Novel bispecific polypeptide complex

Abstract

To design a bispecific molecule with a desired expression level and affinity for an antigen. [Solution] The polypeptide complex comprises heavy chain and light chain antibody variable regions, respectively, fused to the TCR constant region. The bispecific antigen-binding polypeptide complex comprises a first antigen-binding portion and a second antigen-binding portion of the polypeptide complex. The method comprises preparing the polypeptide complex or the bispecific antigen-binding polypeptide complex. The method for treating a disease or disorder comprises using the polypeptide complex or the bispecific antigen-binding polypeptide complex. The polynucleotide encodes the polypeptide complex and / or the bispecific antigen-binding polypeptide complex. The vector or host cell contains the polynucleotide. The composition and pharmaceutical composition contain the polypeptide complex and / or the bispecific antigen-binding polypeptide complex.

Description

[Technical Field]

[0001] (Field of Invention) This disclosure generally relates to soluble polypeptide complexes containing an antibody variable region fused to a constant region of the TCR, and to bispecific polypeptide complexes containing the same. [Background technology]

[0002] (cross reference) This application claims priority to international patent application PCT / CN2017 / 103030, filed on 22 September 2017, the entire contents of which application are incorporated herein by reference.

[0003] (background) Bispecific antibodies are growing as a new category of therapeutic antibodies. They can bind to two different targets or two different epitopes on a target, producing additive or synergistic effects that surpass the effects of individual antibodies. Numerous antibody engineering efforts have designed novel bispecific formats such as DVD-Ig, CrossMab, and BiTE (Spiess et al., Molecular Immunology, 67(2), 95-106 (2015)). However, these formats potentially have various limitations in terms of stability, solubility, short half-life, and immunogenicity.

[0004] Among these bispecific antibody formats, IgG-like bispecific antibodies share a common format in which one arm binds to target A and the other to target B. Structurally, this is constructed from half of antibody A and half of antibody B, which have similar size and shape to natural IgG. To facilitate downstream development, it is desirable that such bispecific molecules have high expression levels and precisely assembled shapes, and can be easily produced from a single host cell like normal IgG. Unfortunately, cognitive light-heavy chain pairing, as well as the assembly of two different half-antibodies, cannot be automatically controlled. Any kind of mispairing in a random manner can result in significant heterogeneity of the product.

[0005] By introducing mutations into the Fc region, such as "knob-into-hole" (Ridgway et al., Protein Engineering, 9(7), 617-21 (1996); Merchant et al., Nature Biotechnology, 16(7), 667-681 (1998)); electrostatic (Gunasekaran et al., Journal of Biological Chemistry, 285(25), 19637-19646 (2010)); or negative state design (Kreudenstein et al., mAbs, 5(5), 646-654 (2013); Leaver-Fay et al., Structure, 24(4), 641-651, (2016)), desirable heterodimerized aggregates of two different heavy chains have been achieved. However, selective pairing of each light-heavy chain of individual antibodies remains a challenge. The light-heavy chain interface contains variable domains (VH-VL) and steady-state domains (CH1-CL). Several strategies have been applied to the design of orthogonal interfaces to facilitate cognitive pair formation. Roche created the CrossMab platform by swapping the CH1 and CL domains (Schaefer et al., Proceedings of the National Academy of Sciences of the United State of America, 108(27), 11187-11192, (2011)), MedImmune introduced a disulfide bond instead (Mazor et al., mAbs, 7(2), 377-389, (2015)), Amgen made the CH1-CL region even more electrostatic (Liu et al., Journal of Biological Chemistry, 290(12), 7535-7562, (2015)), as well as Lilly (Lewis et al., Nature Biotechnology, 32(2), 191-198 (2014)) and Genentech (Dillon et al., mAbs, 9(2)), 213-230, (2017)) introduced mutations into both the variable domain and the constant domain.

[0006] Therefore, there is a great need to design bispecific molecules that possess the desired expression level and affinity for the antigen. [Overview of the project]

[0007] (Brief summary of the invention) In one embodiment, the present disclosure provides a polypeptide complex comprising a first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from N-terminus to C-terminus to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising a first light chain variable domain (VL) of a first antibody functionally linked from N-terminus to C-terminus to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer containing at least one non-natural interchain linkage between C1 and C2, the non-natural interchain linkage being capable of stabilizing the dimer, and the first antibody provides a polypeptide complex having first antigen specificity.

[0008] In one embodiment, the present disclosure relates to a bispecific polypeptide complex comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein the first antigen-binding moiety comprises a first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from N-terminus to C-terminus to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising a first light chain variable domain (VL) of a first antibody functionally linked from N-terminus to C-terminus to a second TCR constant region (C2), wherein C1 and C2 are the first antigen-binding moiety included in C1. The present invention provides a bispecific polypeptide complex in which a dimer can be formed between one mutated residue and a second mutated residue contained in C2, the dimer can be formed with at least one non-natural interchain linkage, the non-natural interchain linkage can stabilize the dimer, the first antibody has a first antigen specificity, the second antigen-binding moiety has a second antigen specificity different from the first antigen specificity, and the first and second antigen-binding moieties are less prone to mispairing than in other cases where both the first and second antigen-binding moieties are natural Fab counterparts.

[0009] In one embodiment, the Disclosure provides a bispecific polypeptide complex comprising a first antigen-binding moiety having a polypeptide complex provided herein having a first antigen-specificity, which is associated with a second antigen-binding moiety having a second antigen-specificity distinct from the first antigen-specificity, and the first and second antigen-binding moieties are less prone to mispairing than in other cases where both the first and second antigen-binding moieties are natural Fab counterparts.

[0010] In one embodiment, the disclosure provides a bispecific fragment of a bispecific polypeptide complex provided herein.

[0011] In one embodiment, the Disclosure provides a conjugate comprising a polypeptide complex provided herein, or a bispecific polypeptide complex provided herein, conjugated in part.

[0012] In one embodiment, the Disclosure provides, in this Specification, isolated polynucleotides encoding a polypeptide complex or a bispecific polypeptide complex provided herein.

[0013] In one embodiment, this disclosure provides, in this specification, an isolated vector comprising a polynucleotide provided herein.

[0014] In one embodiment, the Disclosure provides a host cell comprising an isolated polynucleotide or an isolated vector provided herein.

[0015] In one embodiment, the present disclosure provides a method for expressing a polypeptide complex or a bispecific polypeptide complex provided herein, comprising culturing a host cell provided herein under conditions in which the polypeptide complex or bispecific polypeptide complex is expressed.

[0016] In one embodiment, the present disclosure provides a method for preparing a polypeptide complex provided herein, comprising: a) introducing into a host cell a first polynucleotide encoding a first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from N-terminus to C-terminus to a first TCR constant region (C1), and a second polynucleotide encoding a second polypeptide comprising a first light chain variable domain (VL) of a first antibody functionally linked from N-terminus to C-terminus to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer containing at least one non-natural interchain linkage between a first mutated residue in C1 and a second mutated residue in C2, the non-natural interchain linkage being capable of stabilizing the dimer of C1 and C2, and the first antibody having first antigen specificity; and b) expressing the polypeptide complex in a host cell.

[0017] In one embodiment, the present disclosure provides a method for preparing a bispecific polypeptide complex provided herein, comprising: a) a host cell, a first polynucleotide encoding a first polypeptide containing a first heavy chain variable domain (VH) of a first antibody functionally linked from N-terminus to C-terminus to a first TCR constant region (C1); a second polynucleotide encoding a second polypeptide containing a first light chain variable domain (VL) of a first antibody functionally linked from N-terminus to C-terminus to a second TCR constant region (C2); and the VH of the second antibody The present invention provides a method comprising: a) introducing a third polynucleotide encoding a third polypeptide and a fourth polynucleotide encoding a fourth polypeptide containing the VL of a second antibody, wherein C1 and C2 are capable of forming a dimer containing at least one non-natural interchain linkage between C2, the non-natural interchain linkage being capable of stabilizing the dimer, and the first antibody having first antigen specificity and the second antibody having second antigen specificity; b) expressing a bispecific polypeptide complex in a host cell.

[0018] In certain embodiments, the method for producing a bispecific polypeptide complex provided herein further includes the step of isolating the polypeptide complex.

[0019] In one embodiment, the Disclosure provides compositions comprising a polypeptide complex provided herein, or a bispecific polypeptide complex provided herein.

[0020] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a polypeptide complex provided herein, or a bispecific polypeptide complex provided herein, and a pharmaceutically acceptable carrier.

[0021] In one embodiment, the Disclosure provides a method for treating a condition in a subject requiring such treatment, comprising administering a therapeutically effective amount of the polypeptide complex or bispecific polypeptide complex provided herein to the subject. In certain embodiments, the condition may be alleviated, eliminated, treated, or prevented when both the first and second antigens are modulated.

[0022] In certain embodiments, a non-natural interchain bond is formed between a first mutated residue in C1 and a second mutated residue in C2. In certain embodiments, at least one of the first and second mutated residues is a cysteine ​​residue.

[0023] In certain embodiments, the unnatural interchain bond is a disulfide bond.

[0024] In a particular embodiment, the first mutated residue is contained within the contact interface of C1, and / or the second mutated residue is contained within the contact interface of C2.

[0025] In certain embodiments, at least one native cysteine ​​residue is either absent or present at C1 and / or C2. In certain embodiments, a native cysteine ​​residue is either absent or present at position C74 of the manipulated CBeta. In certain embodiments, native C74 is absent in the CBeta.

[0026] In certain embodiments, at least one native N-glycosylation site is either absent or present within C1 and / or C2. In certain embodiments, the native N-glycosylation site is absent within C1 and / or C2.

[0027] In certain embodiments, the dimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more unnatural interchain bonds. In certain embodiments, at least one unnatural interchain bond is a disulfide bond. In certain embodiments, the dimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more disulfide bonds.

[0028] In a particular embodiment, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 contains manipulated CGamma and C2 contains manipulated CDelta; or f) C1 contains manipulated CDelta and C2 contains manipulated CGamma.

[0029] In a particular embodiment, the first VH is functionally connected to C1 in a first connection domain, and the first VL is functionally connected to C2 in a second connection domain. In a particular embodiment, the first VH meets C1 in the first connection domain via a connector, and the first VL meets C2 in the second connection domain via a connector.

[0030] In certain embodiments, the first and / or second connection domains include a suitable length of C-terminal fragment of the antibody V / C connection (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) and a suitable length of N-terminal fragment of the TCR V / C connection (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues).

[0031] In certain embodiments, the manipulated CBeta contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86, and 122-127; and / or, the manipulated CAlpha contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 6-29, 37-67, and 86-95.

[0032] In certain embodiments, the manipulated CBeta contains a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from S56C, S16C, F13C, V12C, E14C, L62C, D58C, S76C, and R78C, and / or the manipulated CAlpha contains a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C, and S62C.

[0033] In certain embodiments, the manipulated CBeta and manipulated CAlpha are S16C in CBeta and Y11C in CAlpha, F13C in CBeta and L13C in CAlpha, S16C in CBeta and L13C in CAlpha, V12C in CBeta and S16C in CAlpha, E14C in CBeta and S16C in CAlpha, F13C in CBeta and V23C in CAlpha, L62C in CBeta and Y44C in CAlpha, D58C in CBeta and The present invention comprises a pair of mutated cysteine ​​residues that substitute for a pair of amino acid residues selected from the group consisting of T46C in CAlpha, S76C in CBeta, T46C in CAlpha, S56C in CBeta, T49C in CAlpha, S56C in CBeta, L51C in CAlpha, S56C in CBeta, S62C in CAlpha, and R78C in CAlpha, and S62C in CAlpha, wherein the pair of cysteine ​​residues is capable of forming a non-natural interchain disulfide bond.

[0034] In certain embodiments, at least one native glycosylation site is either absent or present in the manipulated CBeta and / or manipulated CAlpha.

[0035] In a particular embodiment, the native glycosylation site in the manipulated CBeta is N69, and / or the native glycosylation site(s) in the manipulated CAlpha are selected from N34, N68, N79, and any combination thereof.

[0036] In certain embodiments, the manipulated CBeta lacks or retains the FG loop and / or the DE loop, which encompass amino acid residues 101-117 and / or amino acid residues 66-71 of the native CBeta.

[0037] In certain embodiments, the manipulated CAlpha includes one of sequence numbers 43-48, and / or the manipulated CBeta includes one of sequence numbers 33-41 and 306.

[0038] In a particular embodiment, C1 includes an operated CBeta, and C2 includes an operated CAlpha; and here, the first connection domain includes or is sequence number 49 or 50, and / or the second connection domain includes or is sequence number 51 or 52.

[0039] In a particular embodiment, C1 includes an operated CAlpha, and C2 includes an operated CBeta; and the first connection domain includes or is sequence number 129 or 130, and / or the second connection domain includes or is sequence number 49 or 50.

[0040] In certain embodiments, the manipulated CBeta contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86, and 122-127; and / or the manipulated CPre-Alpha contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 7-19, 26-34, 56-75, and 103-106.

[0041] In certain embodiments, the manipulated CBeta contains a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from S16C, A18C, E19C, F13C, A11C, S56C, and S76C, and / or the manipulated CPre-Alpha contains a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from S11C, A13C, I16C, S62C, T65C, and Y59.

[0042] In certain embodiments, the manipulated CBeta and manipulated CPre-Alpha include pairs of mutated cysteine ​​residues that substitute for pairs of amino acid residues selected from the group consisting of S16C in CBeta and S11C in CPre-Alpha, A18C in CBeta and S11C in CPre-Alpha, E19C in CBeta and S11C in CPre-Alpha, F13C in CBeta and A13C in CPre-Alpha, S16C in CBeta and A13C in CPre-Alpha, A11C in CBeta and I16C in CPre-Alpha, S56C in CBeta and S62C in CPre-Alpha, S56C in CBeta and T65C in CPre-Alpha, and S76C in CBeta and Y59C in CPre-Alpha, wherein the pairs of mutated cysteine ​​residues are capable of forming unnatural interchain disulfide bonds.

[0043] In certain embodiments, at least one native glycosylation site is absent in the manipulated CBeta and / or the manipulated CPre-Alpha.

[0044] In a particular embodiment, the absent or present glycosylation site in the manipulated CBeta is N69, and / or the absent glycosylation site in the manipulated CPre-Alpha is N50.

[0045] In certain embodiments, the manipulated CBeta lacks or retains the FG loop encompassing amino acid residues 101-107 of the native CBeta and / or the DE loop at the position encompassing amino acid residues 66-71 of the native CBeta.

[0046] In a particular embodiment, the manipulated CPre-Alpha includes one of sequence numbers 82, 83, and 311-318; and / or the manipulated CBeta includes one of sequence numbers 84, 33-41, and 319-324.

[0047] In a particular embodiment, C1 includes an operated CBeta, and C2 includes an operated CPre-Alpha; and here, the first connection domain includes sequence number 49 or 50, and / or the second connection domain includes sequence number 81 or 131.

[0048] In a particular embodiment, C1 includes an operated CPre-Alpha, and C2 includes an operated CBeta; and herein, the first connection domain includes sequence number 132 or 133, and / or the second connection domain includes sequence number 49 or 50.

[0049] In certain embodiments, the manipulated CDelta contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 8-26, 43-64, and 84-88; and / or the manipulated CGamma contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 11-35 and 55-76.

[0050] In certain embodiments, the manipulated CGamma comprises a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and / or the manipulated CDelta comprises a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from F12C, M14C, N16C, D46C, V50C, F87C, and E88C.

[0051] In certain embodiments, the manipulated CGamma and manipulated CDelta include pairs of mutated cysteine ​​residues that substitute for pairs of amino acid residues selected from the group consisting of S17C in CGamma and F12C in CDelta, E20C in CGamma and F12C in CDelta, F14C in CGamma and M14C in CDelta, T12C in CGamma and N16C in CDelta, M62C in CGamma and D46C in CDelta, Q57C in CGamma and V50C in CDelta, A19C in CGamma and E88C in CDelta, and A19C in CGamma and F87C in CDelta, wherein the introduced pairs of cysteine ​​residues are capable of forming interchain disulfide bonds.

