by a ligand capture method using a BIACORE® T200 or BIACORE® 4000 (GE Healthcare, Uppsala, Sweden) that uses surface plasmon resonance analysis as the measurement principle. The BIACORE® Control Software is used to operate the instrument. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, an anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with a 10 mM sodium acetate solution of appropriate pH and injected at an appropriate flow rate and injection time. The binding activity is measured using a buffer containing 0.05% polysorbate 20 (also known as Tween (registered trademark)-20) as the measurement buffer, at a flow rate of 10-30 μL / min, and at a measurement temperature of preferably 25°C or 37°C. When the measurement is performed by having the ligand capture molecule capture an antibody as a ligand, the antibody is injected to capture a desired amount, and then a serial dilution (analyte) of the antigen and / or Fc receptor prepared using the measurement buffer is injected. When the measurement is performed by having the ligand capture molecule capture an antigen and / or Fc receptor as a ligand, the antigen and / or Fc receptor is injected to capture a desired amount, and then a serial dilution (analyte) of the antibody prepared using the measurement buffer is injected.

[0172] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. For example, kinetic parameters are calculated by simultaneously fitting the sensorgrams of binding and dissociation using a 1:1 binding model, and the binding rate (kon or ka), dissociation rate (koff or kd), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, particularly when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (KD) may be calculated using a steady state model. As another parameter of binding activity, the "amount of analyte bound per unit amount of ligand" can also be calculated by dividing the amount of analyte bound (RU) at a specific concentration by the amount of ligand captured (RU).

[0173] 2. Measurement of antigen-binding molecules in the cytoplasm In one embodiment, the antigen-binding molecule in the cytoplasm is measured at any time point after the antigen-binding molecule is contacted with the cell. The antigen-binding molecule is contacted with the cell by any method, including incubation. When the antigen-binding molecule is contacted with the cell by incubation, the incubation time and the time until the antigen-binding molecule in the cytoplasm is measured after incubation are arbitrarily determined. For example, when the antigen-binding molecule present in the cytoplasm is measured only once after the antigen-binding molecule is contacted with the cell, the antigen-binding molecule and the cell are incubated for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours, and then the medium containing the antigen-binding molecule is removed, and the antigen-binding molecule in the cytoplasm is immediately measured. For example, when measuring the antigen-binding molecule present in the cytoplasm multiple times after contacting the antigen-binding molecule with cells, the antigen-binding molecule and cells may be incubated for 1, 2, 3, 4, 5, or 6 hours, the medium containing the antigen-binding molecule may be removed, and the antigen-binding molecule may be incubated with new medium containing no antigen-binding molecule for 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, and / or 16 hours, after which the antigen-binding molecule in the cytoplasm may be measured. The cytoplasmic invading ability of the antigen-binding molecule can also be detected or evaluated by detecting the antigen-binding molecule in the cytoplasm at any time point after contacting the antigen-binding molecule with cells. Therefore, it will be appreciated that the present disclosure also provides a method for detecting the cytoplasmic entry ability of an antigen-binding molecule.

[0174] a) BirA assay In one embodiment, the antigen-binding molecular weight present in the cytoplasm at any time point can be evaluated by detecting a fusion of a biotinylated Avi tag and an antigen-binding molecule with a labeled biotin-binding protein in a BirA assay. The biotinylated Avi tag is measured by using an avidin peroxidase conjugate and an appropriate substrate. For example, when the biotinylated Avi tag is detected with streptavidin-HRP, the antigen-binding molecular weight present in the cytoplasm can be represented by the intensity of the luminescence signal of HRP. The BirA assay is a method known to those skilled in the art, and is described, for example, in WPR Verdurmen et al., Journal of Controlled Release 200 (2015) 13-22, and in the Examples of the present disclosure. The conditions used in the assay for determining the antigen-binding molecular weight present in the cytoplasm can be appropriately selected by those skilled in the art and are therefore not particularly limited. In addition, the fusion of a biotinylated Avi tag and an antigen-binding molecule in the cytoplasm can also be labeled with other labeling substances that can be detected or measured. Specifically, radioactive labeling or fluorescent labeling is known. In the BirA assay, detection of the labeled biotin-binding protein (e.g., detection of HRP luminescence signal in streptavidin-HRP) can be appropriately performed by a method known to those skilled in the art. Specifically, Western blotting or capillary immunoassay (ProteinSimple) is known.

[0175] b) Imaging with a fluorescent microscope In another embodiment, the antigen-binding molecular weight present in the cytoplasm at any time can be evaluated by detecting it with a labeled protein that binds to the antigen-binding molecule. For example, when the antigen-binding molecule contains an IgG antibody, the antigen-binding molecule present in the cytoplasm can be detected with a labeled anti-IgG antibody. The label is, for example, a radioactive label or a fluorescent label. The detection of the labeled antigen-binding molecule can be appropriately performed by a method known to those skilled in the art. For example, as described herein and in the examples of the present disclosure, the labeled antigen-binding molecule can be detected by imaging the fluorescent signal using a fluorescent microscope.

[0176] c) Split-GFP In another embodiment, the antigen-binding molecular weight present in the cytoplasm at any time point can be evaluated by detecting the GFP fluorescent signal in a split-GFP complementary system. Specific methods are known to those skilled in the art and are described, for example, in WO2016 / 013870. Specifically, when GFP, a green fluorescent protein, is divided into fragments 1-10 and fragment 11, the property of showing fluorescence is eliminated, and if the distance between the two fragments becomes close and they bind, the property of showing fluorescence can be restored (Cabantous et al., 2005). Using such characteristics, the GFP fragments 1-10 are expressed in the cytoplasm, and the GFP fragment 11 is fused to an arbitrary site of the antigen-binding molecule. In this method, if GFP fluorescence is observed, it indicates that the two fragments of GFP are bound, that is, that the antigen-binding molecule is present in the cytoplasm.

[0177] d) Improved Split Protein System In another embodiment, the antigen-binding molecular weight present in the cytoplasm at any time point can be evaluated by detecting luminescence and / or fluorescence signals in a split protein system. In one embodiment, the split protein system includes (i) a peptide comprising a mitochondrial outer membrane protein and a first fragment of a luminescent protein, and (ii) a second fragment of a luminescent protein, where the first fragment of the luminescent protein and the second fragment of the luminescent protein as a whole contain a complete complement of the luminescent protein. In the split protein system, the first fragment of the luminescent protein and the second fragment of the luminescent protein do not have the property of showing luminescence, but when the first fragment of the luminescent protein contained in the peptide binds to the second fragment of the luminescent protein, the property of showing luminescence is restored. A method for evaluating cytoplasmic invasion ability using Nanoluc complementation has been reported (Schaub et al., Cancer Res. (2015) 75(23):5023, Dixon et al., ACS Chem Biol. (2016) 11(2):400), but the background luminescence and / or fluorescence signal of the method is still high. With these considerations in mind, in one embodiment, the inventors considered using mitochondrial outer membrane proteins in a split protein system to reduce background signal.

[0178] In one embodiment, the split photoprotein is Split NanoLuc®. NanoLuc is a small 19 kDa luciferase enzyme engineered from deep-sea luminescent shrimp. Split NanoLuc assay utilizes NanoLuc, which is cleaved into two subunits: a small 1.3 kDa Small BiT (SmBiT) (SEQ ID NO: 202) and a larger 18 kDa Large BiT (LgBiT) (SEQ ID NO: 203). Complementation of LgBiT and SmBiT is observed as luminescence from the enzyme reaction on the substrate in live cells using the NanoLuc Live Cell Assay System (Promega). In the Split NanoLuc assay, HeLa cells were genetically modified to stably express NanoLuc LgBiT in the cells. Usually, NanoLuc LgBiT can be expressed as a free cytoplasmic protein. However, in one embodiment, NanoLuc LgBiT is anchored to an intracellular structure in the cytoplasm, such as the outer mitochondrial membrane. The inventors have surprisingly found that anchoring NanoLuc LgBiT to the outer mitochondrial membrane results in significantly lower background signals and better assay sensitivity than when NanoLuc LgBiT is expressed as a free cytoplasmic protein. On the other hand, the SmBiT conjugate can be fused to any site on the antigen-binding molecule. Preferably, the antigen-binding molecule is an antibody, and the second fragment of the light-emitting protein is fused to the C-terminus of the heavy chain of the antibody. The antigen-binding molecule-SmBiT conjugate enters the cytoplasm, thereby complementing NanoLuc LgBiT to form a luciferase enzyme, which can be detected by enhanced light emission.

[0179] In one example, the disclosure provides an improved split protein system comprising (i) a peptide comprising an outer mitochondrial membrane protein and a first fragment of a luminescent protein, and (ii) a second fragment of a luminescent protein, wherein the first fragment and the second fragment of the luminescent protein collectively contain a full complement of the luminescent protein. In another example, the disclosure provides a peptide for use in an improved split protein system, wherein the peptide comprises an outer mitochondrial membrane protein and a first fragment of a luminescent protein, and wherein the first fragment and the second fragment of the luminescent protein collectively contain a full complement of the luminescent protein.

[0180] An example of the first fragment of a luminescent protein is NanoLuc Large BiT (LgBiT). An example of the second fragment of a luminescent protein is NanoLuc SmBiT (SmBiT). In a further embodiment, the peptide comprising a mitochondrial outer membrane protein and a first fragment of a luminescent protein further comprises green fluorescent protein (GFP) or a variant thereof. Preferably, the peptide comprising a mitochondrial outer membrane protein, a first fragment of a luminescent protein, and a GFP variant comprises the amino acid sequence of SEQ ID NO: 196.

[0181] The mitochondrial outer membrane protein used in the improved split protein system can be any protein that is localized on the mitochondrial outer membrane. The mitochondrial outer membrane protein can be, for example, AKAP1, BAK1, FIS1, CYB5B, FISS, FUNDC1, GDAP1, MARC1, MARC2, MFN1, MFN2, MFN3, MIEF1, MIEF2, MID49, MID51, MIGA1, MIGA2, MIRO1, MIRO2, MSTO1, MTX1, MTX2, MTX3, PGAM5, PLD6, RAB32, RMDN3, SYNJ2BP, TOM5, TOM6, TOM7, TOM20, TOM22, TOM34, TOM70, TSPO, and / or UBP30. In a preferred embodiment, the mitochondrial outer membrane protein is Additional Mitochondrial Kinase Anchoring Protein 1 (AKAP1; also known as A-kinase anchoring protein 1). In a preferred embodiment, the mitochondrial outer membrane protein comprises the amino acid sequence of SEQ ID NO: 197. In a preferred embodiment, the peptide comprising the first fragment of the photoprotein comprises the amino acid sequence of SEQ ID NO: 196 (AKAP1-eGFP-NanoLuc LgBit).

[0182] In one aspect, the disclosure provides a nucleic acid encoding a peptide comprising a mitochondrial outer membrane protein and a first fragment of a fluorescent protein, a vector comprising the nucleic acid, a cell comprising the nucleic acid or the vector, or a kit comprising the protein, nucleic acid, vector, or cell. In a further aspect, the disclosure provides a method for detecting a target molecule present in a cytoplasm, comprising: (i) providing a cell comprising a nucleic acid encoding a peptide comprising a mitochondrial outer membrane protein and a first fragment of a fluorescent protein; (ii) providing a target molecule conjugated to a second fragment of a fluorescent protein; (iii) contacting the cell of (i) and the target molecule of (ii); and (iv) detecting a luminescence signal in the cell of (iii), wherein the first fragment of the fluorescent protein and the second fragment together contain a full complement of the fluorescent protein. The method provided herein has a lower background luminescence signal compared to a similar method except that the peptide of (i) does not comprise a mitochondrial outer membrane protein. In a preferred aspect, the target molecule is a cytoplasmic entry antigen-binding molecule.

[0183] In another embodiment, in the improved split protein system, any protein localized on any intracellular structure in the cytoplasm can be used instead of the mitochondrial outer membrane protein. An example of a protein localized on an intracellular structure is a nuclear outer membrane protein, which can be any protein localized on the nuclear outer membrane. The nuclear outer membrane protein can be, for example, NUP37, NUP43, NUP96, NUP85, NUP88, NUP107, NUP133, NUP160, NUP214, and / or NUP358.

[0184] E. Methods for producing and screening antigen-binding molecules and methods for imaging cytoplasmic antigens 1. Method for producing antigen-binding molecules In one aspect, the present disclosure provides a method for producing an antigen-binding molecule of the present disclosure.

[0185] In one embodiment, the method comprises the steps of: (a) obtaining an expression vector comprising a suitable promoter operably linked to a gene encoding an antigen-binding molecule; (b) introducing the vector into a host cell and culturing the host cell to produce the antigen-binding molecule; (c) recovering the antigen-binding molecule from the host cell culture. Includes.

[0186] In one embodiment, the method comprises: (d) obtaining an expression vector comprising a gene encoding the cytoplasmic penetrating antibody or antigen-binding fragment thereof to be produced and a suitable promoter operably linked thereto; (e) introducing the vector into a host cell and culturing the host cell to produce the cytoplasmic penetrating antibody or antigen-binding fragment thereof; and (f) recovering said cytoplasmic penetrating antibody or antigen-binding fragment thereof from the host cell culture. Further includes:

[0187] In another embodiment, the method comprises the steps of: (a) providing a parent cytoplasmic-entering antigen-binding molecule; (b) introducing one or more amino acid substitutions in at least one selected from CDRL1 and CDRL3 in the light chain variable region of the parent cytoplasmic entry antigen binding molecule; and (c) identifying and / or selecting a cytoplasmic entry antibody or antigen-binding fragment thereof containing the one or more amino acid substitutions that exhibits enhanced cytoplasmic entry ability compared to the parent cytoplasmic entry antigen-binding molecule. In a preferred embodiment, the parent cytoplasmic entry antigen binding molecule comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 having the amino acid sequence shown in SEQ ID NO: 4. In another preferred embodiment, the parent cytoplasmic entry antigen binding molecule comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 4.

[0188] In one embodiment, the one or more amino acid substitutions introduced into the light chain variable region of the parent cytoplasmic penetration antigen-binding molecule are located at positions 24 to 34 and 89 to 97 according to the Kabat numbering in the amino acid sequence shown in SEQ ID NO: 4. In a preferred embodiment, the one or more amino acid substitutions are located at positions 24 to 32 and 93 to 97 according to the Kabat numbering in the amino acid sequence shown in SEQ ID NO: 4.

[0189] In one embodiment, the one or more amino acid substitutions introduced into the light chain variable region of the parent cytoplasmic entry antigen binding molecule are selected from the group consisting of the following (positions according to Kabat numbering): In the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid K at position 24 with A, D, E, F, G, H, I, L, N, Q, R, S, T, V, or Y; (b) substitution of amino acid S at position 25 with A, D, E, F, I, M, N, T, V, or Y; (c) substitution of amino acid S at position 26 with A, D, E, F, G, H, I, K, M, N, P, R, T, or V; (d) substitution of amino acid Q at position 27 with A, D, E, F, H, I, L, M, S, or Y; (e) substitution of amino acid S at position 27a with D; (f) substitution of amino acid L at position 27b with I, P, Q, T, or V; (g) substitution of amino acid F at position 27c with A, D, E, I, M, N, S, W, or Y; (h) substitution of amino acid N at position 27d with A, D, E, F, H, N, or Q; (i) substitution of amino acid S at position 27e with A, D, E, I, L, M, N, P, Q, T, V, or Y; (j) substitution of amino acid R at position 27f with A, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y; (k) substitution of amino acid T at position 28 with E, H, I, Q, S, V, W, or Y; (l) substitution of amino acid R at position 29 with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; (m) substitution of amino acid K at position 30 with A, D, E, F, H, I, L, M, N, P, Q, S, T, V, W, or Y; (n) substitution of amino acid N at position 31 with D, M, S, or T; (o) substitution of amino acid Y at position 32 with D, E, G, H, or Q; (p) substitution of amino acid Q at position 89 with H, I, T, or Y; (q) substitution of amino acid Y at position 91 with E, F, or N; (r) substitution of amino acid Y at position 93 with I, L, M, N, P, Q, S, T, V, or W; (s) substitution of amino acid H at position 94 with A, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, or Y; (t) substitution of amino acid M at position 95 with A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; (u) substitution of amino acid Y at position 96 with A, D, E, F, G, H, K, M, N, P, Q, S, T, or V; and (v) Substitution of amino acid T at position 97 with A, D, E, G, H, K, P, Q, R, S, or V.

[0190] In one embodiment, one or more amino acid substitutions introduced into the light chain variable region of the parent cytoplasmic entry antigen binding molecule enhance the cytoplasmic entry activity of the antibody or fragment thereof compared to a reference antibody or fragment thereof that does not contain the substitution and comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:4.