[0052] In certain embodiments, at least one native glycosylation site is either absent or present in the manipulated CGamma and / or manipulated CDelta.

[0053] In a particular embodiment, the native glycosylation site in the manipulated CGamma is N65, and / or the native glycosylation site(s) in the manipulated CDelta are one or both of N16 and N79.

[0054] In a particular embodiment, the manipulated CGamma includes sequence numbers 113, 114, 333, 334, 335, 336, 337, 338, 339, or 340, and / or the manipulated CDelta includes sequence numbers 115, 116, 310, 325, 326, 327, 328, 329, 330, 331, or 332.

[0055] In a particular embodiment, C1 comprises an operated CGamma, and C2 comprises an operated CDelta; and here, the first connection domain comprises sequence number 117 or 118, and / or the second connection domain comprises sequence number 119 or 120.

[0056] In a particular embodiment, C1 comprises an operated CDelta, and C2 comprises an operated CGamma; and here, the first connection domain comprises sequence number 123 or 124, and / or the second connection domain comprises sequence number 125 or 126.

[0057] In certain embodiments, the first polypeptide further comprises an antibody CH2 domain and / or an antibody CH3 domain.

[0058] In certain embodiments, the first antigen specificity and the second antigen specificity are directed to two different antigens, or to two different epitopes on one antigen.

[0059] In a particular embodiment, the first antigen-binding moiety binds to CD3. In a particular embodiment, the second antigen-binding moiety binds to CD19. In a particular embodiment, the first antigen-binding moiety binds to CD19. In a particular embodiment, the second antigen-binding moiety binds to CD3.

[0060] In a particular embodiment, the first antigen-binding moiety binds to CTLA-4. In a particular embodiment, the second antigen-binding moiety binds to PD-1. In a particular embodiment, the first antigen-binding moiety binds to PD-1. In a particular embodiment, the second antigen-binding moiety binds to CTLA-4.

[0061] In certain embodiments, association occurs via connectors, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophobic-hydrophilic interactions, or a combination thereof.

[0062] In certain embodiments, the second antigen-binding moiety includes a heavy chain variable domain and a light chain variable domain of a second antibody having second antigen specificity.

[0063] In a particular embodiment, the second antigen-binding portion includes Fab.

[0064] In certain embodiments, the first and second antigen specificities are directed to two different antigens, or to two different epitopes on a single antigen.

[0065] In certain embodiments, one of the first and second antigen specificities is directed toward a T cell specific receptor molecule and / or a natural killer cell (NK cell) specific receptor molecule, and the other is directed toward a tumor-associated antigen.

[0066] In certain embodiments, one of the first and second antigen specificities is directed toward CD3, and the other is directed toward tumor-associated antigens.

[0067] In certain embodiments, one of the first and second antigen specificities is directed toward CD3, and the other is directed toward CD19.

[0068] In a particular embodiment, the first antigen-binding domain further comprises a first dimer-forming domain, and the second antigen-binding domain further comprises a second dimer-forming domain, where the first and second dimer-forming domains are associated.

[0069] In certain embodiments, association occurs via connectors, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophobic-hydrophilic interactions, or a combination thereof.

[0070] In certain embodiments, the first and / or second dimer-forming domains optionally include at least a portion of an antibody hinge region derived from IgG1, IgG2, or IgG4.

[0071] In certain embodiments, the first and / or second dimer-forming domain further comprises a dimer-forming domain. In certain embodiments, the dimer-forming domain comprises at least a portion of the antibody hinge region, an antibody CH2 domain, and / or an antibody CH3 domain.

[0072] In a particular embodiment, the first dimer-forming domain is functionally linked to the first TCR constant region (C1) by a third connecting domain.

[0073] In certain embodiments, a) C1 includes an operated CBeta and the third connection domain is included in SEQ ID NO: 53 or 54; b) C1 includes an operated CAlpha and the third connection domain is included in SEQ ID NO: 134, 135, 140 or 141; c) C1 includes an operated CPre-Alpha and the third connection domain is included in SEQ ID NO: 134, 135, 140 or 141; d) C1 includes an operated CGamma and the third connection domain is included in SEQ ID NO: 121 or 122; or e) C1 includes an operated CDelta and the third connection domain is included in SEQ ID NO: 127 or 128.

[0074] In a particular embodiment, the second dimer-forming domain is functionally linked to the heavy chain variable domain of the second antigen-binding moiety.

[0075] In certain embodiments, the first and second dimer-forming domains associate in a different manner, inhibiting homodimer formation and / or favoring heterodimer formation.

[0076] In certain embodiments, the first and second dimer-forming domains can associate with the heterodimer via knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased mobility.

[0077] In a particular embodiment, the first antigen-binding moiety comprises a first polypeptide containing VH functionally linked to a chimeric constant region, and a second polypeptide containing VL functionally linked to C2, where the chimeric constant region and C2 are sequence numbers: 177 / 176, 179 / 178, 184 / 183, 185 / 183, 180 / 176, 181 / 178, 182 / 178, 184 / 186, 185 / 186, 188 / 187, 196 / 187, 190 / 189, 192 / 191, 192 / 193, 195 / 194, 198 / Includes pairs of sequences selected from the group consisting of 197, 200 / 199, 202 / 201, 203 / 201, 203 / 204, 205 / 204, 206 / 204, 208 / 207, 208 / 209, 211 / 210, 213 / 212, 213 / 215, 213 / 151, 214 / 212, 214 / 151, 232 / 231, 216 / 215, 218 / 217, 220 / 219, 222 / 221, 224 / 223, 226 / 225, 227 / 223, 229 / 228, 229 / 230, 236 / 235, and 238 / 237.

[0078] In a particular embodiment, the first antigenicity is directed toward CD3, and the first and second polypeptides are sequence numbers: 2 / 1, 3 / 4 / , 5 / 1, 6 / 3, 7 / 3, 9 / 8, 10 / 8, 9 / 11, 10 / 11, 13 / 12, 15 / 14, 17 / 16, 17 / 18, 20 / 19, 21 / 12, 65 / 64, 67 / 66, 69 / 68, 70 / 68, 70 / 71, 72 / Includes pairs of sequences selected from the group consisting of 71, 73 / 71, 75 / 74, 75 / 76, 78 / 77, 86 / 85, 90 / 89, 91 / 92 / , 94 / 93, 96 / 95, 98 / 97, 99 / 95, 101 / 100, 101 / 102, 106 / 105, 108 / 107, 110 / 109, 112 / 111, 137 / 136, 138 / 136, 137 / 139, and 138 / 139.

[0079] In a particular embodiment, the first antigen-binding moiety and the second antigen-binding moiety include four sequence combinations selected from the group consisting of SEQ ID NOs: 22 / 12 / 24 / 23, 25 / 12 / 26 / 23, and 25 / 12 / 27 / 23, wherein the first antigen-binding moiety is capable of binding to CD3 and the second antigen-binding moiety is capable of binding to CD19.

[0080] In certain embodiments, the polypeptide complexes provided herein can be prepared in the form of Fab, (Fab)2, vibody, tribody, triFab, tandem-linked Fab, Fab-Fv, tandem-linked V-domain, tandem-linked scFv, and other formats.

[0081] In another embodiment, the Disclosure provides a kit comprising a polypeptide complex provided herein for the detection, diagnosis, prognosis, or treatment of a disease or condition.

[0082] The features and advantages of the present invention described above and elsewhere will become more apparent from the detailed description of some embodiments described below, which will proceed with reference to the accompanying drawings. [Brief explanation of the drawing]