[0191] In one embodiment, the one or more amino acid substitutions introduced into the light chain variable region of the parent cytoplasmic entry antigen binding molecule are selected from the group consisting of the following (positions according to Kabat numbering): In the amino acid sequence shown in SEQ ID NO: 4, (a) Substitution of amino acid K at position 24 with N; (b) substitution of amino acid Q at position 27 with E; (c) substitution of amino acid S at position 27a with D; (d) substitution of amino acid N at position 27d with D or E; (e) substitution of amino acid S at position 27e with D or E; (f) substitution of amino acid R at position 27f with D, H, K, L, S, or V; (g) substitution of amino acid T at position 28 with D; (h) substitution of amino acid R at position 29 with E, G, M, or S; (i) substitution of amino acid K at position 30 with L or Q; (j) substitution of amino acid Y at position 32 with E; (k) substitution of amino acid Q at position 89 with H, I, T, or Y; (l) substitution of amino acid Y at position 91 with E, F, or N; (m) substitution of amino acid Y at position 93 with E; (n) substitution of amino acid H at position 94 with E, P, or Q; (o) substitution of amino acid M at position 95 with D, N, or P; and (p) Substitution of amino acid Y at position 96 with E or P.

[0192] In one embodiment, the one or more amino acid substitutions introduced into the light chain variable region of the parent cytoplasmic entry antigen binding molecule comprise a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: (a) S27eD / R27fK; (b) S27eD / R27fH; (c) N27dE / S27eD; (d) S27eD / T28D; (e) S27eD / R29E; (f) S27eD / R29S; (g) K24N / S27eD; (h) N27dD / R27fK; (i) N27dE / R27fK; (j) R27fK / T28D; (k) R27fK / R29E; (l) R27fK / R29S; (m) K24N / R27fK; (n) K24N / N27dD; (o) N27dD / T28D; (p)N27dD / R29E; (q) N27dD / R29S; (r) S27eD / Q89H (s) N27dD / Q89H; (t) R27fK / Q89H; (u) S27eD / Y91E; (v) N27dD / Y91E; (w) R27fK / Y91E; (x) Q89H / H94P; (y) Y91E / H94P; (z) H94P / M95N; (aa) H94P / M95D; (bb) H94P / M95P; (cc) H94E / Y96P; (dd) H94E / M95N; (ee) H94E / M95D; (ff) H94E / M95P; (gg) N27dE / R27fK / Q89H / H94E / M95P; (hh) N27dE / R27fK / Q89Y / H94E / M95P; (ii) N27dE / R27fK / Y91E / H94E / M95P; (jj) N27dE / R27fK / Y91F / H94E / M95P; (kk) N27dE / R27fS / Q89H / H94E / M95P; (ll) N27dE / R27fS / Q89Y / H94E / M95P; (mm) N27dE / R27fS / Y91E / H94E / M95P; (nn) N27dE / R27fS / Y91F / H94E / M95P; (oo) N27dE / R27fS / Q89H / H94Q / M95P; (pp) N27dE / R27fS / Q89Y / H94Q / M95P; (qq) N27dE / R27fS / Y91E / H94Q / M95P; (rr) N27dE / R27fS / Y91F / H94Q / M95P; (ss) N27dE / R27fV / Q89H / H94E / M95P; (tt) N27dE / R27fV / Q89Y / H94E / M95P; (uu) N27dE / R27fV / Y91E / H94E / M95P; (vv) N27dE / R27fV / Y91F / H94E / M95P; (ww) N27dE / R29S / Q89H / H94E / M95P; (xx) N27dE / R29S / Q89Y / H94E / M95P; (yy) N27dE / K30L / Q89H / H94E / M95P; (zz) N27dE / K30L / Q89Y / H94E / M95P; (aaa) S27eD / R27fK / Y91E / H94E / M95P; (bbb) S27eD / R27fK / Y91F / H94E / M95P; (ccc) S27eD / R27fK / Y91E / H94Q / M95P; (ddd) S27eD / R27fK / Y91F / H94Q / M95P; (eee) S27eD / R27fK / Y91E / H94Q; (fff) S27eD / R27fK / Y91F / H94Q; (ggg) S27eE / R27fK / Y91E / H94E / M95P; (hhh) S27eE / R27fK / Y91F / H94E / M95P; (iii) S27eE / R27fK / Y91E / H94Q / M95P; (jjj) S27eE / R27fK / Y91F / H94Q / M95P; (kkk) R27fS / R29S / Q89H / H94E / M95P (lll) R27fS / R29S / Q89Y / H94E / M95P; (mmm) R27fS / R29S / Y91E / H94E / M95P; (nnn) R27fS / R29S / Y91F / H94E / M95P; (ooo) R27fS / R29S / Q89H / H94Q / M95P; (ppp) R27fS / R29S / Q89Y / H94Q / M95P; (qqq) R27fS / R29S / Y91E / H94Q / M95P; (rrr) R27fS / R29S / Y91F / H94Q / M95P; (sss) N27dE / R27fS / R29S / Q89Y / H94Q / M95P; and (ttt) N27dE / R27fS / R29S / Y91F / H94Q / M95P.

[0193] In one embodiment, the method further comprises replacing the framework regions (FR) of the light chain variable region with human framework regions (FR).

[0194] In one embodiment, said method further comprises introducing a substitution of the amino acid L at position 2 with I and / or a substitution of the amino acid V at position 3 with Q according to the Kabat numbering.

[0195] In a preferred embodiment, the FR1 sequence of the light chain variable region is replaced with a human Vκ1, Vκ2, or Vκ3 FR1 sequence, more preferably with a human Vκ1 FR1 sequence. In a preferred embodiment, the FR2 sequence of the light chain variable region is replaced with a human Vκ1, Vκ2, or Vκ3 FR2 sequence, more preferably with a human Vκ1 FR2 sequence. In a preferred embodiment, the FR3 sequence of the light chain variable region is replaced with a human Vκ1, Vκ2, or Vκ3 FR3 sequence, more preferably with a human Vκ3 FR3 sequence. In one embodiment, the light chain variable region comprises human Vκ1 FR1, human Vκ1 FR2, and human Vκ3 FR3 sequences.

[0196] In one embodiment, the light chain variable region of the antibody with improved cytoplasmic entry ability comprises one, two, three, or four of the FR domains (FR1, FR2, FR3, and FR4) contained in SEQ ID NO:123.

[0197] In one embodiment, the method further comprises introducing one, two, three or more amino acid substitutions into CDRL2 of the light chain variable region of the parent cytoplasmic entry antigen binding molecule. In a preferred embodiment, the substitutions introduced into CDRL2 are selected from the group consisting of the following (positions according to Kabat numbering): In CDRL2 of the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid W at position 50 with A, G, I, T, or V; (b) substitution of amino acid S at position 52 with F or I; (c) substitution of amino acid T at position 53 with N or Y; and (d) Substitution of amino acid R at position 54 with K or V.

[0198] In a preferred embodiment, the amino acid substitutions introduced into the light chain variable region of the parent cytoplasmic entry antigen binding molecule comprise a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence shown in SEQ ID NO: 4, (a) L2I / V3Q / S27eD / R27fK / W50A / Y91F / H94Q; (b) L2I / V3Q / S27eD / R27fK / W50G / Y91F / H94Q; (c) L2I / V3Q / S27eD / R27fK / W50I / Y91F / H94Q; (d) L2I / V3Q / S27eD / R27fK / W50T / Y91F / H94Q; (e) L2I / V3Q / S27eD / R27fK / W50V / Y91F / H94Q; (f) L2I / V3Q / S27eD / R27fK / S52F / Y91F / H94Q; (g) L2I / V3Q / S27eD / R27fK / S52I / Y91F / H94Q; (h) L2I / V3Q / S27eD / R27fK / T53N / Y91F / H94Q; (i) L2I / V3Q / S27eD / R27fK / T53Y / Y91F / H94Q; (j) L2I / V3Q / S27eD / R27fK / R54K / Y91F / H94Q; (k) L2I / V3Q / S27eD / R27fK / W50I / S52F / Y91F / H94Q; (l) L2I / V3Q / S27eD / R27fK / W50I / T53N / Y91F / H94Q; and (m) L2I / V3Q / S27eD / R27fK / W50I / R54V / Y91F / H94Q.

[0199] In one embodiment, the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 150 and 161 to 173. In one embodiment, the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 123 to 150. In one embodiment, the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161 to 173. In one embodiment, the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced is in the form of Fab or scFv. In one embodiment, the cytoplasmic penetrating antibody or antigen-binding fragment thereof produced further comprises a heavy chain variable region.

[0200] 2. Screening method for cytoplasmic-penetrating antigen-binding molecules In one aspect, the present disclosure provides a method for screening for a cytoplasmic entry antigen-binding molecule. In one embodiment, the screening method comprises: (a) providing a parent cytoplasmic penetration antigen-binding molecule comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO:4; (b) introducing one or more amino acid substitutions in at least one selected from CDRL1 and CDRL3 in the light chain variable region of the parent cytoplasmic entry antigen binding molecule; and (c) identifying and / or selecting a cytoplasmic entry antibody or antigen-binding fragment thereof containing the one or more amino acid substitutions that exhibits enhanced cytoplasmic entry ability compared to the parent cytoplasmic entry antigen-binding molecule. Includes.

[0201] In one embodiment, the method comprises: (c) obtaining an expression vector comprising a gene encoding the cytoplasmic penetrating antibody or antigen-binding fragment thereof to be produced and a suitable promoter operably linked thereto; (d) introducing the vector into a host cell and culturing the host cell to produce the cytoplasmic penetrating antibody or antigen-binding fragment thereof; and (e) recovering said cytoplasmic penetrating antibody or antigen-binding fragment thereof from the host cell culture. Further includes:

[0202] 3. Imaging methods for cytoplasmic antigens In another aspect, the present disclosure provides a method for imaging a cytoplasmic antigen, comprising using an antigen-binding molecule of the present disclosure, an antigen-binding molecule produced by the above-described production method, or a cytoplasmic-penetrating antigen-binding molecule identified as a suitable molecule by the above-described screening method. In another aspect, the present disclosure provides an antigen-binding molecule of the present disclosure, an antigen-binding molecule produced by the above-described production method, or a cytoplasmic-penetrating antigen-binding molecule identified as a suitable molecule by the above-described screening method, for use in imaging of a cytoplasmic antigen in a sample cell. In yet another aspect, the present disclosure provides use of an antigen-binding molecule of the present disclosure, an antigen-binding molecule produced by the above-described production method, or a cytoplasmic-penetrating antigen-binding molecule identified as a suitable molecule by the above-described screening method, in the manufacture of an imaging agent for a cytoplasmic antigen. In certain embodiments of these aspects, the antigen binding molecule used is labeled.

[0203] F. Immunoconjugates The present disclosure also provides an immunoconjugate comprising the antigen-binding molecule of the present invention conjugated to a bioactive molecule.In one embodiment, the bioactive molecule is selected from the group consisting of peptides, proteins, toxins, antibodies, antibody fragments, RNA, DNA, small molecule drugs, nanoparticles, and liposomes.In one embodiment, the immunoconjugate comprises the antigen-binding molecule of the present invention conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant, or animal origin, enzymatically active toxins, or fragments thereof), or radioisotopes.

[0204] G. Methods for specifically delivering antigen-binding molecules into the cytoplasm of target cells and methods for improving the cytoplasmic entry ability of cytoplasmic-entering antigen-binding molecules In one aspect, the present disclosure provides a method for specifically delivering an antigen-binding molecule into the cytoplasm of a target cell, wherein the cell surface antigen to which the antigen-binding molecule binds is an antigen specifically expressed in the target cell. In one embodiment, the method includes contacting the antigen-binding molecule with the target cell. In one embodiment, the antigen-binding molecule is a multifunctional antigen-binding molecule that combines a cytoplasm-penetrating antibody or a fragment thereof, an antibody or a fragment thereof that binds to a cell surface antigen of the target cell, and / or an antibody or a fragment thereof that binds to an antigen in the cytoplasm. In a particular embodiment, the method does not substantially deliver the antigen-binding molecule to a cell that does not express the cell surface antigen. In a particular embodiment, the antigen-binding molecule specifically internalizes into the target cell by binding to a cell surface antigen specific to the target cell, and translocates to the cytoplasm. In a particular embodiment, the antigen-binding molecule forms a multimer and exhibits high cytoplasm-penetrating ability. In another aspect, the present disclosure provides a method for improving the cytoplasmic entry ability of a cytoplasmic entry antigen-binding molecule, comprising introducing one or more amino acid modifications into the Fc region that promote multimer formation, as compared to a parent cytoplasmic entry antigen-binding molecule comprising a parent Fc region that does not contain the one or more amino acid modifications.

[0205] In another aspect, the present disclosure provides a method for specifically delivering a heterologous moiety into the cytoplasm of a target cell, the heterologous moiety being conjugated to an antigen-binding molecule comprising a cell surface antigen-binding domain and a cytoplasmic entry domain. Preferably, the antigen-binding molecule comprises a first and a second Fab region, (a) the first Fab region specifically binds to a cell surface antigen, and (b) the second Fab region has a cytoplasmic entry capability, and the antigen-binding molecule is conjugated to a heterologous moiety. More preferably, the antigen-binding molecule is a bifunctional antibody and does not comprise a cytoplasmic antigen-binding domain. In one embodiment, the cell surface antigen to which the antigen-binding molecule binds is an antigen specifically expressed on a target cell. In one embodiment, the method comprises contacting the target cell with the antigen-binding molecule conjugated to the heterologous moiety. In a preferred embodiment, the method comprises specifically delivering a heterologous moiety into the cytoplasm of the target cell. In a preferred embodiment, the method does not substantially deliver the heterologous moiety to cells that do not express the cell surface antigen.

[0206] H. Methods for Targeting Cytoplasmic Antigens In certain embodiments, the antigen binding molecule of the present disclosure has an increased ability to remove or suppress cytoplasmic antigens in target cells.Therefore, the antigen binding molecule provided herein is useful for knocking down cytoplasmic antigens in target cells at the protein level.The term "knockdown" as used herein means that the expression or abundance of a particular protein is reduced.Specifically, when cytoplasmic antigens are knocked down at the protein level, the amount of cytoplasmic antigens per cell is reduced.In another specific embodiment, the antigen binding molecule of the present disclosure has an increased ability to activate cytoplasmic antigens in target cells.

[0207] I. Methods and Compositions for Diagnosis and Detection In certain embodiments, the antigen-binding molecules provided herein are useful for detecting the presence of a cytoplasmic antigen in a target cell in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection.

[0208] In one embodiment, the antigen-binding molecule is provided for use in diagnostic or detection methods.In a further aspect, a method is provided for detecting the presence of cytoplasmic antigen in target cells in biological samples.In a particular embodiment, the method comprises contacting a biological sample with the antigen-binding molecule described herein under conditions that allow the antigen-binding molecule to bind to the cytoplasmic antigen, and detecting whether a complex is formed between the antigen-binding molecule and the cytoplasmic antigen.Such a method can be an in vitro method or an in vivo method.In a different embodiment, the antigen-binding molecule of the present disclosure is useful for detecting cytoplasmic antigen by methods such as imaging.

[0209] In certain embodiments, the antigen-binding molecule of the present disclosure is provided as being labeled. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and indirectly detectable moieties (e.g., enzymes or ligands) through, for example, enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, the following: radioisotopes 32 P, 14 C. 125 I, 3 H and 131I, fluorophores such as rare earth chelates or those linked to fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0210] J. Pharmaceutical Preparations Pharmaceutical formulations of the antigen-binding molecules described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antigen-binding molecules having the desired purity with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, the following: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.; small (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of their use (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

[0211] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0212] The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated. Those with complementary activities that do not adversely affect each other are preferred. Such active ingredients are present in suitable combinations in amounts that are effective for the intended purpose.

[0213] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0214] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antigen-binding molecules, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0215] Preparations to be used for in vivo administration are typically sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0216] K. Therapeutic Methods and Compositions In one aspect, the antigen binding molecules provided herein may be used in therapeutic methods. In one embodiment, the antigen-binding molecule of the present disclosure is provided for use as a medicament. In a particular embodiment, the antigen-binding molecule of the present disclosure is provided for use in a method of treatment. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., as described below). The "individual" according to any of the above embodiments is preferably a human.

[0217] In a further aspect, the present disclosure provides the use of the antigen-binding molecule of the present disclosure in the manufacture or preparation of a medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments may be a human.

[0218] In a further aspect, the present disclosure provides a pharmaceutical formulation comprising any of the antigen-binding molecules provided herein (e.g., for use in any of the therapeutic methods described above).In one embodiment, the pharmaceutical formulation comprises any of the antigen-binding molecules provided herein and a pharma- ceutically acceptable carrier.In another embodiment, the pharmaceutical formulation comprises any of the antigen-binding molecules provided herein and at least one additional therapeutic agent.

[0219] The antigen-binding molecules of the present disclosure (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, such as by injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or repeated administration over various time periods, bolus administration, and pulse infusion.

[0220] The antigen-binding molecules of the present disclosure are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antigen-binding molecules are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the formulation, the type of disorder or treatment, and other factors discussed above. These are typically used in the same dosages and routes of administration as described herein, or about 1 to 99% of the dosages described herein, or any dosage and any route deemed empirically / clinically appropriate.

[0221] For the prevention or treatment of a disease, the appropriate dose of the antigen-binding molecule of the present disclosure (when used alone or with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody if the antigen-binding molecule of the present disclosure is an antibody, the severity and course of the disease, whether the antigen-binding molecule is administered for prophylactic or therapeutic purposes, medical history, the patient's clinical history and response to the antigen-binding molecule, and the discretion of the attending physician. The antigen-binding molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the antigen-binding molecule may be the initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion. One typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or longer, depending on the circumstances, treatment is usually maintained until the desired suppression of disease symptoms occurs. One exemplary dose of the antigen-binding molecule is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives about 2 to about 20, or for example about 6 doses of the antigen-binding molecule). A high initial loading dose may be followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and measurements.