[0083] [Figure 1] Figure 1 shows a schematic diagram of the antibody formats tested. Both anti-CD3 antibody T3 and anti-CD19 antibody U4 were developed. The constant region (CL and CH1) of T3 was replaced with the constant domain of the TCR, designing a unique light-heavy chain interface orthogonal to that of a normal antibody. Using TCR-modified T3 and native U4 with a "knob-into-hole" mutation in the Fc domain, bispecific antibody formats E17 and F16 were designed. [Figure 2A-2D]Figures 2A-2D show superimposed poses of antibody Fv models and TCR structures, providing guidance for the fusion of antibody Fv and the TCR constant region. Figure 2A shows an antibody Fv structural model constructed based on the sequence of the anti-CD3 antibody T3 developed in the laboratory. Figure 2B shows the TCR structure derived from PDB 4L4T. Figure 2C shows antibody Fv structural models superimposed on the TCR variable region in different orientations. Rough chimeric proteins were prepared by removing the TCR variable domain from the superimposed poses, as shown in Figure 2D. The overlapping residues in this connection region assisted in the design of the connection region. The antibody VL chain and TCR alpha chain are colored white. The VH chain and beta chain are colored black. [Figure 3A-3B] Figures 3A-3B show a comparison between the constant region of the TCR and the constant region of the antibody Fab. Figure 3A shows the crystal structure of the TCR derived from PDB 4L4T. Figure 3B shows the antibody Fab structural model prepared using the Fv domain of the T3 model and the constant domain of the antibody derived from PDB 5DK3. Clear differences in the FG loop and DE loop between the constant domain of the TCR and the constant domain of the antibody Fab are marked by displaying all side chain residues. [Figure 4] Figure 4 shows the SDS-PAGE results of deglycosylation mutants of chimeric antibodies of TCR-antibodies with CAlpha and CBeta chains. All samples were supernatants collected from products expressing Expi293. Lanes 1, 3, 5, 7, and 9 are the unreduced PAGEs for Design_2-QQQQ, Design_2-AAAA, Design_2-QSKE, Design_2-ASKE, and Design_2-QQQQQ, respectively. Lanes 2, 4, 6, 8, and 10 are the corresponding reduced PAGEs. [Figure 5] Figure 5 shows the dose-dependent FACS binding of all deglycosylated mutants to CD3-expressing Jurkat cells. All samples were supernatants collected from deglycosylated mutants expressed in Expi293. Wild-type anti-CD3 antibody (T3-IgG1) was used as a positive control. [Figure 6A-6B]Figures 6A-6B show the SDS-PAGE results of the chain mispairing test for antibody T3 and U4 in IgG1 (Figure 6A) and IgG4 (Figure 6B) isotypes. Lanes 1-2 represent the T3_light-U4_heavy and T3_heavy-U4_light pairs, respectively. Lanes 3-4 show the same pairing order as lanes 1-2, but with T3 modified using the TCR constant region. In both figures, lanes 1-4 are the non-reduced samples, and lanes 5-8 are the corresponding reduced samples. [Figures 7A-7B] Figures 7A-7B show the SDS-PAGE results for purified bispecific antibodies E17-Design_2-QQQQ in (Figure 7A) IgG1 and (Figure 7B) IgG4. The IgG1 isotype was purified by a three-step process: protein A chromatography, ion exchange chromatography (IEC), and size exclusion chromatography (SEC). IgG4 was obtained after a two-step purification process: protein A chromatography and SEC. Figures 7C-7D show SEC-HPLC data for purified samples of IgG1 (Figure 7C) and IgG4 (Figure 7D) to determine the purity of the samples. [Figure 8] Figure 8 shows the SDS-PAGE results of Fab fragments of 6×His-tagged chimeric T3 purified by Ni Sepharose® Excel chromatography. Lanes 1 and 3 are bands of T3-Fab-Design_2.his1, and lanes 2 and 4 are bands of T3-Fab-Design_2.his2. [Figure 9] Figure 9 illustrates the dose-dependent FACS binding of the Fab fragment of TCR-modified chimeric T3. A monovalent wild-type T3 antibody (T3-Fab-IgG4) was used as the positive control. A standard human IgG4 antibody was used as the negative control. [Figure 10A-10B]Figures 10A-10B show the dose-dependent FACS binding of the designed bispecific antibody E17-Design_2-QQQQ to CD3+ Jurkat cells. Wild-type antibodies T3 and U4, as well as their monovalent forms, were used as positive controls (Figure 10A), and Figure 10B is for CD19+ Ramos cells. Both IgG1 and IgG4 isotypes were tested. Unrelated human IgG1 or IgG4 antibodies were used as negative controls. [Figure 11A-11B] Figures 11A-11B show a comparison of FACS binding of two designed bispecific antibodies, E17-Design_2-QQQQ and F16-Design_2-QQQQ, to CD3 expressed on Jurkat cells (Figure 11A) and CD19 expressed on Ramos cells (Figure 11B). Bispecific antibodies in both IgG1 and IgG4 isotypes were tested. Standard human IgG1 or IgG4 antibodies were used as negative controls. [Figure 12] Figure 12 illustrates a cytotoxicity assay of T cells directed to kill malignant B cells, mediated by the bispecific antibody E17-Design_2-QQQQ, designed for both IgG1 and IgG4. Parental monospecific anti-CD3 antibody (T3-IgG4), anti-CD19 (U4-IgG) antibody, and unrelated human IgG1 antibody were used as negative controls. [Figure 13] Figure 13 compares the activity of two designed bispecific antibodies, E17-Design_2-QQQQ and F16-Design_2-QQQQ, in mediating T cell engagement and malignant B cell death. An unrelated human IgG antibody was used as a negative control. [Figure 14A-14B]Figures 14A and 14B show the deconvoluted mass spectra of the bispecific antibody E17-Design_2-QQQQ under non-reducing conditions (Figure 14A) and reducing conditions (Figure 14B). The peak at 148180.53 in Figure 14A is the exact molecular weight of the intact WuXiBody. The peak at 22877Da indicates the light chain observed in the reduced mass spectrum of Figure 14B. The small peak at 149128.45Da in Figure 14A is estimated to be an O-glycosylation located on the light chain (approximately greater than 947.92Da), shown in Figure 14B. [Figures 15A-15B] Figures 15A-15B illustrate the role of interchain disulfide bonds in antibody expression at the alpha / beta interface as characterized by SDS-PAGE. Figure 15A shows an antibody containing an interchain disulfide bond between CAlpha and CBeta; Figure 15B shows an antibody without an interchain disulfide bond between CAlpha and CBeta; lanes 1 and 3 are the unreduced PAGE results for Design_2-QQQQ-IgG4 with and without the introduced disulfide bond, respectively. Lanes 2 and 4 are the reduced PAGE results for Design_2-QQQQ-IgG4 with and without the introduced disulfide bond, respectively. [Figure 16] Figure 16 shows the SDS-PAGE of disulfide bonds designed at the pre-alpha / beta interface. Lanes 1 and 2 are "Design_5_Pre_TCR_Conjunction'1_Cys13" and "Design_6_Pre_TCR_Conjunction'1_Cys14", respectively, treated under non-reducing conditions. Lanes 4 and 5 are "Design_5_Pre_TCR_Conjunction'1_Cys13" and "Design_6_Pre_TCR_Conjunction'1_Cys14", respectively, treated under reducing conditions. [Figures 17A-17B]Figures 17A and 17B show SDS-PAGEs of disulfide bonds designed at the delta / gamma interface. Lanes 6 and 8 represent "Design_2_Cys5_no_Glyco" and "Design_2_hypeCys2_no_Glyco," respectively. Figure 17A is the non-reducing SDS-PAGE. Figure 17B is the reducing SDS-PAGE. [Figures 18A-18E] Figure 18A shows the sequence of the native TCR alpha chain and its corresponding sequence with mutated cysteine ​​residues. TRAC_Human is the native sequence of the alpha chain constant region. 4L4T_Alpha_Crystal is the sequence of the crystal structure with the S55C mutation that can form interchain disulfide bonds (PDB code 4L4T). The gray region is the constant region used as the backbone of the chimeric protein in the present invention. Figure 18B shows the sequence of the native TCR beta chain and its corresponding sequence with mutated cysteine ​​residues. TRBC1_Human and TRBC2_Human are the native sequences of the beta chain constant region. Figure 18C shows the native TCR pre-alpha chain sequence. PTCRA_Human is the native sequence of the pre-alpha chain constant region (only the pre-alpha chain does not have a variable region). 3OF6_PreAlpha_Crystal is the sequence of the crystal structure (PDB code 3OF6). The gray areas are the steady regions previously used to define the numbering. Figure 18D shows the sequence of the natural TCR delta chain. TRA@_Human is the natural sequence of the delta steady region. 4LFH_Delta_Crystal is the steady region of the delta chain sequence in the crystal structure (PDB code 4LFH). The gray areas are the steady regions previously used to define the numbering. Figure 18E shows the sequence of the natural TCR gamma chain. TRGC1_Human and TRGC2_Human are the natural sequences of the gamma steady region. 4LFH_Gamma_Crystal is the steady region of the gamma chain sequence in the crystal structure (PDB code 4LFH). The gray areas are the steady regions previously used to define the numbering. [Figures 19A-19E]Figures 19A-19E show the sequences and numbering of the TCR constant region. Figure 19A shows the sequences and numbering of the TCR alpha constant region. Figure 19B shows the sequences and numbering of the TCR beta constant region. Figure 19C shows the sequences and numbering of the TCR pre-alpha constant region. Figure 19D shows the sequences and numbering of the TCR delta constant region. Figure 19E shows the sequences and numbering of the TCR gamma constant region. [Figures 20A-20D] Figures 20A-20D show the sequences and numbering of knob-into-hole mutations in IgG1 and IgG4. Figure 20A shows the sequences and numbering of IgG1 "knob" mutations. Figure 20B shows the sequences and numbering of IgG4 "knob" mutations. Figure 20C shows the sequences and numbering of IgG1 "hole" mutations. Figure 20D shows the sequences and numbering of IgG4 "hole" mutations. [Figures 21A-21B] Figures 21A-21B show the binding of E17-Design_2-QQQQ to human C1Q in both IgG4 (Figure 21A) and wild-type IgG1 (Figure 21B) formats by ELISA. Human IgG1 antibody was used as a control. [Figure 22] Figure 22 provides a schematic description of four symmetrical WuXiBody formats: G19, G19R, G25, and G25R. For formats G19 and G25, two TCR-containing chimeric Fab-like domains were grafted at the C-terminus and N-terminus of a normal antibody, respectively. Rectangles represent the constant TCR domain, while ovals represent the variable and constant domains of the antibody. The difference between formats G19 and G19R or G25 and G25R lies in the switched positions of the normal and chimeric Fab. These formats can accommodate different variable regions from different antibody pairs and typically have molecular weights of approximately 240–250 kD. [Figures 23A-23B]Figures 23A and 23B show the SDS-PAGE (Figure 23A) and SEC-HPLC (Figure 23B) characteristics of two purified bispecific antibodies in G19 format. The lane numbers in the SDS-PAGE correspond to the labeling numbers in the SEC-HPLC figure. Lanes 1 and 2 represent the T1U6 and U6T1 antibody pairs, respectively. In T1U6, T1 (anti-CTLA-4) is located at the N-terminus of this format, while in U6T1, U6 (anti-PD-1) is located at the N-terminus of this format. Both bispecific molecules were purified by protein A chromatography to achieve a purity of approximately 90%. [Figures 24A-24B] Figures 24A-24B show dose-dependent FACS binding of purified U6T1 and T1U6 antibodies in G19 format to human PD-1 (Figure 24A) and CTLA-4 (Figure 24B) engineered cells. IgG4 antibody was used as a negative control. [Figures 25A-25B] Figures 25A-25B show the SDS-PAGE (Figure 25A) and SEC-HPLC (Figure 25B) characteristics of purified bispecific antibodies of protein A in different symmetry formats. Lanes 1-3 represent U6T1 antibody pairs in G19R, G25, and G25R formats, respectively. PC is a control protein known to have a molecular weight of 250 kD. All three bispecific molecules had a purity of 90% or higher. The lane numbers in the SDS-PAGE correspond to the labeling numbers in the SEC-HPLC figure. [Figures 26A-26B] Figures 26A-26B show dose-dependent FACS binding of purified U6T1 bispecific antibodies in G19R, G25, and G25R formats to human PD-1 (Figure 26A) and CTLA-4 (Figure 26B) engineered cells. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as a negative control. [Figures 27A-27B]Figures 27A and 27B show FACS competitive assays of bispecific antibodies designed in G19R, G25, and G25R formats to block human PD-L1 binding to PD-1 (Figure 27A) and CD80 binding to CTLA-4 (Figure 27B), respectively. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as a negative control. [Figures 28A-28B] Figures 28A and 28B show the SDS-PAGE (Figure 28A) and SEC-HPLC (Figure 28B) features of purified bispecific antibodies of protein A in different symmetry formats. Lanes 1-4 represent U6T5 antibody pairs in G19, G19R, G25, and G25R formats, respectively. PC is a control protein with a molecular weight of 250 kD. All three bispecific molecules had a purity of 90% or higher. The lane numbers in the SDS-PAGE correspond to the labeling numbers in the SEC-HPLC figure. [Figures 29A-29B] Figures 29A-29B show dose-dependent FACS binding of purified bispecific antibodies in G19, G19R, G25, and G25R formats to human PD-1 (Figure 29A) and CTLA-4 (Figure 29B) engineered cells. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as a negative control. [Figure 30] Figure 30 shows the ELISA dual-conjugation assay of two molecules, U6T5.G25 and U6T1.G25R. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as a negative control. [Figure 31A-31B] Figures 31A and 31B show competitive FACS assays of the designed bispecific antibodies U6T5.G25 and U6T1.G25R for blocking the binding of human PD-L1 to PD-1 (Figure 31A) and the binding of CD80 to CTLA-4 (Figure 31B), respectively. A benchmark bispecific anti-CTAL-4 × PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as a negative control. [Figure 32] Figure 32 provides a schematic description of three symmetry formats G26, G27, and G26R, which have light-chain-heavy-chain switched chimeric Fab-like domains. [Figures 33A-33B] Figures 33A and 33B show the SDS-PAGE (Figure 33A) and SEC-HPLC (Figure 33B) characteristics of protein A-purified bispecific antibodies in G27 and G26R formats. Lanes 1 and 2 represent T4U6 antibody pairs in G27 and G26R formats, respectively. Only the G26R format antibody achieved a purity of 90% after purification. The lane numbers in the SDS-PAGE correspond to the labeling numbers in the SEC-HPLC figure. [Figures 34A-34B] Figures 34A-34B show dose-dependent FACS binding of purified bispecific T4U6 antibody pairs in G26R format to human PD-1 (Figure 34A) and CTLA-4 (Figure 34B) engineered cells. A benchmark bispecific anti-CTAL-4×PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as a negative control. [Figures 35A-35B] Figures 35A and 35B show the SDS-PAGE (Figure 35A) and SEC-HPLC (Figure 35B) characteristics of the G26 format protein A-purified bispecific U6T4 antibody pair. A purity of 90% was achieved after purification. [Figures 36A-36D] Figures 36A–36D show dose-dependent ELISA conjugation of purified bispecific U6T4 antibody pairs in G26 format to human PD-1 (Figure 36A) and CTLA-4 (Figure 36B) engineered cells, and dose-dependent FACS conjugation of purified bispecific U6T4 antibody pairs in G26 format to human PD-1 (Figure 36C) and CTLA-4 (Figure 36D) engineered cells. A benchmark bispecific anti-CTAL-4 × PD-1 antibody (BMK1.IgG1) was used as a control, and an IgG4 antibody was used as a negative control. [Figure 37] Figure 37 shows flow cytometry histograms of the cynomolgus monkey-CD19 transfused cell line WBP701.CHO-K1.cpro1.FL.C9 and the CHO-K1 parent cell line. [Figure 38] Figure 38 shows the SDS-PAGE of W3438-T3U4.F16-1.uIgG4.SP. M: Protein marker; Lane 1: W3438-T3U4.F16-1.uIgG4.SP, unreduced; Lane 3: W3438-T3U4.F16-1.uIgG4.SP, reduced. [Figure 39] Figure 39 shows the SEC-HPLC of W3438-T3U4.F16-1.uIgG4. [Figure 40] Figure 40 shows the SDS-PAGE of W3438-T3U4.E17-1.uIgG4.SP. M: Protein marker; Lane 1: W3438-T3U4.E17-1.uIgG4.SP, unreduced; Lane 2: W3438-T3U4.E17-1.uIgG4.SP, reduced. [Figure 41] Figure 41 shows the SEC-HPLC of W3438-T3U4.E17-1.uIgG4.SP. [Figures 42A-42B] Figures 42A-42B show the binding of W3438-T3U4.E17-1.uIgG4.SP to Ramos cells (Figure 42A) and Jurkat cells (Figure 42B) as determined by FACS. [Figures 43A-43B] Figures 43A and 43B show the binding of W3438-T3U4.F16-1.uIgG4.SP to Ramos cells (Figure 43A) and Jurkat cells (Figure 43B) as determined by FACS. [Figure 44] Figure 44 shows the binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus monkey-CD19 expressing cells as determined by FACS. [Figure 45] Figure 45 shows the binding of W3438-T3U4.E17-1.uIgG4.SP to cynomolgus monkey CD3 by ELISA. [Figures 46A-46B] Figures 46A-46B show the affinity of W3438-T3U4.E17-1.uIgG4.SP to human CD19 and CD3, as measured by binding to Ramos cells (Figure 46A) and Jurkat cells (Figure 46B). [Figures 47A-47B]Figures 47A-47B show the binding of W3438-T3U4.E17-1.uIgG4.SP-mediated CD3+ cells to CD19+ cells (Figure 47A). Unrelated IgG was used as a negative control (Figure 47B). [Figures 48A-48B] Figures 48A and 48B show the cytotoxic activity of W3438-T3U4.E17-1.uIgG4.SP-mediated T cells that kill Raji cells (Figure 48A) and the cytotoxic activity of W3438-T3U4.F16-1.uIgG4.SP-mediated T cells that kill Raji cells (Figure 48B). [Figures 49A-49D] Figures 49A-49D show the expression of CD69 and CD25 on T cells in the presence or absence of CD19+ target cells. Percentage of CD69+-expressing T cells in the CD4+ T cell subset (Figure 49A); Percentage of CD69-expressing T cells in the CD8+ T cell subset (Figure 49B); Percentage of CD25-expressing T cells in the CD4+ T cell subset (Figure 49C); Percentage of CD25-expressing T cells in the CD8+ T cell subset (Figure 49D). [Figures 50A-50D] Figures 50A-50D show the release of IFN-γ and TNF-α cytokines from T cells in the presence or absence of CD19+ target cells. IFN-γ release in CD4+ T cell subsets (Figure 50A); TNF-α release in CD4+ T cell subsets (Figure 50B); IFN-γ release in CD8+ T cell subsets (Figure 50C); TNF-α release in CD8+ T cell subsets (Figure 50D). [Figures 51A-51B] Figures 51A-51B show the stability of W3438-T3U4.E17-1.uIgG4.SP in human serum. Binding of serum-incubated W3438-T3U4.E17-1.uIgG4.SP samples to Ramos on the specified day (Figure 51A); binding of serum-incubated W3438-T3U4.E17-1.uIgG4.SP samples to Jurkat on the specified day (Figure 51B). [Figure 52] Figure 52 shows the binding of W3438-T3U4.E17-1.uIgG4.SP to C1Q by ELISA. IgG1 antibody was used as a control. [Figure 53] Figure 53 shows tumor volume traces in mixed-type PBMC humanized mice with Raji xenograft tumors after administration of W3438-T3U4.E17-1.uIgG4.SP at different doses. Data points represent the group mean, and error bars represent the standard error (SEM) of that mean. IgG4 antibody was used as a negative control. [Figure 54] Figure 54 shows the pharmacokinetics of W3438-T3U4.E17-1.uIgG4.SP in cynomolgus monkeys. Serum samples from two monkeys were detected by ELISA. [Figures 55A-55B] Figures 55A-55B show anti-drug antibodies (ADA detected by ELISA) in serum samples from monkey #1 (Figure 55A) and monkey #2 (Figure 55B), including both pre- and post-administration results of W3438-T3U4.E17-1.uIgG4.SP. [Figures 56A-56B] Figures 56A and 56B show the SDS-PAGE characteristics of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP (M: protein marker, PC: positive control of a bispecific antibody of approximately 250 kDa) (Figure 56A), and the SEC-HPLC characteristics of W3248-U6T1.G25R-1.uIgG4.SP and W3248-U6T5.G25-1.uIgG4.SP (Figure 56B). [Figure 57] Figure 57 shows the melting temperatures of W3248-U6T1.G25R-1.uIgG4.SP, W3248-U6T5.G25-1.uIgG4.SP, and the benchmark bispecific anti-CTLA-4×PD-1 antibody WBP324-BMK1.uIgG1.KDL. [Figure 58]Figure 58 shows FACS binding of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to human PD-1 engineered cells. WBP324-BMK1.uIgG1.KDL, W324-BMK2.uIgG4, and W324-BMK3.uIgG4 are different types of the benchmark bispecific anti-CTLA-4×PD-1 antibody. WBP305-BMK1.IgG4 is an anti-PD-1 antibody. An IgG4 antibody was used as a negative control. [Figure 59] Figure 59 shows the FACS binding of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to cynomolgus monkey PD-1 engineered cells. WBP3055_1.153.7.hAb and WBP305-BMK1.IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as a negative control. [Figure 60] Figure 60 shows FACS binding of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to human CTLA-4 engineered cells. WBP324-BMK1.uIgG1.KDL, W324-BMK2.uIgG4, and W324-BMK3.uIgG4 are bispecific anti-CTLA-4×PD-1 antibodies with different benchmarks. WBP316-BMK1.IgG4 is an anti-CTLA-4-1 antibody. An IgG4 antibody was used as a negative control. [Figure 61] Figure 61 shows the FACS binding of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to cynomolgus monkey CTLA-4 engineered cells. WBP324-BMK1.uIgG1.KDL is the benchmark bispecific anti-CTLA-4 × PD-1 antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as a negative control. [Figure 62]Figure 62 summarizes the binding affinities of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to CTLA-4 and PD-1, as measured by SPR. WBP316-BMK1.IgG4 is an anti-CTLA-4-1 antibody. The parental anti-PD-1 antibody was used as a control. [Figure 63] Figure 63 shows a competitive FACS assay of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP for blocking human PD-L1 protein binding to PD-1 engineered cells. WBP324-BMK1.uIgG1.KDL is the benchmark bispecific anti-CTLA-4×PD-1 antibody. WBP3055_1.153.7.hAb and WBP305-BMK1.IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as a negative control. [Figure 64] Figure 64 shows a competitive FACS assay of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block human CTLA-4 protein binding to CD80-engineered cells. WBP324-BMK1.uIgG1.KDL is the benchmark bispecific anti-CTLA-4 × PD-1 antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as a negative control. [Figure 65] Figure 65 shows a competitive FACS assay of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block cynomolgus monkey CTLA-4 protein binding to CD80-engineered cells. WBP324-BMK1.uIgG1.KDL is the benchmark bispecific anti-CTLA-4 × PD-1 antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as a negative control. [Figure 66]Figure 66 shows the ELISA dual-conjugation assay of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP. WBP324-BMK1.uIgG1.KDL is the benchmark bispecific anti-CTLA-4×PD-1 antibody. An IgG4 antibody was used as a negative control. [Figure 67] Figure 67 shows the FACS dual conjugation of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to CTLA-4 and PD-1. An IgG4 antibody was used as a negative control. [Figures 68A-68B] Figures 68A and 68B show the stability of W3248-U6T5.G25-1.uIgG4.SP in serum over 14 days, as measured by ELISA dual conjugation to human CTLA-4 and PD-1 (Figure 68A), and the stability of W3248-U6T1.G25R-1.uIgG4.SP in serum over 14 days, as measured by ELISA dual conjugation to human CTLA-4 and PD-1 (Figure 68B). [Modes for carrying out the invention]

[0084] (Detailed description of the invention) The following descriptions of this disclosure are intended solely to illustrate various embodiments of this disclosure. Therefore, the specific modifications considered are not intended to limit the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, modifications, and modifications can be made without departing the scope of this disclosure, and such equivalent embodiments should be understood to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0085] (definition)

[0086] Articles (a, an, and the) are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “a polypeptide complex” means one polypeptide complex or two or more polypeptide complexes.