[0222] It will be understood that any of the above-described formulations or therapeutic methods may be practiced using an immunoconjugate of the present disclosure in place of, or in addition to, an antigen-binding molecule.

[0223] L.Product In another aspect of the disclosure, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition is an antigen-binding molecule of the present disclosure. The label or package insert indicates that the composition is used to treat the condition of choice. The article of manufacture may further include (a) a first container with a composition comprising an antigen-binding molecule of the present disclosure contained therein; and (b) a second container with a composition comprising an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture in this aspect of the disclosure may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0224] It will be understood that any of the above-mentioned articles of manufacture may contain an immunoconjugate of the present disclosure instead of or in addition to an antigen-binding molecule. EXAMPLES

[0225] The following are examples of the methods and compositions of the present disclosure. In light of the general description above, it will be understood that various other embodiments may be practiced.

[0226] The foregoing invention has been described in detail by way of illustration and example for purposes of facilitating a clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

[0227] Example 1: A comprehensive mutagenesis approach to improve the cytoplasmic transport ability and antibody expression levels of the parent light chain variable region (VL) hT4VL.FRv4 The present invention provides a cytoplasmic penetration antibody, particularly a VL, having improved cytoplasmic permeability over the previously reported cytoplasmic penetration antibody h3D8, particularly the light chain variable region (VL) of h3D8 disclosed in WO2016013870 (i.e., hT4VL); and confirmed that the antigen-binding molecule or antibody has improved permeability to the cytoplasm.

[0228] The inventors first converted each framework (FR) of hT4VL (SEQ ID NO: 174) to the germline sequence of human VK1 or VK3 as listed in Table 2. As a result, hT4VL.FRv4 (SEQ ID NO: 4), which differs from hT4VL only by having human VK3 (VK3) FR3, was finally selected for further improvement as the best VL candidate in terms of the combination of expression yield, aggregation profile revealed by a single SEC peak, and cytoplasmic entry activity. Subsequently, hT4VL.FRv4 (SEQ ID NO: 4) was selected as the parent VL to comprehensively introduce amino acid modifications other than cysteine ​​at all positions in CDRL1 and CDRL3 to find mutations that can improve cytoplasmic transport ability and antibody expression level. Genes encoding VL variants were transiently expressed in combination with a light chain constant region (KT0, SEQ ID NO: 12) and a heavy chain expression vector (3D8H-SG1.SmBiTb, SEQ ID NO: 13) using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Expression and purification were performed by methods known to those skilled in the art. The expression levels of the variants are summarized in Table 3. The variants with improved expression levels compared to the parent antibody with hT4VL.FRv4 are shown in bold. To evaluate the cytoplasmic transport ability, the inventors performed the Split Nanoluc (Split Nluc) assay described in Reference Example 1.

[0229] [Table 2]

[0230] [Table 3]

[0231] [Table 4]

[0232] Example 2: Mutants with Amino Acid Mutations that Improve Cytoplasmic Entry and Other Properties of hT4VL.FRv4 Based on the results of Example 1, several mutants were selected to introduce further amino acid mutations (Table 5), and Ab expression level, cytoplasmic transport ability, and extracellular matrix (ECM) binding were tested. Antibodies with low ECM binding were considered more suitable because high ECM binding can worsen antibody pharmacokinetics (US2014 / 0080153). ECM binding studies were performed as described in Reference Example 2. The results were summarized in Table 5 and Tables 6-1 and 6-2. Mutants with better antibody expression level, higher split Nanoluc signal, and lower ECM binding compared to those of parent antibody hT4VL.FRv4 are shown in bold. Acidic residues (D or E) appear to be better at positions 27, 27a, 27d, 27e, and 28 (Kabat numbering), while lysine or serine at position 27f (Kabat numbering), and proline or glutamine at position 94 (Kabat numbering) appear to be promising amino acid residues at the respective positions. Importantly, most of the combination mutants listed in Tables 6-1 and 6-2 showed better profiles than the parent Ab (hT4VL.FRv4). In particular, the mutation combinations S27eD / R27fK, N27dE / R27fK, N27dD / Q89H, R27fK / Y91E, H94E / M95P, N27dE / R27fK / Q89Y / H94E / M95P, N27dE / R27fK / Y91F / H94E / M95P, N27dE / R27fS / Q89Y / H94E / M95P, and N27d E / R27fS / Q89Y / H94Q / M95P, S27eD / R27fK / Y91F / H94Q / M95P, S27eD / R27fK / Y91F / H94Q, and S27eE / R27fK / Y91F / H94Q / M95P (Kabat numbering) showed lower ECM binding while exhibiting higher Split Nluc signals compared to other combinations.

[0233] [Table 5]

[0234] [Table 6-1]

[0235] [Table 6-2] TIFF2024500267000008.tif234170

[0236] Example 3: Mutants with FR1 mutations In a previous report, hT4VL is a humanized VL with two non-human germline residues (Leucine at position 2 and Valine at position 3 corresponding to FR1 of mouse VK1) in FR1 of human VK1, and it was reported that the mouse residue Leucine at position 2 is required for cytoplasmic transport activity (Dong-Ki Choi et al., mAbs 6:6, 1402--1414; November / December 2014; WO2016013870). In Example 1 and Example 2 of the present application, the same modified human VK1 FR1 was used during FR reselection.

[0237] The 28 mutants identified in Example 2 were selected to additionally introduce a substitution of leucine at position 2 to isoleucine (L2I) and a substitution of valine at position 3 to glutamine (V3Q) in FR1, such that the mutants have the wild-type human germline VK1 FR1 sequence (i.e., back-mutation of mouse residues to the corresponding human residues in human VK1 FR1) (Table 7). The inventors envisioned that such antibody mutants with fully human VK1 FR1 would be closer to human germline and, as a result, have a lower immunogenicity risk. Ab expression levels, cytoplasmic transport ability, ECM binding, and also SEC (size exclusion chromatography) profiles were evaluated for the mutants shown in Table 7.

[0238] The aggregation profile was evaluated by size-exclusion chromatography (SEC) analysis using UPLC (Acquity H-Class bio, Waters) for each mutant. 50 mM phosphate / 300 mM NaCl (pH 7.0) was used as the running buffer and a BEH200 column (Waters, #186005225) was used as the analytical column. Chromatograms were recorded by 220 nM UV absorption. Data were processed using Empower3 software (Waters) to calculate the monomer peak percentage and full width at half maximum of the monomer peak. A high monomer peak percentage and a sharp symmetrical peak shape (narrow peak width) are considered as good physicochemical properties. The results of the SEC analysis are summarized in Table 7 (mutants that showed a better profile compared to the original hT4VL are shown in bold). While the monomer peak percentage of the original hT4VL was lower than 70% and the peak width could not be calculated due to its asymmetric peak shape, all of the mutants with the mutations listed in Table 7 showed better monomer peak percentages and sharper peak shapes, as shown in Figure 1.

[0239] Notably, in contrast to the case of hT4VL, which requires non-human germline residues in VK1 FR1 to retain its cytoplasmic transport ability, mutants with the L2I / V3Q mutation (corresponding to the wild-type fully human VK1 FR1 sequence) surprisingly exhibit improved or strong cytoplasmic transport ability (Table 7), as well as improved Ab expression levels, lower ECM binding, and better SEC (size exclusion chromatography) profiles.

[0240] Based on all the results, hT4VL.FRv40563 (L2I / V3Q / S27eD / R27fK / Y91F / H94Q, SEQ ID NO: 145) was selected as the template antibody for further CDRL2 optimization. As a result of comprehensive mutagenesis of the CDRL2 region, several mutations (e.g., W50A / G / I / T / V, S52F / I, T53N / Y, R54K / V, and combinations thereof) significantly improved the Split Nluc signal compared to the template antibody while retaining low ECM binding (Table 8).

[0241] [Table 7] TIFF2024500267000010.tif23456

[0242] [Table 8]

[0243] Example 4: Preparation of bifunctional antibodies containing a cytoplasmic entry domain and a cell surface antigen-binding domain; and imaging analysis of the cytoplasmic transport of the bifunctional antibodies in ASGPR-expressing cell lines In order to specifically deliver the bifunctional antibody to the cytoplasm of target cells in a cell surface antigen-dependent manner as reported in WO2020022262, an attempt was made to construct a bifunctional and multivalent antibody from the cytoplasmic penetrating antibody identified in Example 3 and an antibody against a cell surface antigen.

[0244] Imaging analysis was carried out to evaluate the cytoplasmic entry ability of the bifunctional antibody in a receptor-dependent manner. The antibody was transiently expressed using Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) and purified by methods known to those skilled in the art. The antibodies listed in Table 9 were used to prepare the bifunctional antibodies listed in Table 10. The bifunctional antibodies were prepared by methods known to those skilled in the art. To target ASGPR, anti-ASGPR antibody AGA0078 (VH: SEQ ID NO: 159, VL: SEQ ID NO: 156) was used.

[0245] In the imaging analysis, the ASGPR-expressing HeLa cell line (HeLa / ASGPR / BirA) described in Reference Example 3 was used. HeLa / ASGPR / BirA cells were cultured in Minimum Essential Medium Eagle (Sigma, Cat# M4655-500ML) containing 10% FBS (Sigma Aldrich, Cat# 172012-500ML), Penicillin-Streptomycin (Gibco, Cat# 15140-122), 750 μg / mL Geneticin (Thermo Fisher Scientific, Cat# 10131-027), and 100 μg / mL zeocin (Thermo Fisher Scientific, Cat# R25001). The HeLa / ASGPR / BirA suspension was added to BioCoat Collagen I Multiwell Plates 96-well Black / clear (Corning, Cat# 354649) at 50 μL / well (6 × 10 3The cells were seeded with 100 μL / well of 200 mM glycine (Wako Pure Chemical Industries, Cat# 077-00735)-HCl (Wako Pure Chemical Industries, Cat# 083-01095), 150 mM NaCl (Wako Pure Chemical Industries, Cat# 191-01665), pH 2.5. The cells were then cultured overnight in a humidified 5% CO2, 37°C incubator. After removing the culture supernatant, the cells were incubated with 3 μM of antibody in 50 μL / well of culture medium for 3 hours in a humidified 5% CO2, 37°C incubator. After incubation, the cells were washed twice in 200 mM glycine (Wako Pure Chemical Industries, Cat# 077-00735)-HCl (Wako Pure Chemical Industries, Cat# 083-01095), 150 mM NaCl (Wako Pure Chemical Industries, Cat# 191-01665), pH 2.5. The cells were then fixed with 4% paraformaldehyde-phosphate buffer (Nacalai Tesque, Cat# 09154-56) for 15 min at room temperature and permeabilized with PBS containing 0.5% Triton X-100 (Bio-Rad, Cat# 161-0407) and 5% FBS. The solution used for permeabilization was also used for subsequent incubation with secondary antibodies and washing steps. The cells were incubated with 1 / 500 diluted Goat Anti-Human IgG-AF488 (SouthernBiotech, Cat# 2040-30) and 1 / 2000 diluted Hoechst33342 (Thermo Fisher Scientific, Cat# H3570) for 30 min at room temperature. After washing twice, the cells were subjected to imaging analysis using an Operetta CLS High-Content Analysis System (Perkin Elmer). The fluorescence intensity of Alexa488 was evaluated by using Harmony software (Perkin Elmer).

[0246] As a result, the antibody in the bifunctional molecule format with the anti-ASGPR antibody (AGA0078) selected in Example 3 showed higher Alexa488 fluorescence intensity than that in the bifunctional molecule format with the anti-KLH antibody (IC17) (Figure 2). This indicates that the bifunctional antibody can be delivered into HeLa / ASGPR / BirA cells in an ASGPR-dependent manner, supporting the receptor-dependent cytoplasmic transport observed by the Split-NanoLuc assay in Example 3.

[0247] [Table 9] TIFF2024500267000013.tif234170

[0248] [Table 10] TIFF2024500267000015.tif216157TIFF2024500267000016.tif216157TIFF2024500267000017.tif198157

[0249] Example 5: Cytoplasmic transport ability was confirmed using a heavy chain other than 3D8 Although the same 3D8 heavy chain (SEQ ID NO: 13) was used in Examples 1-4 above, the inventors further evaluated whether the newly identified light chain (VL) variants in Examples 1-4 would retain their cytoplasmic transport ability when combined with other heavy chains other than 3D8. To this end, the anti-IL6R heavy chain, AL1W8P226 (SEQ ID NO: 151), was used as a control and combined with the VL variants identified in Examples 1-4. A bifunctional antibody comprising an anti-IL6R heavy chain and the VL variants identified in Examples 1-4 was prepared and tested for cytoplasmic transport ability by Split Nluc assay using HepG2 cells that endogenously express IL6R on the cell surface. As a result, the antibody comprising the VL variant together with AL1W8P226 showed a higher Split Nluc signal compared to a bivalent anti-IL6R antibody (heavy chain SEQ ID NO: 152, light chain SEQ ID NO: 153) and also compared to an antibody comprising AL1W8P226 together with the negative control light chain IC17L (SEQ ID NO: 154), suggesting that the VL variant exhibits cytoplasmic transport activity and that target cell (IL6R)-specific cytoplasmic transport may be mediated by IL6R binding (Figure 3).

[0250] Example 6: Antibody Pharmacokinetic (PK) Profiles of Optimized Variants As demonstrated in Example 2 and Example 3, the inventors have succeeded in identifying cell-penetrating antibodies with dramatically improved physicochemical properties, such as ECM binding and SEC profile, which may affect antibody PK. One of the selected mutants, 3D8H / hT4VL.FRv40563 (VH: SEQ ID NO: 158, VL: SEQ ID NO: 145), was subjected to a mouse intravenous administration test. After antibody administration, blood was sampled over time and plasma was obtained. The plasma concentration of the administered antibody was then measured by ELISA. The time course data of the antibody concentration in plasma was analyzed with WinNonlin ver 7.0 (Certara) to calculate the antibody clearance (CL). As a result, the antibody PK profile of the newly identified mutant (3D8H / hT4VL.FRv40563) was significantly improved compared to 3D8 (3D8H / hT4VL) (Figure 12). In addition, the PK of a bifunctional antibody containing a cytoplasmic entry domain (FRv40563) and a cell surface antigen binding domain (anti-ASGPR antibody, AGA0078) was also evaluated. ASGPR is mainly expressed in the liver, and the bivalent anti-ASGPR antibody showed very fast excretion. As shown in Figure 13, FRv40563n / / AGA0078p showed faster clearance compared to its control antibody FRv40563n / / IC17p, suggesting that the bifunctional antibody FRv40563n / / AGA0078p can be delivered to the liver in an ASGPR-dependent manner. In addition, FRv40563n / / AGA0078p showed faster clearance than IC17n / / AGA0078p. This could support ASGPR-dependent cytoplasmic transport in vivo.

[0251] Reference Example 1: Split NanoLuc assay for evaluating cytoplasmic transport ability (1) Concept of the Split NanoLuc Assay NanoLuc is a small 19 kDa luciferase enzyme engineered from deep-sea luminescent shrimp. The Split NanoLuc assay utilizes NanoLuc that is cleaved into two subunits: the small 1.3 kDa Small BiT (SmBiT) and the larger 18 kDa Large BiT (LgBiT). Complementation of LgBiT and SmBiT is observed as luminescence from the enzymatic reaction on a substrate in live cells using the NanoLuc Live Cell Assay System (Promega). In the Split NanoLuc assay, HeLa cells were genetically engineered to stably express NanoLuc LgBiT intracellularly. NanoLuc LgBiT was either expressed as a free cytoplasmic protein or anchored to an intracellular structure in the cytoplasm, e.g., the outer mitochondrial membrane. Anchoring NanoLuc LgBiT to the outer mitochondrial membrane resulted in significantly lower background signal and better assay sensitivity compared to when NanoLuc LgBiT was expressed as a free cytoplasmic protein. This was despite the fact that the tethered NanoLuc LgBiT had lower overall expression as measured by GFP. SmBiT was fused or conjugated to the antibody tested. Entry of the antibody-SmBiT conjugate into the cytoplasm would complement NanoLuc LgBiT to form the luciferase enzyme, which could be detected by enhanced light emission.