[0087] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of reference. In certain embodiments, when the terms “about” or “approximately” precede a number, they indicate a value within a range of plus or minus 15%, 10%, 5%, or 1%.

[0088] Throughout this disclosure, unless circumstances require otherwise, it will be understood that the phrases “including,” “including,” and “containing” imply that they encompass one or more processes or elements, or groups of processes or elements, but do not imply that they exclude any other one or more processes or elements, or groups of processes or elements. “Consists of” means that it includes and is limited to what follows the phrase “consists of.” Thus, the phrase “consists of” indicates that the enumerated elements are necessary or mandatory, and that other elements are absent. “Essentially consists of” means that it includes any elements enumerated after the phrase and is limited to other elements that do not interfere with or contribute to the activity or action identified in the disclosure relating to the enumerated elements. Thus, the phrase “essentially consists of” indicates that the enumerated elements are necessary or mandatory, while other elements are optional or may or may not exist depending on whether they affect the activity or action of the enumerated elements.

[0089] Any references throughout this disclosure, such as “one embodiment,” “a certain embodiment,” “a special embodiment,” “a related embodiment,” “a specific embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, mean that the special characteristics, structures, or features described in conjunction with that embodiment are included in at least one embodiment of this disclosure. Accordingly, the appearance of preceding phrases in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, the special characteristics, structures, or features may be combined in any preferred manner in one or more embodiments.

[0090] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues, or aggregates of polymers of multiple amino acid residues. These terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those subsequently modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., compounds having an alpha carbon, carboxyl group, amino group, and R group bonded to hydrogen, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but still retain the same basic chemical structure as naturally occurring amino acids. The alpha carbon refers to the first carbon atom bonded to a functional group such as a carbonyl. The beta carbon refers to the second carbon atom bonded to the alpha carbon, and this system continues to name the carbons alphabetically by Greek letters. Amino acid mimes refer to compounds that have a different structure from the general chemical structure of amino acids but function in a similar manner to naturally occurring amino acids. The term “protein” typically refers to a large polypeptide. The term “peptide” typically refers to a short polypeptide. Polypeptide sequences are usually described as having an amino terminus (N-terminus) at the left end of the polypeptide sequence; and a carboxyl terminus (C-terminus) at the right end of the polypeptide sequence. As used herein, “polypeptide complex” refers to a complex comprising one or more polypeptides associated to perform a particular function. In certain embodiments, this polypeptide is related to immunity.

[0091] As used herein, the term “antibody” encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. Intact antibodies of the natural type contain two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") and first, second, and third constant regions (CH1, CH2, and CH3), while each light chain consists of a variable region ("LCVR") and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. Antibodies have a “Y” structure, where the Y-shaped stem consists of the second and third constant regions of two heavy chains linked together via disulfide bonds. Each arm of this Y-shape contains a variable region and first constant region of a single heavy chain, linked to a variable region and constant region of a single light chain. The variable regions of the light and heavy chains contribute to antigen binding. The variable regions of both chains generally contain three hypervariable loops, referred to as complementarity-determining regions (CDRs) (the light (L) chain CDRs include LCDR1, LCDR2, and LCDR3, while the heavy (H) chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundary of an antibody is defined or identified according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). These three types of CDRs are sandwiched between flanking sequences (stretches) known as framework regions (FRs), which are more highly conserved than CDRs and form scaffolds to support hypervariable loops.Each of the HCVR and LCVR contains four FRs, and the CDRs and FRs are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains do not participate in antigen binding but exert various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains. Some of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0092] As used herein, the antibody term "variable domain" refers to an antibody variable region or fragment thereof containing one or more CDRs. A variable domain may include an intact variable region (such as an HCVR or LCVR), but it may also include a sub-intact variable region that still retains the ability to bind to an antigen or form an antigen-binding site.

[0093] As used herein, the term “antigen-binding moiety” refers to an antibody fragment formed from an antibody moiety containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain an intact, native antibody structure. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding moiety is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, the antigen-binding moiety may contain one or more CDRs derived from a specific human antibody grafted onto a framework region derived from one or more different human antibodies. More detailed formats of the antigen-binding moiety are described in the papers by Spiess et al. (2015 (op. above)) and Brinkman et al. (mAbs, 9(2), pp.182-212 (2017)), which are incorporated herein by reference in their entirety.

[0094] In relation to the antibody, "Fab" refers to the portion of the antibody consisting of a single light chain (both variable and constant regions) associated by disulfide bonds to a variable region and a first constant region of a single heavy chain. In certain embodiments, both the constant regions of the light and heavy chains are replaced by a TCR constant region.

[0095] "Fab" refers to a Fab fragment that includes the hinge region.

[0096] "F(ab')2" refers to the dimer of Fab'.

[0097] "Vibody" refers to a fusion protein formed by the fusion of scFv to the C-terminus of either the light chain (Fab-L-scFv) or Fd (Fab-H-scFv).

[0098] A "tribody" refers to a fusion protein formed by the fusion of scFv to both its light and heavy chains (Fab-(scFv)2).

[0099] "WuXiBody" is a bispecific antibody containing a soluble chimeric protein along with the antibody's variable domain and the constant domain of the TCR, where the subunits of the TCR constant domain (such as the alpha and beta domains) are linked by manipulated disulfide bonds.

[0100] In the context of antibodies, "fragment difficult (Fd)" refers to the amino-terminal half of a heavy chain fragment that can be combined with a light chain to form a Fab.

[0101] In the context of antibodies, "Fc" refers to the portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of the first heavy chain, which are bound to the second and third constant regions of the second heavy chain by disulfide bonds. The Fc portion of an antibody contributes to various effector functions, such as ADCC and CDC, but does not function in antigen binding.

[0102] The "hinge region" of an antibody contains the heavy chain molecule that links the CH1 domain to the CH2 domain. This hinge region consists of approximately 25 amino acid residues, is mobile, and therefore allows the two N-terminal antigen-binding regions to move independently.

[0103] As used herein, the term "CH2 domain" refers to a portion of a heavy chain molecule, extending from approximately 244 to 360 amino acids in an IgG antibody, using a conventional numbering scheme (amino acids 244 to 360, Kabat numbering system; and amino acids 231 to 340, EU numbering system; see Kabat, E. et al., US Department of Health and Human Services, (1983)).

[0104] The "CH3 domain" extends from the CH2 domain of the IgG molecule to the C-terminus and contains approximately 108 amino acids. Certain immunoglobulin classes, such as IgM, also contain a CH4 region.

[0105] In relation to antibodies, "Fv" refers to the smallest fragment of an antibody that possesses a complete antigen-binding site. An Fv fragment consists of a variable domain of a light chain bound to a variable domain of a heavy chain. Numerous Fv designs have been provided, including dsFv, where the association between the two domains is enhanced by an introduced disulfide bond; and scFv, which can be formed as a single polypeptide using a peptide linker to join the two domains together. Fv constructs containing the corresponding variable domain of an immunoglobulin heavy or light chain and the variable domain of an immunoglobulin heavy or light chain associated with a constant domain have also been constructed. Fv can also be polymerized to form diabodies and tribodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0106] "scFab" refers to a fusion polypeptide containing Fd linked to a light chain via a polypeptide linker, resulting in the formation of a single-stranded Fab fragment (scFab).

[0107] "TriFab" refers to a trivalent, bispecific fusion protein with Fab function, composed of three units. TriFab houses two normal Fabs fused to an asymmetric Fab-like region.

[0108] "Fab-Fab" refers to a fusion protein formed by the fusion of the Fd chain of the first Fab arm to the N-terminus of the Fd chain of the second Fab arm.

[0109] "Fab-Fv" refers to a fusion protein formed by fusing the Fd chain of HCVR to the C-terminus and the light chain of LCVR to the C-terminus. The "Fab-dsFv" molecule can be formed by introducing an interdomain disulfide bond between the HCVR domain and the LCVR domain.

[0110] "mAb-Fv" or "IgG-Fv" refers to a fusion protein formed by the fusion of one Fc chain of an HCVR domain to its C-terminus, and to an LCVR domain, either individually expressed or fused to the other C-terminus, resulting in a bispecific trivalent IgG-Fv (mAb-Fv) fusion protein with Fv stabilized by interdomain disulfide bonds.

[0111] "scFab-Fc-scFv2" and "scFab-Fc-scFv" refer to fusion proteins formed by the fusion of a single-strand Fab with the Fc and disulfide-stabilized Fv domains.

[0112] "Appended IgG" refers to a fusion protein with Fab arms fused to IgG to form a bispecific (Fab)2-Fc format. This can form "IgG-Fab" or "Fab-IgG" with Fab fused to the C-terminus or N-terminus of the IgG molecule, with or without a connector. In certain embodiments, the appended IgG can be further modified into the IgG-Fab4 format (see Brinkman et al., 2017, op. cit.).

[0113] "DVD-Ig" refers to a dual-variable-domain antibody formed by the fusion of additional HCVR and LCVR domains of second specificity to the IgG heavy and light chains. "CODV-Ig" refers to a related format in which two HCVR domains and two LCVR domains are linked in a manner that allows for the formation of variable HCVR-LCVR domain crossover pairs, which are arranged (from N-terminus to C-terminus) in either sequential HCVRA-HCVRB and LCVRB-LCVRA, or sequentially HCVRB-HCVRA and LCVRA-LCVRB.

[0114] "CrossMab" refers to a technique for pairing unmodified light chains with corresponding unmodified heavy chains, and for pairing modified light chains with corresponding modified heavy chains, resulting in antibodies with reduced mispairing in the light chains.

[0115] "BiTE" is a bispecific T cell engager molecule that contains a first scFv with primary antigen specificity in LCVR-HCVR orientation, linked to a second scFv with secondary specificity in HCVR-LCVR orientation.

[0116] "Percent (%) sequence identity" for an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after sequence alignment and the introduction of gaps as necessary to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for the purpose of determining the % identity of amino acid (or nucleic acid) sequences can be achieved using publicly available tools such as BLASTN, BLASTp (available from the website of the National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the website of the European Institute for Bioinformatics (EBI), see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or they may customize the parameters to suit the sorting process, for example, by selecting a suitable algorithm.

[0117] As used herein, “antigen” or “Ag” refers to a compound, composition, peptide, polypeptide, protein, or substance that can stimulate antibody production or T cell response in cell cultures or animals, and includes compositions that are added to cell cultures (e.g., hybridomas) or injected into or absorbed by animals (e.g., those containing cancer-specific proteins). Antigens react with products of specific humoral or cellular immunity (e.g., antibodies), including those induced by heterologous antigens.

[0118] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a conjugate (such as an antibody) binds. An epitope can be formed from both contiguous amino acids (also referred to as linear or continuous epitopes) or non-contiguous amino acids juxtaposed by the three-dimensional folding of a protein (also referred to as conformational or higher-order structure epitopes). Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on a protein, and small segments of contiguous amino acids can be digested from an antigen bound to a major histocompatibility complex (MHC) molecule or retained upon exposure to a denaturing solvent, whereas epitopes formed by three-dimensional folding are typically lost upon treatment with a denaturing solvent. An epitope typically contains at least 3, more generally at least 5, about 7, or about 8 - 10 amino acids in its unique spatial higher-order structure.

[0119] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the polypeptide complexes and bispecific polypeptide complexes provided herein have a binding affinity (K -9 , ,

[0120] , -8 , , -8 , -7 , , -8 ,

[0119] , -7 , off , , -7 , on , <00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The term “functionally linked” or “functionally linked” refers to two or more biological sequences of interest being proximal to each other, with or without spacers or linkers, in a relationship that enables them to function in the intended manner. When used in relation to polypeptides, it is intended to mean that the polypeptide sequence is linked in such a way that the linked product will have the intended biological function. For example, an antibody variable region may be functionally linked to a constant region to provide a stable product with antigen-binding activity. The term is also used in relation to polynucleotides. For example, when a polynucleotide encoding a polypeptide is functionally linked to a regulatory sequence (e.g., a promoter, enhancer, or silencer sequence), it is intended to mean that the polynucleotide sequence is linked in such a way that it enables the regulated expression of the polypeptide derived from the polynucleotide.

[0121] When used in relation to amino acid sequences (e.g., peptides, polypeptides, or proteins), the terms “fusion” or “fused” refer to a combination of two or more amino acid sequences that do not exist in nature, formed by, for example, chemical bonding or recombinant means to a single amino acid sequence. Fusion amino acid sequences may be produced by genetic recombination of two encoding polynucleotide sequences and may be expressed by introducing a construct containing the recombinant polynucleotides into a host cell.

[0122] As used herein, the term "spacer" refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or 5 to 15, 20, 30, 50, or more amino acid residues in length, which is linked by peptide bonds and used to link one or more polypeptides. Spacers may or may not have a secondary structure. Spacer sequences are known in the art; see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994). Any suitable spacer known in the art may be used. For example, spacers useful in this disclosure may be rich in glycine and proline residues. Examples of spacers include single or repeat sequences composed of threonine / serine and glycine, such as TGGGG (SEQ ID NO: 266), GGGGS (SEQ ID NO: 267), or SGGGG (SEQ ID NO: 268), or tandem repeat sequences thereof (e.g., 2, 3, 4, or more repeat sequences). Alternatively, the spacer may be a long peptide chain containing one or more consecutive or tandem repeat sequences of the amino acid sequence GAPGGGGAAAAAGGGGG (SEQ ID NO: 269). In a particular embodiment, the spacer contains 1, 2, 3, 4, or more consecutive or tandem repeat sequences of SEQ ID NO: 269.

[0123] The term "antigen specificity" refers to a particular antigen or its epitope that is selectively recognized by an antigen-binding molecule.

[0124] As used herein, the term "substitution" in relation to amino acid residues refers to the naturally occurring or induced substitution of one or more amino acids by another amino acid in a peptide, polypeptide, or protein. Substitutions in polypeptides may result in a reduction, enhancement, or loss of polypeptide function.

[0125] Substitutions can also be “conservative substitutions” with respect to the amino acid sequence, which refers to substitutions of amino acid residues with different amino acid residues having side chains with similar physicochemical properties, or substitutions of those amino acids that are not important to the activity of the polypeptide. For example, conservative substitutions can be made between amino acid residues with nonpolar side chains (e.g., Met, Ala, Val, Leu, and Ile, Pro, Phe, Trp), between residues with uncharged polar side chains (e.g., Cys, Ser, Thr, Asn, Gly, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), between amino acids with beta-branched side chains (e.g., Thr, Val, and Ile), between amino acids with sulfur-containing side chains (e.g., Cys and Met), or between residues with aromatic side chains (e.g., Trp, Tyr, His, and Phe). In certain embodiments, substitutions, deletions, or additions can also be considered “conservative substitutions.” The number of amino acids inserted or deleted can range from approximately 1 to 5. Conservative substitutions usually do not cause significant changes to the higher-order structure of the protein, and as a result, the biological activity of the protein can be preserved.

[0126] As used herein, the terms "mutation" or "mutated" in relation to amino acid residues refer to the substitution, insertion, or addition of an amino acid residue.

[0127] As used herein, "homologous sequence" and "homologous sequence" refer to polynucleotide sequences (or their complementary chains) or amino acid sequences that are interchangeable and, when arbitrarily aligned, have at least 80% (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with respect to another sequence.

[0128] As used herein, "T cell" refers to a type of lymphocyte that plays a crucial role in cell-mediated immunity, including helper T cells (e.g., CD4). +T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 3 T cells, T helper type 17 T cells), cytotoxic T cells (e.g., CD8) + T cells), memory T cells (e.g., central memory T cells (TCM cells), effector memory T cells (TEM cells and TEMRA cells), and CD8 + or CD4 + This includes tissue-resident memory T cells (TRMs), natural killer T (NKT) cells, and suppressor T cells, which are any of the following:

[0129] The native "T cell receptor" or native "TCR" is a heterodimeric T cell surface protein that associates with variant CD3 chains to form a complex capable of mediating signal transduction. The TCR belongs to the immunoglobulin superfamily and is similar to a semi-antibody with one heavy chain and one light chain. The native TCR has an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of the TCR has a constant region proximal to the membrane and a variable region distal to the membrane.

[0130] As used herein, the terms “subject,” “individual,” “animal,” or “patient” refer to humans or non-human animals, including mammals or primates, who require diagnosis, prognosis, improvement, prevention, and / or treatment for a disease or disorder. Mammal subjects include humans, domestic animals, farm animals, and animals in zoos, sports, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0131] (A. Polypeptide complex)

[0132] A novel polypeptide complex is provided herein, comprising an antibody heavy chain variable domain functionally linked to a first T cell receptor (TCR) constant region and an antibody light chain variable domain functionally linked to a second TCR constant region, wherein the first and second TCR constant regions are associated via at least one non-natural interchain linkage. The polypeptide complex comprises at least two polypeptide chains, each comprising an antibody-derived variable domain and a TCR-derived constant region. The two polypeptide chains of the polypeptide complex comprise a pair of heavy chain variable domains and a light chain variable domain, each functionally linked to a pair of TCR constant regions. Examples of TCR constant region pairs include, for example, alpha / beta, pre-alpha / beta, and gamma / delta TCR constant regions. The TCR constant regions in the polypeptide complex provided herein may be full-length or fragmentary, and the pairs of TCR constant regions can be manipulated insofar as they can associate with each other to form a dimer.

[0133] Surprisingly, polypeptide complexes provided herein, accompanied by at least one non-natural interchain linkage (particularly a non-natural disulfide linkage), can be recombinantly expressed and assembled into desired higher-order structures, which have been found to stabilize the TCR constant region dimer while simultaneously providing good antigen-binding activity of the antibody variable region. Furthermore, the polypeptide complexes have been found to tolerate conventional antibody manipulations well, such as modification of glycosylation sites and removal of some native sequences. Moreover, the polypeptide complexes provided herein can be incorporated into bispecific formats, which are readily expressed and assembled, and due to the presence of the TCR constant region in the polypeptide complex, mispairing of antigen-binding sequences is minimal or virtually nonexistent. Additional advantages of the polypeptide complexes and constructs provided herein will become more apparent in the following disclosures.

[0134] In one embodiment, the present disclosure provides a polypeptide complex comprising a first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from N-terminus to C-terminus to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising a first light chain variable domain (VL) of a first antibody functionally linked from N-terminus to C-terminus to a second TCR constant region (C2), wherein C1 and C2 are capable of forming a dimer comprising at least one non-natural interchain linkage between C1 and C2, and this non-natural interchain linkage is capable of stabilizing the dimer, and the first antibody has first antigen specificity.

[0135] (i. TCR steady region)

[0136] The polypeptide complex provided herein includes a constant region derived from the TCR.

[0137] Native TCRs consist of two polypeptide chains and generally have two types: one consisting of an alpha-beta chain (i.e., alpha / beta TCR), and the other consisting of a gamma-delta chain (i.e., gamma / delta TCR). These two types are structurally similar but have different configurations and functions. Approximately 95% of human T cells have alpha / beta TCRs, while the remaining 5% have gamma / delta TCRs. A precursor of the alpha chain is also recognized and is called the pre-alpha chain. Each of the two TCR polypeptide chains contains an immunoglobulin domain and a membrane-proximal region. The immunoglobulin region contains a variable region and a constant region and is characterized by the presence of an immunoglobulin-type fold. Each TCR polypeptide chain has cysteine ​​residues (e.g., at the C-terminus of the constant domain or the N-terminus of the membrane-proximal region), which together can form a disulfide bond that links the two TCR chains together.

[0138] Figures 18A–18E show the amino acid sequences of the native TCR constant regions of the TCR alpha, pre-alpha, beta, gamma, and delta chains. For clarity and consistency, each amino acid residue in these sequences is numbered in Figures 19A–19E, and such numbering is used throughout this disclosure to refer to specific amino acid residues on specific TCR constant regions.

[0139] The constant region of the human TCR alpha chain is known as TRAC and is the amino acid sequence of NCBI deposit number P01848, or sequence number 254.

[0140] The human TCR beta-chain constant region has two distinct variants known as TRBC1 and TRBC2 (IMGT nomenclature), the corresponding sequences of which are shown in SEQ ID NO: 256 and SEQ ID NO: 257, respectively (see also Toyonaga B, et al., PNAs, Vol. 82, pp. 8624-8628, Immunology (1985)). These two beta-constant domains differ in the amino acid residues at positions 4, 5, and 37 of exon 1. Specifically, TRBC1 has 4N, 5K, and 37F in exon 1, while TRBC2 has 4K, 5N, and 37Y in exon 1.

[0141] Specifically, the native TCR beta chain contains a native cysteine ​​residue at position 74 (see Figure 19B), which does not form a pair and consequently does not form a disulfide bond in the native alpha / beta TCR. In certain embodiments, in the polypeptide complex provided herein, this native cysteine ​​residue is either absent or mutated to another residue. This is useful to avoid incorrect intra-chain or inter-chain pairing. In certain embodiments, the native cysteine ​​residue is substituted with another residue, such as serine or alanine. In certain embodiments, substitutions in certain embodiments, in vitro, can improve TCR refolding efficiency.

[0142] The human TCR gamma chain constant region has two known variants, TRGC1 and TRGC2 (see Lefranc et al., Eur. J. Immunol. 19:989-994 (1989)), which have the amino acid sequences of NCBI deposit numbers A26659 and P03986, respectively, or sequence numbers 263 and 265, respectively.

[0143] The human TCR delta chain constant region is known as TRDC and has the amino acid sequence of NCBI deposit number A35591, sequence number 261.

[0144] The constant region of the TCR in the polypeptide complex provided herein may also be derived from a pre-T cell antigen receptor (pre-TCR). The pre-TCR is expressed by immature thymocytes and plays a central role in early T cell development. The pre-TCR has a normal beta chain but possesses a special pre-alpha chain that has a sequence and structure different from that of a normal alpha chain, and is accompanied only by an available constant region (see Harald von Boehmer, Nat Rev Immunol, Jul;5(7):571-7 (2005)). The sequence of the human pre-alpha chain constant region (PTCRA) has the amino acid sequence of NCBI deposit number AAF89556.1, or sequence number: 259.

[0145] In this disclosure, the first and second TCR constant regions of the polypeptide complex provided herein are capable of forming a dimer between the TCR constant regions, comprising at least one non-natural interchain linkage capable of stabilizing the dimer.

[0146] As used herein, the term “dimer” refers to an associated structure formed by two molecules, such as polypeptides or proteins, through covalent or non-covalent interactions. Homodimerization is formed by two identical molecules, while heterodimerization is formed by two different molecules. A dimer formed by a first and second TCR constant region is a heterodimer.

[0147] Interchain bonds are formed between one amino acid residue on one TCR constant region and another amino acid residue on another TCR constant region. In certain embodiments, unnatural interchain bonds can be any bond or interaction that allows two TCR constant regions to associate into a dimer. Suitable examples of unnatural interchain bonds include disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophobic-hydrophilic interactions, knob-into-holes, or combinations thereof.

[0148] A "disulfide bond" refers to a covalent bond with the structure RSS-R'. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond with a second thiol group derived from another cysteine ​​residue, for example. Disulfide bonds are formed between the thiol groups of two cysteine ​​residues present in each of two polypeptide chains, thereby forming interchain bridges or interchain bonds.

[0149] Electrostatic interactions are non-covalent interactions that are important for protein folding, stability, mobility, and function, and include ionic interactions, hydrogen bonds, and halogen bonds. Electrostatic interactions can form in polypeptides, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp in the first chain and Arg, Val, or Thr in the second chain.

[0150] Salt bridges are short-range electrostatic interactions primarily arising from an anionic carboxylate (either Asp or Glu) and a cationic ammonium (Lys) or guanidinium (Arg), which are spatially close pairs of oppositely charged residues in the native protein structure. Charged and polar residues within highly hydrophobic interfaces can act as binding hotspots. Among these, residues with ionizable side chains, such as His, Tyr, and Ser, can also be involved in salt bridge formation.

[0151] Hydrophobic interactions can be formed between one or more Val, Tyr, and Ala atoms in the first chain and one or more Val, Leu, and Trp atoms in the second chain, or between His and Ala atoms in the first chain and Thr and Phe atoms in the second chain (see Brinkmann et al., 2017, op. cit.).

[0152] Hydrogen bonds are formed by the electrostatic attraction between two polar groups when a hydrogen atom is covalently bonded to a highly electronegative atom such as nitrogen, oxygen, or fluorine. Hydrogen bonds can be formed in polypeptides between the skeletal oxygen (e.g., chalcogen group) and amide hydrogen (nitrogen group) of two residues, such as the nitrogen group of Asn and the oxygen group of His, or the oxygen group of Asn and the nitrogen group of Lys. Hydrogen bonds are stronger than van der Waals interactions but weaker than covalent or ionic bonds, and are important for maintaining secondary and tertiary structures. For example, an alpha-helix is ​​formed when amino acid residue spacing occurs regularly between positions i and i+4, and a beta-sheet is a peptide chain sequence of 3 to 10 amino acids in length, formed when two peptides are linked by at least two or three skeletal hydrogen bonds, forming a twisted pleated sheet.

[0153] As used herein, “knob-into-hole” refers to an interaction between two polypeptides, where one polypeptide has a projection (i.e., a “knob”) due to the presence of an amino acid residue with a bulky side chain (e.g., tyrosine or tryptophan), and the other polypeptide has a cavity (i.e., a “hole”) containing an amino acid residue with a small side chain (e.g., alanine or threonine), and the projection can be positioned within the cavity to facilitate the interaction between the two polypeptides and form a heterodimer or complex. Methods for producing polypeptides in a knob-into-hole manner are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0154] In certain embodiments, the TCR constant region dimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unnatural interchain bonds. Optionally, at least one of the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unnatural interchain bonds is a disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction, or any combination thereof.

[0155] As used herein, “unnatural” interchain junctions refer to interchain junctions not observed in the natural association of the TCR constant region of the natural counterpart. For example, unnatural interchain junctions may be formed between a mutated amino acid residue and a natural amino acid residue present on each TCR constant region; or between two mutated amino acid residues present on each TCR constant region. In certain embodiments, at least one unnatural interchain junction is formed between a first mutated residue in the first TCR constant region of the polypeptide complex and a second mutated residue in the second TCR constant region.

[0156] A "mutated" amino acid residue is one that has been substituted, inserted, or added and is different from its native counterpart in the corresponding native TCR constant region. For example, if an amino acid residue at a particular position in the wild-type TCR constant region is referred to as a "native" residue, its mutated counterpart is any residue that is different from the native residue but exists at the same position in the TCR constant region. A mutated residue can be a different residue that either substitutes for the native residue at the same position or is inserted before the native residue and thus occupies its original position.

[0157] In certain embodiments, the mutated residue may be a naturally occurring amino acid residue. In certain embodiments, at least one of the first and second non-natural amino acid residues is a mutated cysteine ​​residue. In certain embodiments, one or more of the non-natural interchain bonds are disulfide bonds. In certain embodiments, the non-natural disulfide bonds may be formed between two mutated cysteine ​​residues, each contained within the first and second TCR constant regions.

[0158] In certain embodiments, at least one of the first and second mutated residues is a non-naturally occurring amino acid residue. A non-naturally occurring amino acid residue refers to an amino acid residue that is not naturally found in human proteins but can be expressed via nucleic acid codons that can be introduced into encoding polynucleotides. For example, a non-naturally occurring amino acid such as L-3,4-dihydroxyphenylalanine (L-DOPA) can react with and crosslink with natural amino acids such as cysteine, histidine, and lysine through periodic acid-induced oxidation. It has been shown that the incorporation of L-DOPA into an antibody allows the non-natural amino acid to effectively crosslink residues on the antigen, resulting in the formation of a covalently bonded antibody-antigen complex (Xu, J. et al., 2014, "Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex," Journal of Structural Biology, 185(2), pp.215-222). In this specification, the mutated amino acid residues in the first and / or second TCR constant regions may include unnatural amino acid residues, such as L-DOPA, which can crosslink with natural amino acid residues (or amino acid residues that do not occur naturally) to form unnatural interchain covalent bonds.

[0159] In certain embodiments, at least one unnatural disulfide bond is formed between a mutated cysteine ​​residue and a native cysteine ​​residue. In certain embodiments, an unnatural disulfide bond is formed between two mutated cysteine ​​residues. In certain embodiments, at least one of the cysteine ​​residues forming the unnatural disulfide bond is a mutated cysteine ​​residue. In certain embodiments, both cysteine ​​residues forming the unnatural disulfide bond are mutated cysteine ​​residues on the first and second TCR constant regions, respectively.

[0160] In certain embodiments, the first and / or second TCR constant regions may be manipulated to include one or more mutated amino acid residues that contribute to the formation of non-native interchain bonds. To introduce such mutated residues into the TCR constant regions, the coding sequence of the TCR region may be manipulated, for example, to replace a codon encoding a native residue with a codon encoding a mutated residue, or to insert a codon encoding a mutated residue before a codon encoding a native residue. One or more desired mutated amino acid residues may be introduced into the TCR constant regions as one or more amino acid residues (e.g., cysteine ​​residues) that can, for example, form disulfide bonds, lead to electrostatic interactions between two TCR constant regions, increase the mobility of the TCR constant regions, position at least one amino acid that forms a covalent bond away from the TCR constant domain, such as a hydrogen bond, or contribute to the formation of a salt bridge; hydrophobic amino acid residues that can lead to hydrophobic interactions; and hydrophilic amino acid residues that can lead to hydrophilic interactions.

[0161] In certain embodiments, the first and / or second TCR constant regions can be manipulated to include one or more mutated cysteine ​​residues. For example, a non-cysteine ​​residue can be replaced with a cysteine ​​residue, or a cysteine ​​residue can be inserted between two original adjacent native non-cysteine ​​residues. The location of the replacement can be determined such that, after the replacement with the cysteine ​​residue, a non-native interchain disulfide bond is formed between the two TCR constant regions. To this end, several factors can be considered, including, for example, that the cysteine ​​residues forming the disulfide bond are sufficiently close, have appropriate alpha-beta bond orientation, that the thiol groups of the cysteine ​​residues are oriented facing each other, that the residue to be replaced has a side chain with chemical properties relatively similar to those of cysteine, and / or that the replacement does not substantially disrupt the tertiary structure of the TCR constant region or the polypeptide complex itself.

[0162] Those skilled in the art can determine the distance and angle between two amino acid residues to be replaced using, for example, non-limitingly, preferred methods known in the art, such as distance mapping by photodetection, computer modeling, NMR spectroscopy, or X-ray crystallography. In illustrative examples, the protein crystal structure of a polypeptide of interest (such as a TCR constant region) can be obtained from a publicly available database, such as the PDB database, or elucidated using methods such as X-ray crystallography. The distance and angle between amino acid residues can be determined based on the protein crystal structure data using suitable computer software. In certain embodiments, disulfide bonds in the polypeptide complex provided herein can be formed between mutated cysteine ​​residues having each beta-carbon that are sufficiently close, for example, with a distance of less than 8 Å, 7 Å, 6 Å, 5 Å, 4 Å, 3 Å, 2 Å, 1 Å or less, provided the complex is folded correctly.