[0252] (2) Construction of cell lines To generate HeLa cells stably expressing human ASGPR, cells were transfected with plasmids encoding human ASGPR-H1 (SEQ ID NO: 198) and ASGPR-H2 (SEQ ID NO: 199) subunits, respectively, at a ratio of 2:1 using Lipofectamine 3000 (Invitrogen). ASGPR-H1 and ASGPR-H2 were cloned into the plasmid pCXND3, which uses the CAG promoter and encodes the neomycin antibiotic resistance gene. Antibiotic-resistant cells were further enriched for high expression of ASGPR by cell sorting on a FACSAria III (BD). Enriched cells were then isolated by limiting dilution and plating, and identified as single cell clones by microscopic observation (Solentim, Cell Metric CLD). To generate cells expressing both ASGPR and eGFP-Nanoluc LgBiT, and cells expressing ASGPR and AKAP1-eGFP-NanoLuc LgBiT, HeLa cells stably expressing ASGPR were nucleofected using the Cell Line Optimization 4D-Nucleofector™ X Kit (Lonza) and the 4D-Nucleofector Core unit and 4D-Nucleofector X unit (Lonza) using the preset nucleofection program for HeLa. The LgBiT constructs were cloned into the plasmid pCXZD1, which uses the CAG promoter and encodes the zeocin antibiotic resistance gene. The first was NanoLuc LgBiT conjugated to the C-terminus of eGFP (eGFP-NanoLuc LgBit; SEQ ID NO: 200). Next was one with an additional mitochondrial kinase anchoring protein 1 (AKAP1) conjugated to the N-terminus of eGFP (AKAP1-eGFP-NanoLuc LgBit; SEQ ID NO: 196). Antibiotic-resistant cells were further enriched for high expression of eGFP by cell sorting on a FACSAria III (BD).The enriched cells were then isolated by limiting dilution and plating and identified as single cell clones by microscopic observation (Solentim, Cell Metric CLD). HeLa cells were cultured in standard culture medium of Minimum Essential Media (Gibco) containing 10% v / v fetal bovine serum, 1% v / v MEM Non-Essential Amino Acids Solution (Gibco), 1% v / v sodium pyruvate (Gibco), and 1% v / v Penicillin-Streptomycin (Gibco). HeLa clones expressing asialoglycoprotein receptor (ASGPR) were cultured in standard culture medium supplemented with 1 mg / mL Geneticin (G418 sulfate) (Gibco). HeLa cells expressing ASGPR and eGFP-NanoLuc LgBiT, or ASGPR and AKAP1-eGFP-NanoLuc LgBiT were cultured in standard culture medium containing 1 mg / mL Geneticin and 600 μg / mL Zeocin (Invivogen). All cell types were cultured in a humidified 5% CO2, 37 °C incubator.

[0253] (3) Assay set conditions HeLa cells expressing ASGPR and eGFP-NanoLuc LgBiT (hereafter referred to as HeLa-Nluc) and HeLa cells expressing ASGPR and AKAP1-eGFP-NanoLuc LgBiT (hereafter referred to as HeLa-AKAP1_Nluc) were rinsed with DPBS, no calcium, no magnesium (Gibco). The cells were then trypsinized with 0.25% Trypsin-EDTA (Gibco) at room temperature until detached from the culture flask. Trypsin was then neutralized in excess culture medium containing 10% FBS. The cells were pelleted by centrifugation at 200 × g for 5 min at 4 °C and rinsed twice with chilled Opti-MEM (Gibco). The cells were then diluted to 1 × 10 6Cells / mL were suspended in chilled Opti-MEM and kept cold for up to 30 minutes prior to use. Antibodies to be tested were diluted 11x the test concentration in histidine buffer, 20 mM His-HCl + 150 mM NaCl (Nacalai Tesque). 5x substrate from the NanoLuc Live Cell Assay System (Promega, Cat#N2011) was prepared at room temperature. 1x10 5 Cells were transferred to 100 μL per well (Perkin Elmer, Cat#6005688) and 10 μL of 11x antibody was added directly to the cells and mixed with a pipette. 5x substrate was then added to the cell and antibody mixture immediately after mixing.

[0254] The plate was immediately placed in a pre-warmed 37°C luminescence plate reader (Promega, Glomax Explorer). Luminescence was read after 12 cycles of a two-step read cycle, 5 min apart, and shaking the plate at 400 rpm for 1 min. Fold changes relative to buffer wells are summarized in Tables 4-8. Mutants showing superior fold changes compared to the parent antibody are shown in bold.

[0255] Reference Example 2: Characterization of antibody extracellular matrix (ECM) binding The binding of each antibody to ECM (extracellular matrix) was evaluated by the following procedure with reference to WO2012093704 A1: ECM Phenol red free (BD Matrigel #356237) was diluted to 2 mg / mL in TBS and 5 μL was added to the center of each well of an ECL assay plate (L15XB-3, MSD KK, high bind) cooled on ice. The coated plate was then sealed and stored overnight at 4°C. The assay was then performed the next day at room temperature. 150 μL of ECL blocking buffer (PBS supplemented with 0.5% BSA and 0.05% Tween 20) was added to each well of the coated plate and the plate was left at room temperature for at least 2 hours. The blocking buffer was then removed. Each antibody sample was diluted to 9 μg / mL in ACES-T and further diluted to 3 μg / mL in ECLDB (PBS supplemented with 0.1% BSA and 0.01% Tween 20). 50 μL of the diluted antibody solution was added to each well and incubated for 1 hour at 30° C. with shaking at 600 rpm. The samples were then removed by gently tapping the inverted plate. Next, 50 μL of glutaraldehyde was added to each well to fix the samples and incubated for 10 minutes at room temperature. The wells were then washed three times with PBS-T (PBS supplemented with 0.05% Tween 20). The secondary antibody was diluted to 1 μg / mL in ECLDB and 50 μL of the secondary antibody solution was added to each well of the plate. The plate was sealed and incubated for 1 hour at 30° C. with shaking at 600 rpm while shielded from light. The secondary antibody solution was then removed. The plates were washed again three times with PBS-T, after which READ buffer (MSD KK) was added at 150 μL / well, followed by immediate detection of luminescent signals using a Sector Imager 2400 (MSD KK).

[0256] Reference Example 3: Construction of cell lines for imaging analysis To generate HeLa cells stably expressing human ASGPR (HeLa / ASGPR), cells were transfected with plasmids encoding human ASGPR-H1 (SEQ ID NO: 198) and ASGPR-H2 (SEQ ID NO: 199) protein subunits, respectively, at a ratio of 2:1 using Lipofectamine 3000 (Theromo Fisher Scientific). ASGPR-H1 and ASGPR-H2 were cloned into the plasmid pCXND3, which uses the CAG promoter and encodes the neomycin antibiotic resistance gene. Antibiotic-resistant cells were further enriched for high expression of ASGPR by cell sorting on a FACSAria III (Beckton Dickinson). Enriched cells were then isolated by limiting dilution and plating, and identified as single-cell clones by microscopic observation (Solentim, Cell Metric CLD).

[0257] The gene encoding the BirA protein (SEQ ID NO:201) was cloned into the plasmid pCXZD1, which uses the CAG promoter and encodes the zeocin antibiotic resistance gene. HeLa / ASGPR cells were transfected with the pCXZD1 BirA plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). Antibiotic-resistant cells were further enriched for high expression of BirA by cell sorting on a FACSAria IIu sorter (Beckton Dickinson). The enriched cells were then isolated by limiting dilution and plating, and identified as single-cell clones by microscopic observation. Selected clones were then expanded. ASGPR expression was confirmed by flow cytometry analysis using a FACS Verse (Beckton Dickinson) with anti-ASGPR antibody and Goat Anti-Human IgG-Alexa Fluor 647 (Southern Biotech, Cat# 2040-31). BirA expression was also confirmed by capillary immunoassay using Simple Western Wes (Protein Simple). A HeLa cell line stably expressing ASGPR and BirA (HeLa / ASGPR / BirA) was used for imaging analysis. For the assay, HeLa / ASGPR / BirA cells were cultured in Minimum Essential Medium Eagle (Sigma, Cat# M4655-500ML) containing 10% FBS (Sigma Aldrich, Cat# 172012-500ML), Penicillin-Streptomycin (Gibco, Cat# 15140-122), 750 μg / mL Geneticin (Thermo Fisher Scientific, Cat# 10131-027), and 100 μg / mL zeocin (Thermo Fisher Scientific, Cat# R25001).

Claims

1. A cytoplasmic penetration antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDRL1, CDRL2, and CDRL3 of the amino acid sequence set forth in SEQ ID NO:4, wherein the light chain variable region comprises one or more amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid S at position 27e with D, A, E, I, L, M, N, P, Q, T, V, or Y; (b) substitution of amino acid K at position 24 with A, D, E, F, G, H, I, L, N, Q, R, S, T, V, or Y; (c) substitution of amino acid S at position 25 with A, D, E, F, I, M, N, T, V, or Y; (d) substitution of amino acid S at position 26 with A, D, E, F, G, H, I, K, M, N, P, R, T, or V; (e) substitution of amino acid Q at position 27 with A, D, E, F, H, I, L, M, S, or Y; (f) substitution of amino acid S at position 27a with D; (g) substitution of amino acid L at position 27b with I, P, Q, T, or V; (h) substitution of amino acid F at position 27c with A, D, E, I, M, N, S, W, or Y; (i) substitution of amino acid N at position 27d with A, D, E, F, H, N, or Q; (j) substitution of amino acid R at position 27f with A, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, or Y; (k) substitution of amino acid T at position 28 with E, H, I, Q, S, V, W, or Y; (l) substitution of amino acid R at position 29 with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; (m) substitution of amino acid K at position 30 with A, D, E, F, H, I, L, M, N, P, Q, S, T, V, W, or Y; (n) substitution of amino acid N at position 31 with D, M, S, or T; (o) substitution of amino acid Y at position 32 with D, E, G, H, or Q; (p) substitution of amino acid Q at position 89 with H, I, T, or Y; (q) substitution of amino acid Y at position 91 with E, F, or N; (r) substitution of amino acid Y at position 93 with I, L, M, N, P, Q, S, T, V, or W; (s) substitution of amino acid H at position 94 with A, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, or Y; (t) substitution of amino acid M at position 95 with A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; (u) substitution of amino acid Y at position 96 with A, D, E, F, G, H, K, M, N, P, Q, S, T, or V; and (v) Substitution of amino acid T at position 97 with A, D, E, G, H, K, P, Q, R, S, or V.