[0163] Further preferred locations for manipulation of the first and / or second TCR constant region can be selected from publicly available crystal structure data for this complex, between TCR alpha and beta (Boulter, JM et al., Protein Engineering, 16(9), pp.707-711 (2003)) or between gamma and delta (Allison, TJ et al., Nature, 411(6839), pp.820-824 (2001); Uldrich, AP et al., Nature Immunology, 14(11), pp.1137-1145 (2013)). Once the residue to be replaced is determined, those skilled in the art can easily identify the codon of interest to be mutated (e.g., through sequence sorting using existing software such as ClustalW (EBI website (www.ebi.ac.uk / index.html)) and then mutate it to a cysteine ​​codon by methods known in the art, such as PCR mutagenesis.

[0164] The formation of interchain disulfide bonds can be determined by preferred methods known in the art. For example, expressed protein products can be subjected to reduced and unreduced SDS-PAGE, and the resulting bands can be compared to identify differences that may indicate the presence of interchain disulfide bonds.

[0165] Non-native interchain links can stabilize polypeptide complexes. Such stabilization can be carried out in various ways. For example, the presence of mutated amino acid residues or non-native interchain links can cause polypeptide complexes to be stably expressed and / or expressed at high levels and / or associated with stable complexes having desired biological activity (e.g., antigen-binding activity) and / or expressed and assembled at high levels with desired stable complexes having desired biological activity. The ability of interchain links to stabilize the first and second TCR constant regions can be evaluated using appropriate methods known in the art, such as molecular weight indicated on an SDS-PAGE or thermal stability measured by differential scanning calorimetry (DSC) or differential scanning fluorescence (DSF). In illustrative examples, the formation of stable polypeptide complexes provided herein can be confirmed by SDS-PAGE if the product exhibits a molecular weight equivalent to the molecular weight of the first and second polypeptides combined. In certain embodiments, the polypeptide complexes provided herein are stable in such a way that their thermal stability is approximately 50%, 60%, 70%, 80%, or 90% greater than that of natural Fab. In certain embodiments, the polypeptide complexes provided herein are stable in such a way that their thermal stability is equivalent to that of natural Fab.

[0166] While we do not wish to link this to any particular theory, it is thought that the non-native interchain bonds (such as disulfide bonds) formed between the first and second TCR constant regions of the polypeptide complex stabilize the TCR constant region heterodimer, thereby enhancing the level of precise folding, structural stability, and / or expression of the heterodimer and polypeptide complex. Unlike the native TCR anchored to the membrane on the T cell surface, the heterodimer of the native TCR extracellular domain is observed to be far less stable, despite its similarity to antibody Fab in 3D structure. In fact, the instability of the native TCR under soluble conditions has been used as a significant obstacle to elucidating its crystal structure (see Wang, Protein Cell, 5(9), 649-652, (2014)). By introducing a cysteine ​​(Cys) mutation pair within the TCR constant region and thereby enabling the formation of non-native interchain disulfide bonds, this polypeptide complex is expressed stably while the antigen-binding ability of the antibody variable region is retained.

[0167] A TCR constant region containing mutated residues is also referred to herein as an "engineered" TCR constant region. In certain embodiments, the first TCR constant region (C1) of the polypeptide complex contains an engineered TCR alpha chain (CAlpha), and the second TCR constant region (C2) contains an engineered TCR beta chain (CBeta). In certain embodiments, C1 contains an engineered CBeta, and C2 contains an engineered CAlpha. In certain embodiments, C1 contains an engineered TCR pre-alpha chain (CPre-Alpha), and C2 contains an engineered CBeta. In certain embodiments, C1 contains an engineered CBeta, and C2 contains an engineered CPre-Alpha. In certain embodiments, C1 contains an engineered TCR gamma chain (CGamma), and C2 contains an engineered TCR delta chain (CDelta). In certain embodiments, C1 contains an engineered CDelta, and C2 contains an engineered CGamma.

[0168] In certain embodiments, the manipulated TCR constant region contains one or more mutated cysteine ​​residues. In certain embodiments, one or more mutated residues are contained within the contact interface of the first and / or second manipulated TCR constant region.

[0169] As used herein, the term "contact interface" refers to a specific region(s) on a polypeptide where polypeptides interact / associate with each other. A contact interface contains one or more amino acid residues capable of interacting with corresponding amino acid residues(s) so as to come into contact or associate when an interaction occurs. The amino acid residues in a contact interface may or may not be in a continuous sequence. For example, if the interface is three-dimensional, the amino acid residues within the interface may be separated into different positions on a linear sequence.

[0170] In certain embodiments, the manipulated CBeta contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86, and 122-127. In certain embodiments, the manipulated CAlpha contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 6-29, 37-67, and 86-95. Unless otherwise specified, the numbering of amino acid residues within the constant region of the TCR in this disclosure is as shown in Figures 19A-19E.

[0171] In certain embodiments, one or more disulfide bonds may be formed between the manipulated CAlpha and the manipulated CBeta. The mutated cysteine ​​residue in CBeta may be a substitution selected from the group consisting of S56C, S16C, F13C, V12C, E14C, F13C, L62C, D58C, S76C, and R78C, and / or the mutated cysteine ​​residue in CAlpha may be a substitution selected from the group consisting of T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C, and S62C. In certain embodiments, the mutated cysteine ​​residue pairs are S16C in CBeta and Y11C in CAlpha, F13C in CBeta and L13C in CAlpha, S16C in CBeta and L13C in CAlpha, V12C in CBeta and S16C in CAlpha, E14C in CBeta and S16C in CAlpha, F13C in CBeta and V23C in CAlpha, L62C in CBeta and Y44C in CAlpha, and D in CBeta. The substitutions can be selected from the group consisting of 58C and T46C in CAlpha, S76C in CBeta and T46C in CAlpha, S56C in CBeta and T49C in CAlpha, S56C in CAlpha and L51C in CAlpha, S56C in CAlpha and S62C in CAlpha, and R78C in CAlpha and S62C in CAlpha, and the cysteine ​​residue pair can form a non-natural interchain disulfide bond.

[0172] As used herein, "XnY" with respect to the TCR constant region means that the nth amino acid residue X on the TCR constant region (based on the numbering provided in Figures 19A-19E) is replaced by the amino acid residue Y, where X and Y are intended to be one-letter abbreviations of specific amino acid residues. It should be noted that the number n is simply based on the numbering provided in Figures 19A-19E and may differ from its actual position. For illustrative purposes, we use the sequence CBeta(S56C)(N69Q) shown in Sequence ID No. 34. The substitution of S with C occurs at the 48th residue in Sequence ID No. 34, while this residue was designed as the 56th residue based on the numbering system in Figures 19A-19E, and as a result the substitution of S with C is designated as S56C, not S48C. Similarly, the substitution of N with Q is designated as N69Q based on the numbering system in Figures 19A-19E. This amino acid residue substitution specification rule applies to all TCR constant regions in this disclosure unless otherwise specified. Similarly, when used in relation to the Fc region, "XnY" means that the nth amino acid residue X on the Fc constant region (based on the numbering in Figures 20A–20D provided herein) is replaced by amino acid residue Y.

[0173] In a particular embodiment, the manipulated CBeta includes or is one of sequence numbers 33-41, and the manipulated CAlpha includes or is one of sequence numbers 43-48.

[0174] In certain embodiments, one or more unnatural disulfide bonds may be formed within the contact interface between CPre-Alpha and CBeta. In certain embodiments, the contact interface on CPre-Alpha is selected from substitutions at amino acid residues 7-19, 26-34, 56-75 and 103-106. In certain embodiments, the contact interface on CBeta is selected from substitutions at amino acid residues 9-35, 52-66, 71-86 and 122-127.

[0175] In certain embodiments, one or more disulfide bonds may be formed between the manipulated pre-TCR alpha constant region (CPre-Alpha) and the beta chain constant region (CBeta). The mutated cysteine ​​residue in CBeta may be a substitution selected from the group consisting of S16C, A18C, E19C, F13C, A11C, S56C, and S76C, and / or the mutated cysteine ​​residue in CPre-Alpha may be a substitution selected from the group consisting of S11C, A13C, I16C, S62C, T65C, and Y59. In certain embodiments, the mutated cysteine ​​residue pair can be a substitution selected from the group consisting of S16C in CBeta and S11C in CPre-Alpha, A18C in CBeta and S11C in CPre-Alpha, E19C in CBeta and S11C in CPre-Alpha, F13C in CBeta and A13C in CPre-Alpha, S16C in CBeta and A13C in CPre-Alpha, A11C in CBeta and I16C in CPre-Alpha, S56C in CBeta and S62C in CPre-Alpha, S56C in CBeta and T65C in CPre-Alpha, and S76C in CBeta and Y59C in CPre-Alpha, where the mutated cysteine ​​residue pair can form a non-natural interchain disulfide bond.

[0176] In a particular embodiment, the manipulated CBeta includes or is one of sequence numbers 33-41, and the manipulated CPre-Alpha includes or is one of sequence numbers 82 and 83.

[0177] In certain embodiments, one or more non-natural disulfide bonds may be formed within the contact interface between CGamma and CDelta. In certain embodiments, the contact interface on CGamma is selected from substitutions at amino acid residues 11-35 and 55-76. In certain embodiments, the contact interface on CDelta is selected from substitutions at amino acid residues 8-26, 43-64, and 84-88.

[0178] In certain embodiments, one or more disulfide bonds may be formed between the manipulated CGamma and CDelta. The mutated cysteine ​​residue in CGamma may be a substitution selected from the group consisting of S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and / or the mutated cysteine ​​residue in CDelta may be a substitution selected from the group consisting of F12C, M14C, N16C, D46C, V50C, F87C, and E88C. In certain embodiments, the mutated cysteine ​​residue pair can be a substitution selected from the group consisting of S17C in CGamma and F12C in CDelta, E20C in CGamma and F12C in CDelta, F14C in CGamma and M14C in CDelta, T12C in CGamma and N16C in CDelta, M62C in CGamma and D46C in CDelta, Q57C in CGamma and V50C in CDelta, A19C in CGamma and F87C in CDelta, and A19C in CGamma and E88C in CDelta, and the mutated cysteine ​​residue pair introduced here is capable of forming an interchain disulfide bond.

[0179] In a particular embodiment, the manipulated CGamma includes or is one of sequence numbers 113 and 114, and the manipulated CDelta includes or is one of sequence numbers 115 and 116.

[0180] In addition to non-natural amino acid residues, the manipulated TCR constant region may, in certain embodiments, further include additional modifications to one or more natural residues in the wild-type TCR constant region sequence. Examples of such additional modifications include modifications to natural cysteine ​​residues, modifications to natural glycosylation sites, and / or modifications to natural loops.

[0181] Certain native TCR constant regions (such as CBeta) may contain native cysteine ​​residues that may be modified (e.g., removed) in some embodiments of this disclosure or retained in some other embodiments. In certain embodiments, a native disulfide bond on the alpha / beta heterodimer TCR may or may not be present between the TRAC and the TRBC1 or TRBC2 constant domain, i.e., between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2 according to IMGT TCR nomenclature.

[0182] In certain embodiments, at least one native cysteine ​​residue may or may not be present in the manipulated CBeta. For example, the native cysteine ​​residue at position C74 of the CBeta may or may not be present in the manipulated CBeta. In certain embodiments, a manipulated CBeta that lacks the native cysteine ​​residue C74 includes or is one of SEQ ID NOs: 32-41.

[0183] While we do not wish to link this to any particular theory, the polypeptide complex provided herein is considered advantageous in that it tolerates both the presence and absence of the native cysteine ​​residue on CBeta. Although the presence of the native cysteine ​​residue on soluble TCR heterodimers has been suggested to impair the ligand-binding ability of TCRs (see, e.g., U.S. Patent No. 7,666,604), the polypeptide complex provided herein can tolerate the presence of this native cysteine ​​residue without negatively affecting its antigen-binding activity. Furthermore, despite the conflicting teaching by Wu et al. (mAbs, 7(2), pp.364-376 (2005)) that the native disulfide bond in TCR heterodimers is beneficial for stabilizing TCR heterodimers, the polypeptide complex provided herein, lacking the native cysteine ​​residue, was expressed at high levels.

[0184] In certain embodiments, one or more naturally occurring glycosylation sites present in the natural TCR constant region are modified (e.g., removed) or maintained within the polypeptide complex provided herein. The term “glycosylation site” as used herein with respect to polypeptide sequences refers to an amino acid residue accompanied by a side chain to which a sugar moiety (e.g., an oligosaccharide structure) can be bound. Glycosylation of polypeptides such as antibodies is typically either N-linked or O-linked. N-linked glycosylation refers to the binding of a sugar moiety to a side chain of an asparagine residue, for example, to an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. O-linked glycosylation refers to the binding of a sugar to a hydroxyamino acid, most commonly serine or threonine, of one of the N-acetyl(aceyl) galactosamine, galactose, or xylose. Conveniently, the removal of the natural glycosylation site can be achieved by altering the amino acid sequence, such that one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites) or one or more serine or threonine residues (for O-linked glycosylation sites) are substituted.

[0185] In certain embodiments, at least one native glycosylation site in the polypeptide complex provided herein is either absent or present in the manipulated TCR constant region, for example, in the first and / or second TCR constant region. Without wishing to be tied to any theory, it is believed that the polypeptide complex provided herein may tolerate the removal of all or part of the glycosylation site without affecting protein expression and stability, in contrast to the existing teaching (see Wu et al., Mabs, 7:2, 364-376, 2015) that the presence of N-linked glycosylation sites on the TCR constant region, such as CAlpha (i.e., N34, N68, and N79) and CBeta (i.e., N69), is essential for protein expression and stability.

[0186] In certain embodiments, at least one of the N-glycosylation sites in the manipulated CAlpha, e.g., N34, N68, N79, and N61, in the polypeptide complex provided herein is either absent or present. In certain embodiments, a manipulated CAlpha sequence without a glycosylation site includes or is one of sequence numbers 44-48. In certain embodiments, at least one of the N-glycosylation sites in the manipulated CBeta, e.g., N69, is either absent or present. A manipulated CBeta sequence (TRBC1) without a glycosylation site includes or is one of sequence numbers 34-36. A manipulated CBeta sequence (TRBC2) without a glycosylation site includes or is one of sequence numbers 38-40.

[0187] In certain embodiments, at least one N-glycosylation site, e.g., N50, in the manipulated CPre-Alpha in the polypeptide complex provided herein is either absent or present. An manipulated CPre-Alpha sequence that lacks a glycosylation site includes or is sequence number 83.

[0188] In certain embodiments, at least one N-glycosylation site, e.g., N65, in the manipulated CGamma complex provided herein is either absent or present. In certain embodiments, a manipulated CGamma sequence without a glycosylation site includes or is SEQ ID NO: 114. In certain embodiments, one of the N-glycosylation sites, e.g., N16 and N79, in the manipulated CDelta sequence is either absent or present. A manipulated CDelta sequence without a glycosylation site includes or is present.

[0189] In certain embodiments, one or more native secondary structures present in the native TCR constant region may be modified (e.g., removed) or maintained in the polypeptide complex provided herein. In certain embodiments, native loops (such as the FG loop and / or DE loop of native CBeta) may be modified (e.g., removed) or maintained in the polypeptide complex provided herein. The terms “FG loop” and “DE loop” refer to structures primarily found in the TCR beta chain constant domain. The FG loop is a typically elongated, solvent-exposed structural element encompassing amino acid residues 101–117 of native CBeta and forming one component of the alpha / beta TCR cavity to CD3. The DE loop encompasses amino acid residues 66–71 of native CBeta. Alignment of the sequence of the TCR beta chain constant region with the sequence of the immunoglobulin CH1 constant region revealed that the FG loop of the TCR beta chain constant region is significantly longer. Figure 3 shows the difference in constant regions between the T cell beta chain and the antibody heavy chain. In certain embodiments, the FG loop sequence (YGLSENDEWTQDRAKPVT, SEQ ID NO: 79) is absent and / or replaced by YPSN (SEQ ID NO: 80). In certain embodiments, the native DE loop sequence (QPALNDSR, SEQ ID NO: 88) is absent and / or replaced by QSGR (SEQ ID NO: 87). In certain embodiments, a CBeta sequence that does not have a native FG loop contains or is one of SEQ ID NOs: 37-40. In certain embodiments, a CBeta sequence that does not have both a native FG loop and a native DE loop contains or is 41.