2. The cytoplasmic entry antibody or antigen-binding fragment thereof of claim 1, wherein the one or more amino acid substitutions enhance the cytoplasmic entry activity of the antibody or fragment thereof compared to a reference antibody or fragment thereof that does not contain the substitutions and comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:

4.

3. The cytoplasmic entry antibody or antigen-binding fragment thereof of claim 1 or 2, wherein each of CDRL1 and CDRL3 in the light chain variable region contains two or more amino acid substitutions selected from the group defined in claim 1(a) to (v).

4. 4. The cytoplasmic penetration antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the light chain variable region comprises a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: (a) S27eD / R27fK; (b) S27eD / R27fH; (c) N27dE / S27eD; (d) S27eD / T28D; (e) S27eD / R29E; (f) S27eD / R29S; (g) K24N / S27eD; (h) N27dD / R27fK; (i) N27dE / R27fK; (j) R27fK / T28D; (k) R27fK / R29E; (l) R27fK / R29S; (m) K24N / R27fK; (n) K24N / N27dD; (o) N27dD / T28D; (p) N27dD / R29E; (q) N27dD / R29S; (r) S27eD / Q89H; (s) N27dD / Q89H; (t) R27fK / Q89H; (u) S27eD / Y91E; (v) N27dD / Y91E; (w) R27fK / Y91E; (x) Q89H / H94P; (y) Y91E / H94P; (z) H94P / M95N; (aa) H94P / M95D; (bb) H94P / M95P; (cc) H94E / Y96P; (dd) H94E / M95N; (ee) H94E / M95D; (ff) H94E / M95P; (gg) N27dE / R27fK / Q89H / H94E / M95P; (hh) N27dE / R27fK / Q89Y / H94E / M95P; (ii) N27dE / R27fK / Y91E / H94E / M95P; (jj) N27dE / R27fK / Y91F / H94E / M95P; (kk) N27dE / R27fS / Q89H / H94E / M95P; (ll) N27dE / R27fS / Q89Y / H94E / M95P; (mm) N27dE / R27fS / Y91E / H94E / M95P; (nn) N27dE / R27fS / Y91F / H94E / M95P; (oo) N27dE / R27fS / Q89H / H94Q / M95P; (pp) N27dE / R27fS / Q89Y / H94Q / M95P; (qq) N27dE / R27fS / Y91E / H94Q / M95P; (rr) N27dE / R27fS / Y91F / H94Q / M95P; (ss) N27dE / R27fV / Q89H / H94E / M95P; (tt) N27dE / R27fV / Q89Y / H94E / M95P; (uu) N27dE / R27fV / Y91E / H94E / M95P; (vv) N27dE / R27fV / Y91F / H94E / M95P; (ww) N27dE / R29S / Q89H / H94E / M95P; (xx) N27dE / R29S / Q89Y / H94E / M95P; (yy) N27dE / K30L / Q89H / H94E / M95P; (zz) N27dE / K30L / Q89Y / H94E / M95P; (aaa) S27eD / R27fK / Y91E / H94E / M95P; (bbb) S27eD / R27fK / Y91F / H94E / M95P; (ccc) S27eD / R27fK / Y91E / H94Q / M95P; (ddd) S27eD / R27fK / Y91F / H94Q / M95P; (eee) S27eD / R27fK / Y91E / H94Q; (fff) S27eD / R27fK / Y91F / H94Q; (ggg) S27eE / R27fK / Y91E / H94E / M95P; (hhh) S27eE / R27fK / Y91F / H94E / M95P; (iii) S27eE / R27fK / Y91E / H94Q / M95P; (jjj) S27eE / R27fK / Y91F / H94Q / M95P; (kkk) R27fS / R29S / Q89H / H94E / M95P; (lll) R27fS / R29S / Q89Y / H94E / M95P; (mmm) R27fS / R29S / Y91E / H94E / M95P; (nnn) R27fS / R29S / Y91F / H94E / M95P; (ooo) R27fS / R29S / Q89H / H94Q / M95P; (ppp) R27fS / R29S / Q89Y / H94Q / M95P; (qqq) R27fS / R29S / Y91E / H94Q / M95P; (rrr) R27fS / R29S / Y91F / H94Q / M95P; (sss) N27dE / R27fS / R29S / Q89Y / H94Q / M95P; and (ttt) N27dE / R27fS / R29S / Y91F / H94Q / M95P.

5. The cytoplasmic penetration antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain variable region comprises a human framework region (FR).

6. The cytoplasmic penetration antibody or antigen-binding fragment thereof of claim 5, wherein the light chain variable region comprises amino acid I at position 2 and / or amino acid Q at position 3 according to the Kabat numbering.

7. The cytoplasmic penetration antibody or antigen-binding fragment thereof of claim 5, wherein the light chain variable region comprises one, two, three, or four of the FR domains (FR1, FR2, FR3, and FR4) contained in SEQ ID NO:

123.

8. The cytoplasmic entry antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the light chain variable region further comprises one, two, three or more amino acid substitutions in CDRL2.

9. The cytoplasmic penetration antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the light chain variable region further comprises one, two, three or more amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In CDRL2 of the amino acid sequence shown in SEQ ID NO: 4, (a) substitution of amino acid W at position 50 with A, G, I, T, or V; (b) substitution of amino acid S at position 52 with F or I; (c) substitution of amino acid T at position 53 with N or Y; and (d) Substitution of amino acid R at position 54 with K or V.

10. 10. The cytoplasmic penetration antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the light chain variable region comprises a combination of amino acid substitutions (positions according to Kabat numbering) selected from the group consisting of: In the amino acid sequence of SEQ ID NO: 4, (a) L2I / V3Q / S27eD / R27fK / W50A / Y91F / H94Q; (b) L2I / V3Q / S27eD / R27fK / W50G / Y91F / H94Q; (c) L2I / V3Q / S27eD / R27fK / W50I / Y91F / H94Q; (d) L2I / V3Q / S27eD / R27fK / W50T / Y91F / H94Q; (e) L2I / V3Q / S27eD / R27fK / W50V / Y91F / H94Q; (f) L2I / V3Q / S27eD / R27fK / S52F / Y91F / H94Q; (g) L2I / V3Q / S27eD / R27fK / S52I / Y91F / H94Q; (h) L2I / V3Q / S27eD / R27fK / T53N / Y91F / H94Q; (i) L2I / V3Q / S27eD / R27fK / T53Y / Y91F / H94Q; (j) L2I / V3Q / S27eD / R27fK / R54K / Y91F / H94Q; (k) L2I / V3Q / S27eD / R27fK / W50I / S52F / Y91F / H94Q; (l) L2I / V3Q / S27eD / R27fK / W50I / T53N / Y91F / H94Q; and (m) L2I / V3Q / S27eD / R27fK / W50I / R54V / Y91F / H94Q.

11. The cytoplasmic penetration antibody or antigen-binding fragment thereof of any one of claims 1 to 10, comprising a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 14-144, 146-150 and 161-173.

12. The cytoplasmic penetration antibody or antigen-binding fragment thereof of claim 11, comprising a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 123-144, and 146-150.

13. The cytoplasmic penetration antibody or antigen-binding fragment thereof of claim 11, comprising a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 161 to 173.

14. A multifunctional antibody comprising a cell surface antigen-binding domain, a cytoplasmic antigen-binding domain, and a cytoplasmic entry domain, wherein the cytoplasmic entry domain comprises a light chain variable region according to any one of claims 1 to 13.

15. A composition or pharmaceutical composition for delivery of a biologically active molecule into the cytoplasm, comprising a cytoplasmic penetrating antibody or antigen-binding fragment thereof described in any one of claims 1 to 13, or a multifunctional antibody described in claim 14.

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