[0190] In the polypeptide complex provided herein, the constant region derived from the TCR is functionally linked to the variable region derived from the antibody. The heavy chain or light chain variable region of the antibody may be functionally linked to the constant region of the TCR with or without a spacer.

[0191] In a particular embodiment, the variable domain (VH) of the first antibody is fused to the first TCR constant region (C1) via a first connecting domain, and the variable domain (VL) of the first antibody is fused to the second TCR constant region (C2) via a second connecting domain.

[0192] As used herein, “connection domain” refers to a boundary or border region where two amino acid sequences are fused or combined. In certain embodiments, the connection domain comprises at least a portion of the C-terminal fragment from a first fusion partner and is fused with or without a spacer to at least a portion of the N-terminal fragment from a second fusion partner. In such embodiments, the connection domain comprises fragments from both fusion partners, and the fusion point is located at the point where the two fragments are linked to each other, for example, directly or via a spacer. In certain other embodiments, the connection domain consists of a fragment from one fusion partner. In such embodiments, the fusion point can be either end of the connection domain.

[0193] In a particular embodiment, the first connection domain includes at least a portion of the C-terminal fragment of the antibody V / C connection and at least a portion of the N-terminal fragment of the TCR V / C connection.

[0194] As used herein, the term "antibody V / C connection" refers to the boundary between the variable domain and the constant domain of an antibody, for example, the boundary between the heavy chain variable domain and the CH1 domain, or between the light chain variable domain and the light chain constant domain. Similarly, the term "TCR V / C connection" refers to the boundary between the TCR variable domain and the constant domain, for example, the boundary between the TCR alpha variable domain and the constant domain, or between the TCR beta variable domain and the constant domain.

[0195] When the Fv region of an immunoglobulin is aligned with the TCR immunoglobulin-like domain, the antibody V / C conjugate and the TCR V / C conjugate are also aligned. An example is shown in Table 1 below, where the antibody heavy chain V / C conjugate (SEQ ID NO: 270) is aligned with the TCR beta V / C conjugate (SEQ ID NO: 271), and the antibody light chain V / C conjugate (SEQ ID NO: 272) is aligned with the TCR beta V / C conjugate (SEQ ID NO: 273).

[0196] The first and / or second junction domains of the polypeptide complex provided herein may be selected to include an appropriate length of the C-terminal fragment of the antibody V / C junction (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) and an appropriate length of the N-terminal fragment of the TCR V / C junction (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues). For example, as shown in Table 1, the junction domains may be selected to have all the sequences derived from the TCR V / C junction (e.g., see SEQ ID NO: 145), most of the sequences (e.g., see SEQ ID NO: 147), or some of the sequences derived from the TCR V / C junction (e.g., see SEQ ID NO: 146). Further using Table 1 as an example, the junction domains may contain more residues derived from the TCR V / C junction than from the antibody V / C junction (e.g., see SEQ ID NO: 147), or vice versa (e.g., see SEQ ID NO: 146).

[0197] In certain embodiments, the first and / or second connection domains of the polypeptide complex provided herein have a full length equivalent to that of an antibody V / C connection or a TCR V / C connection.

[0198] The appropriate connection domain can be determined based on structure. For example, this may be considered when superimposing the three-dimensional structures of the antibody and TCR, determining the overlap of antibody V / C connections and TCR V / C connections on the superimposed structure, and determining the length or ratio of sequences derived from the antibody or TCR V / C connections.

[0199] In certain embodiments, the first and / or second connection domains include a spacer between the antibody V / C connection and the TCR V / C connection fragments. Any suitable spacer sequence or length can be used, as long as it does not negatively affect the antigen binding or stability of the polypeptide complex.

[0200] Examples of sequences for the antibody variable / constant domain boundary, the TCR variable / constant domain boundary, and the antibody variable / TCR constant region boundary are provided in Table 1-6 below.

[0201] In certain embodiments, C1 comprises manipulated CBeta and C2 comprises manipulated CAlpha. For illustrative purposes, Table 1 shows examples of designs for connection domains useful for antibody VH fused to TCR CBeta or antibody VL fused to TCR CAlpha. The antibody VH / constant domain boundary is aligned with the TCR variable / CBeta boundary, and the antibody VL / constant domain boundary is aligned with the TCR variable / CAlpha boundary. Examples of connection domain designs are also provided in an aligned form (see, e.g., SEQ ID NOs: 144, 145, 146, or 147), with the first or second connection domain being underlined. In such embodiments, the first connection domain comprises or includes SEQ ID NOs: 49 or 50. In such embodiments, the second connection domain comprises or includes SEQ ID NOs: 51 or 52.

[0202] [Table 1]

[0203] In certain embodiments, C1 comprises engineered CAlpha and C2 comprises engineered CBeta. Table 2 shows examples of designs for connecting domains useful for antibody VH fused to TCR CAlpha or antibody VL fused to TCR CBeta. The antibody VH / constant domain boundary is aligned with the TCR variable / CAlpha boundary, and the antibody VL / constant domain boundary is aligned with the TCR variable / CBeta boundary. Examples of designs of the connecting domains are also provided in alignment (see, e.g., SEQ ID NO: 148, 149, or 150), and the first or second connecting domain is shown underlined. In such embodiments, the first connecting domain comprises or is SEQ ID NO: 129 or 130. In such embodiments, the second connecting domain comprises or is SEQ ID NO: 49 or 50.

[0204]

Table 2

[0205] In certain embodiments, C1 comprises engineered CBeta and C2 comprises engineered CPre-Alpha. Table 3 shows examples of designs for connecting domains useful for antibody VH fused to TCR CBeta or antibody VL fused to TCR CPre-Alpha. The antibody VH / constant domain boundary is aligned with the TCR variable / CBeta boundary, and the antibody VL / constant domain boundary is aligned with the TCR variable / CPre-Alpha boundary. Examples of designs of the connecting domains are also provided in alignment (see, e.g., SEQ ID NO: 170, 171, 169, or 156), and the first or second connecting domain is shown underlined. In such embodiments, the first connecting domain comprises or is SEQ ID NO: 49 or 50. In such embodiments, the second connecting domain comprises or is SEQ ID NO: 81 or 131.

[0206]

Table 3

[0207] In certain embodiments, C1 comprises manipulated CPre-Alpha, and C2 comprises manipulated CBeta. Table 4 shows examples of designs for connection domains useful for antibody VH fused to TCR CPre-Alpha, or antibody VL fused to TCR CBeta. The antibody VH / constant domain boundary is aligned with the TCR variable / CPre-Alpha boundary, and the antibody VL / constant domain boundary is aligned with the TCR variable / CBeta boundary. Examples of connection domain designs are also provided in an aligned form (see, for example, SEQ ID NOs: 172, 173, 174, or 175), with the first or second connection domain being underlined. In such embodiments, the first connection domain comprises or includes SEQ ID NOs: 81, 131, 132, or 133. In such embodiments, the second connection domain comprises or includes SEQ ID NOs: 49 or 50.

[0208] [Table 4]

[0209] In certain embodiments, C1 contains an engineered CGamma, and C2 contains an engineered CDelta. Table 5 shows examples of designs for connection domains useful for antibody VH fused to TCR CGamma, or antibody VL fused to TCR CDelta. The antibody VH / constant domain boundary is aligned with the TCR variable / CGamma boundary, and the antibody VL / constant domain boundary is aligned with the TCR variable / CDelta boundary. Examples of connection domain designs are also provided in an aligned form (see, for example, SEQ ID NOs: 157, 158, 159, or 160), with the first or second connection domain being underlined. In such embodiments, the first connection domain includes or is SEQ ID NOs: 117 or 118. In such embodiments, the second connection domain includes or is SEQ ID NOs: 119 or 120.

[0210] [Table 5]

[0211] In certain embodiments, C1 comprises an engineered CDelta and C2 comprises an engineered CGamma. Table 6 shows examples of designs for connection domains useful for antibody VH fused to TCR CDelta or antibody VL fused to TCR CGamma. The antibody VH / constant domain boundary is aligned with the TCR variable / CDelta boundary, and the antibody VL / constant domain boundary is aligned with the TCR variable / CGamma boundary. Examples of connection domain designs are also provided in an aligned form (see, for example, SEQ ID NOs: 161, 162, 163, or 164), with the first or second connection domain being underlined. In such embodiments, the first connection domain comprises or includes SEQ ID NOs: 123 or 124. In such embodiments, the second connection domain comprises or includes SEQ ID NOs: 125 or 126.

[0212] [Table 6]

[0213] In a particular embodiment, the first polypeptide comprises a sequence containing functionally linked domains such as formula (I):VH-HCJ-C1, and the second polypeptide comprises a sequence containing functionally linked domains such as formula (II):VL-LCJ-C2, where: VH is the heavy chain variable domain of the antibody; HCJ is the first connection domain as defined above; C1 is the first TCR constant domain as defined earlier; VL is the variable domain of the antibody light chain; LCJ is the second connection domain as defined earlier; C2 is the second TCR constant domain as defined earlier.

[0214] In a particular embodiment, C1 is an manipulated CAlpha containing or being a sequence selected from the group consisting of SEQ ID NOs: 42-48, and C2 is an manipulated CBeta containing or being a sequence selected from the group consisting of SEQ ID NOs: 32-41; HCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 49 and 50; and LCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 51 and 52.

[0215] In a particular embodiment, C1 is an engineered CBeta containing or being a sequence selected from the group consisting of SEQ ID NOs: 32-41, and C2 is an engineered CAlpha containing or being a sequence selected from the group consisting of SEQ ID NOs: 42-48; HCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 129 and 130; and LCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 49 and 50.

[0216] In a particular embodiment, C1 is an engineered CBeta containing or being a sequence selected from the group consisting of SEQ ID NOs: 32-41, 84, 319, 320, 321, 322, 323, and 324; and C2 is an engineered CPre-Alpha containing or being a sequence selected from the group consisting of SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, and 318; HCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 49 and 50; and LCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 81 and 131.

[0217] In a particular embodiment, C1 is an engineered CPre-Alpha containing or being a sequence selected from the group consisting of SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, and 318; and C2 is an engineered CBeta containing or being a sequence selected from the group consisting of SEQ ID NOs: 32-41; HCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 81, 131, 132, and 133; and LCJ contains or being a sequence selected from the group consisting of SEQ ID NOs: 49 and 50.

[0218] In a particular embodiment, C1 is an engineered CGamma containing or being a sequence selected from the group consisting of SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339, and 340; C2 is an engineered CDelta containing or being a sequence selected from the group consisting of SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331, and 332; HCJ contains or is a sequence selected from the group consisting of SEQ ID NOs: 117 and 118; and LCJ contains or is a sequence selected from the group consisting of SEQ ID NOs: 119 and 120.

[0219] In a particular embodiment, C1 is an engineered CDelta containing or being a sequence selected from the group consisting of SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331, and 332; and C2 is an engineered CGamma containing or being a sequence selected from the group consisting of SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339, and 340; HCJ contains or is a sequence selected from the group consisting of SEQ ID NOs: 123 and 124; and LCJ contains or is a sequence selected from the group consisting of SEQ ID NOs: 125 and 126.

[0220] U.S. Patent No. 9,683,052 discloses that specific residues within the contact interface between TCR constant regions can be engineered into the Fc region to facilitate the heterodimer pair formation of two heavy chains. Such residues and / or corresponding residues within the contact interface between the TCR constant regions disclosed herein can also be engineered into the Fab region, such as the CH1 and CL domains, to facilitate the pair formation between the light chain and the heavy chain.

[0221] (ii. Antibody variable region)

[0222] The polypeptide complex provided herein comprises a first heavy chain variable domain (VH) and a first light chain variable domain (VL) of a first antibody. In a normal native antibody, the variable region includes, for example, three CDR regions flanked by framework (FR) regions as shown by the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, from the N-terminus to the C-terminus. The polypeptide complex provided herein can include such normal antibody variable regions, but is not limited thereto. For example, the variable domain can include up to three CDRs and up to four FRs in total of the heavy or light chain of an antibody, as long as the variable domain is capable of specifically binding to an antigen.

[0223] The first antibody has first antigen specificity. In other words, VH and VL form an antigen-binding site that can specifically bind to an antigen or epitope. Antigen specificity can be directed to any suitable antigen or epitope, such as exogenous antigens, endogenous antigens, autoantigens, neoantigens, viral antigens, or tumor antigens. Exogenous antigens enter the body by inhalation, oral ingestion, or infusion, and can be presented by antigen-presenting cells (APCs) by endocytosis or phagocytosis, forming MHC II complexes. Endogenous antigens are produced within normal cells as a result of cellular metabolism, intracellular viral or bacterial infection, and these form MHC I complexes. Autoantigens (e.g., peptides, DNA, or RNA) are recognized by the immune system of patients with autoimmune diseases, whereas under normal conditions, these antigens should not be targets of the immune system. Neoantigens are not present in a normal body overall and are produced as a result of certain diseases such as tumors or cancer. In certain embodiments, this antigen is associated with a specific disease (e.g., tumor or cancer, autoimmune disease, infectious and parasitic disease, cardiovascular disease, neurological disorder, neuropsychiatric condition, trauma, inflammation, coagulation disorder). In certain embodiments, this antigen is associated with the immune system (e.g., immunological cells such as B cells, T cells, NK cells, macrophages).

[0224] In certain embodiments, the first antigen specificity is directed towards an immune-related antigen or its epitope. Examples of immune-related antigens include T cell-specific receptor molecules and / or natural killer cell (NK cell)-specific receptor molecules.

[0225] T cell-specific receptor molecules bind to T cells and, in the presence of additional signals, are activated and react with epitopes / antigens presented by antigen-presenting cells or other cells referred to as APCs. T cell-specific receptor molecules can be TCR, CD3, CD28, CD134 (also known as OX40), 4-1BB, CD5, and CD95 (also known as the Fas receptor).

[0226] Examples of NK cell-specific receptor molecules include CD16, low-affinity Fc receptors and NKG2D, and CD2.

[0227] In certain embodiments, the first antigen specificity is directed towards tumor-associated antigens or their epitopes. The term “tumor-associated antigen” refers to an antigen that is presented on or can be presented on the surface of tumor cells and is located on or within tumor cells. In some embodiments, tumor-associated antigens may be presented only by tumor cells and not by normal cells, i.e., non-tumor cells. In some other embodiments, tumor-associated antigens may be expressed exclusively on tumor cells or exhibit tumor-specific mutations compared to non-tumor cells. In some other embodiments, tumor-associated antigens are found in both tumor and non-tumor cells, but are overexpressed on tumor cells compared to non-tumor cells, or are accessible to antibody binding in tumor cells due to the less compact structure of tumor tissue compared to non-tumor tissue. In some embodiments, tumor-associated antigens are located on the blood vessels of the tumor.

[0228] Exemplary examples of tumor-associated surface antigens include CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), and epidermal growth factor receptor ( Examples include EGFR, Her2neu, Her3, IGFR, IL3R, fibroblast-activating protein (FAP), CDCP1, Delrin 1, Tenacin, Frizzled1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), Endoglin, CLEC14, Tem1-8, and Tie2. Further examples include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carboanhydrase IX (MN / CA IX), de2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), and TAG-72.

[0229] In certain embodiments, the first antigen specificity is directed to an antigen or epitope selected from the group consisting of: CD3, 4.1BB (CD137), OX40 (CD134), CD16, CD47, CD19, CD20, CD22, CD33, CD38, CD123, CD133, CEA, cdH3, EpCAM, epidermal growth factor receptor (EGFR), EGFRvIII (EGFR-derived variant), HER2, HER3, dLL3, BCMA, sialyl-Lea, 5T4, ROR1, melanoma-associated chondroitin sulfate proteoglycan, mesoserine, folate receptor 1, VEGF receptor, EpCAM, HER2 / n eu, HER3 / neu, G250, CEA, MAGE, proteoglycan, VEGF, FGFR, αVβ3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD21, CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, ganglioside GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4 Trp2, gp100, tyrosinase, Muc-1, telomerase, survivalin, G250, p53, CA125 MUC, Wue antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EpHA2, and cell surface targets GC182, GT468, or GT512.

[0230] This antibody-variable domain can be derived from the parent antibody. The parent antibody can be any type of antibody, including, for example, a fully human antibody, a humanized antibody, or an animal antibody (e.g., mouse, rat, rabbit, sheep, cattle, dog, etc.). The parent antibody can be a monoclonal antibody or a polyclonal antibody.

[0231] In certain embodiments, the parent antibody is a monoclonal antibody. Monoclonal antibodies can be produced by various methods known in the art, such as hybridoma technology, recombinant methods, phage display, or any combination thereof.

[0232] Hybridoma technology involves fusing antibody-expressing B cells with immortalized B cell lines to create hybridomas, which are then screened for desired characteristics such as high antibody production levels, good growth of hybridoma cells, and strong antibody binding or good biological activity (see, e.g., Harlow et al., (1988) Antibody: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition)).

[0233] Recombination is another method for producing parental antibodies. Simply put, cells such as lymphocytes secreting the antibody of interest are obtained, identified, and isolated. Subsequently, heavy and light chain variable region cDNAs are produced by reverse transcriptase PCR. Using these variable region cDNA sequences, the coding sequence for the polypeptide complex provided herein can be constructed and subsequently expressed in suitable host cells (for a review, see, for example, U.S. Patent No. 5,627,052; PCT Publication WO 92 / 02551; and Babcock et al., (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848).

[0234] Antibody libraries remain an alternative method for obtaining parent antibodies. Simply put, antibody libraries can be screened to identify antibodies with desired binding specificity. Methods for screening such recombinant antibody libraries are well known in the art, and include, for example, U.S. Patent No. 5,223,409; PCT Publications WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 97 / 29131; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; McCafferty et al., (1990) Nature 348:552-554; Griffiths et al., (1993) EMBO J. 12:725-734; Hawkins et al., (1992) J. Mol. Biol. 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nucl. Acid Res. 19:4133-4137; and Barbas et al., (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and the method described in U.S. Patent Application Publication No. 20030186374.

[0235] Another exemplary method for obtaining parental antibodies is phage display (e.g., Brinkman et al., (1995) J. Immunol. Methods 182:41-50; Ames et al., (1995) J. Immunol. Methods 184:177-186; Kettleborough et al., (1994) Eur. J. Immunol. 24:952-958; Persic et al., (1997) Gene 187 See 9-18; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108). Polynucleotide sequences encoding antibody domains are introduced into phage particles to create a library of phage particles presenting various functional antibody domains. Fd and M13 are commonly used filamentous phages, and the functional antibody domain displayed on the phage particle can be, for example, a Fab, Fv, or disulfide-stabilized Fv antibody domain, which is recombinantly fused to a phage protein encoded by geneIII or geneVIII. The phage library can be screened, for example, using an antigen of interest that is optionally labeled or bound to or captured on a solid substrate (e.g., beads). For selected phages, their polynucleotide sequences encoding the antibody variable domain are obtained and used to construct polypeptide complexes provided herein. Similarly, a yeast library displaying the antibody variable domain can be prepared by ligating the antibody domain to the yeast cell wall (see, for example, U.S. Patent No. 6,699,658), and then screened with the bound antigen to obtain parental antibodies useful for constructing polypeptide complexes provided herein.

[0236] Furthermore, parental antibodies can also be produced by injecting some or all of the human immunoglobulin gene loci of the antigen of interest into transgenic non-human animals, such as OmniRat, OmniMouse (see, for example, Osborn M. et al., Journal of Immunology, 2013, 190: 1481-90; Ma B. et al., Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al., Science, 2009, 325:433; U.S. Patent No. 8,907,157; ​​European Patent No. 2152880B1; European Patent No. 2336329B1), HuMab mouse (see, for details, Lonberg, N. et al., Nature 368(6474): 856 859 (1994)), Xeno mouse (see, for details, Mendez et al., Nat Examples include the Genet., 1997, 15:146-156, the TransChromo mouse (Ishida et al., Cloning Stem Cells, 2002, 4:91-102), the VelocImmunemouse (Murphy et al., Proc Natl Acad Sci USA, 2014, 111:5153-5158), the KyMouse (Lee et al., Nat Biotechnol, 2014, 32:356-363), and the transgenic rabbit (Flisikowska et al., PLoS One, 2011, 6:e21045).

[0237] The parent antibodies described herein can be further modified, for example, by grafting a CDR sequence onto a different framework or scaffold, by substituting one or more amino acid residues within one or more framework regions, or by substituting one or more residues within one or more CDR regions for affinity maturation. These modifications can be achieved by those skilled in the art using conventional techniques.

[0238] This parent antibody may also be a therapeutic antibody known in the art, such as one approved by the FDA for therapeutic or diagnostic use, or one in clinical trials or under research and development for the treatment of a condition. Polynucleotide and protein sequences relating to the variable region of known antibodies can be obtained from public databases such as www.ncbi.nlm nih gov / entrez- / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / ; www.abcam.com / ; www.antibodyresource.com / onlinecomp.html.

[0239] Examples of therapeutic antibodies include, but are not limited to, rituximab (Rituxan, IDEC / Genentech / Roche) (see, e.g., U.S. Patent No. 5,736,137), a chimeric anti-CD20 antibody approved for the treatment of non-Hodgkin lymphoma; HuMax-CD20, an anti-CD20 currently under development by Genmab; the anti-CD20 antibody described in U.S. Patent No. 5,500,362; and AME-133 (Applied Molecular Evolution), hA20 (Immunomedics), HumaLYM (Intracel), and PRO70769 (PCT application PCT / US2003 / 040426); trastuzumab (Herceptin, Genentech) (see, e.g., U.S. Patent No. 5,677,171), a humanized anti-Her2 / neu antibody approved for the treatment of breast cancer; pertuzumab (rhuMab-2C4, Omnitarg), currently under development by Genentech; anti-Her2 antibody described in U.S. Patent No. 4,753,894; cetuximab (Erbitux, Imclone) (U.S. Patent No. 4,943,533; PCT publication WO 96 / 40210), Chimeric anti-EGFR antibodies in clinical trials for various cancers; ABX-EGF (US Patent No. 6,235,883) currently under development by Abgenix-Immunex-Amgen; HuMax-EGFr (US Patent No. 7,247,301) currently under development by Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (US Patent No. 5,558,864; Murthy et al., (1987) Arch. Biochem. Biophys. 252(2):549-60; Rodeck et al., (1987) J. Cell. Biochem. 35(4):315-20; Kettleborough et al., (1991) Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT Public WO 95 / 20045;Modjtahedi et al., (1993) J. Cell Biophys. 22(1-3):129-46;Modjtahedi et al., (1993) Br. J. Cancer 67(2):247-53;Modjtahedi et al., (1996)Br. J. Cancer 73(2):228-35; Modjtahedi et al., (2003) Int. J. Cancer 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (US Patent No. 5,891,996; No. 6,506,883; Mateo et al., (1997) Immunotechnol. 3(1):71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al., (2003) Proc. Natl. Acad. Sci. USA 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT Public WO 0162931); and SC100 (Scancell) (PCT WO 01 / 88138); alemtuzumab (Campus, Millennium), currently approved humanized mAbs for the treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3 (Orthoclon OKT3), an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson; ibritumomab tiuxetan (Zevalin), an anti-CD20 antibody developed by IDEC / Schering AG; gemtuzumab ozogamicin (Mylotarg), an anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth; alefacept (Amevib), an anti-LFA-3 antibody developed by BiogenAbsiximab (Leopro) developed by Centocor / Lilly (Fc fusion), basiliximab (Symlect) developed by Novartis, palivizumab (Synagis) developed by Medimmune, infliximab (Remicade), anti-TNFα antibody adalimumab (Humira) developed by Centocor, anti-TNFα antibody humicade developed by Abbott, anti-TNFα antibody golimumab (CNTO-148) developed by Celltech, fully human TNF antibody etanercept (Enbrel) developed by Centocor, p75 developed by Immunex / Amgen TNF receptor Fc fusion, renercept, p55 TNF receptor Fc fusion developed by Roche, ABX-CBL, anti-CD147 antibody developed by Abgenix, ABX-IL8, anti-IL8 antibody developed by Abgenix, ABX-MA1, anti-MUC18 antibody developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), anti-MUC1 developed by Antisoma, Therex (R1550), anti-MUC1 antibody developed by Antisoma, AngioMab (AS1405) developed by Antisoma, HuBC-1 developed by Antisoma, thioplatin (AS1407) developed by Antisoma, antegrain (natalizumab), anti-α-4-β1 (VLA-4) and α-4-β-7 antibodies developed by Biogen, VLA-1 mAb, anti-VLA-1 integrin antibody developed by Biogen, LTBR mAb, anti-lymphotoxin β receptor (LTBR) antibody developed by Biogen, CAT-152, anti-TGF-β.2 antibody developed by Cambridge Antibody Technology, ABT 874 (J695), anti-IL-12p40 antibody developed by Abbott, CAT-192, anti-TGF-β.1 antibody developed by Cambridge Antibody Technology and Genzyme, CAT-213, anti-eotaxin I antibody developed by Cambridge Antibody Technology, CambridgeLymphostat B anti-Blys antibody developed by Antibody Technology and Human Genome Sciences Inc., TRAIL-RlmAb, anti-TRAIL-R1 antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc., avastin bevacizumab (rhuMAb-VEGF), anti-VEGF antibody developed by Genentech, anti-HER receptor family antibody developed by Genentech, anti-tissue factor (ATF), anti-tissue factor antibody developed by Genentech, Xolair (omalizumab), anti-IgE antibody developed by Genentech, Raptiva (efalizumab), anti-CD11a antibody developed by Genentech and Xoma, MLN-02 antibody (formerly LDP-02) developed by Genentech and Millennium Pharmaceuticals, HuMax CD4, anti-CD4 antibody developed by Genmab, HuMax-IL15, anti-IL15 antibody developed by Genmab and Amgen, HuMax-Inflam, HuMax-Cancer, anti-heparanase I antibody developed by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, HuMax-TAC, IDEC-131 developed by Genmab and Amgen, anti-CD40L antibody, IDEC-151 (crenoliximab) developed by IDEC Pharmaceuticals, anti-CD4 antibody, IDEC-114 developed by IDEC Pharmaceuticals, anti-CD80 antibody, IDEC-152 developed by IDEC Pharmaceuticals, anti-CD23, IDECAnti-macrophage chemoattractant (MIF) antibody developed by Pharmaceuticals, BEC2; anti-idiotype antibody developed by Imclone, IMC-1C11; anti-KDR antibody developed by Imclone, DC101; anti-flk-1 antibody developed by Imclone; anti-VE cadherin antibody developed by Imclone, CEA-Cide (rabetuzumab); anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics, LymphoCide (epratuzumab); anti-CD22 antibody developed by Immunomedics, Im AFP-Cide developed by Immunomedics, MyelomaCide developed by Immunomedics, LkoCide developed by Immunomedics, ProstaCide developed by Immunomedics, MDX-010, anti-CTLA4 antibody developed by Medarex, MDX-060, anti-CD30 antibody developed by Medarex, MDX-070 developed by Medarex, MDX-018 developed by Medarex, Osidem (IDM-1), and Medarex and Immuno-Designed Anti-Her2 antibody developed by Molecules, HuMax-CD4, anti-CD4 antibody developed by Medarex and Genmab, HuMax-IL15, anti-IL15 antibody developed by Medarex and Genmab, CNTO 148, anti-TNFα antibody developed by Medarex and Centocor / J&J, CNTO 1275, anti-cytokine antibody developed by Centocor / J&J, MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibody developed by MorphoSys, MOR201, anti-fibroblast growth factor receptor 3 (FGFR-3) antibody developed by MorphoSys, Nuvion (bicilizumab), anti-CD3 antibody developed by Protein Design Labs, HuZAF, anti-interferon-γ antibody developed by Protein Design Labs, Protein Design Anti-α5β1 integrin, Protein developed by LabsAnti-IL-12 and ING-1 antibodies developed by Design Labs, anti-Ep-CAM antibodies developed by Xoma, Xolair (omalizumab), humanized anti-IgE antibodies developed by Genentech and Novartis, and anti-β2 integrin antibodies developed by MLN01 and Xoma. In another embodiment, the therapeutic agents include KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); NTO95 (αV integrin, Centocor); MEDI-522 (αV.β.3 integrin, Medimmune); volociximab (αV.β.1 integrin, Biogen / PDL); human mAb216 (B cell glycosylated epitope, NCI); BiTE MT103 (bispecific CD19×CD3, Medimmune); 4G7×H22 (bispecific B cell × Fc gamma R1, Medarex / Merck KGa); rM28 (bispecific CD28×MAPG, European Patent No. EP1444268); MDX447 (EMD 82633) (Bispecific CD64 × EGFR, Medarex); Katsumakisomab (Lemomab) (Bispecific EpCAM × Anti-CD3, Trion / Fres); Erzumakisomab (Bispecific HER2 / CD3, Fresenius Biotech); Olegovomab (OvaRex) (CA-125, ViRexx); Rencarex (WX G250) (Carboxylate dehydrogenase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Ciprizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); Vertuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); Lumiliximab (IDEC)152) (CD23, Biogen); Muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1 (CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimubab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campas lh (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093 / D93) (Cut-type collagen, Tracon); HuLuc63 (CS1, PDL Pharma); Ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome Science / Glaxo Smith) Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (Lexatumumab) (DR5 TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8 (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectavix) (EGFR, Amgen); Saltumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANTImmunotherapeutics); Adekatumab (MT201) (Epcam, Merck); Edrecolomab (Panorex, 17-1A) (Epcam, Glaxo / Centocor); MORAb-003 (Folic acid receptor a, Morphotech); KW-2871 (Ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2(DI), Genentech); Apolitumab (HLA-DRβ chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); Anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871 (IGF-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-β-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); anti-KIR (1-7F9) (Killer cell Ig-like receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTBR, ​​Biogen); HuHMFG1 (MUC1, Antisoma / NCI); RAV12 (N-linked glycosphingoplasmic receptape, Raven); CAL (Parathyroid hormone-related protein (PTH-rP), University of California); CT-011 (PD1, CureTech); MDX-1106 (Ono-4538) (P D1, Medarex / Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone); Babituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb(pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFα, Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005 / 111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab) PCT published WO / 2000 / 034337, University of Examples include Texas; IMC-18F1 (VEGFR1, Imclone); and IMC-1121 (VEGFR2, Imclone).

[0240] a) Anti-CD3 binding moiety

[0241] In a particular embodiment, the first or second antigen-binding moiety is an anti-CD3 antibody-derived 0250 anti-CD3 binding moiety comprising one, two, or three heavy chain CDR sequences selected from the group consisting of SEQ ID NOs: 342-344 and / or one, two, or three light chain CDR sequences selected from SEQ ID NOs: 345-347.

[0242] These CDR sequences are derived from the anti-CD3 antibodies shown in Table A below. The CDR sequence of the WBP3311_2.306.4 antibody is provided below.

[0243] [Table 7]

[0244] The sequences of the heavy chain and κ-light chain variable regions of the WBP3311_2.306.4 antibody are provided below.

[0245] WBP3311_2.306.4-VH Amino acid sequence (SEQ ID NO: 348): QVQLQQSGPELVKPGASVRISCKAS GFAFTDYYIH WVKQRPGQGLE WIGWISPGNVNTKYNENFKG RATLTADLSSSTAYMQLSSLTSEDSAVYFCAR DGYSLYYFDY WGQGTTLTVSS Nucleic acid sequence (SEQ ID NO: 349): CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCCGTGAGGATATCCTGCAAGGCTTCTGGCTTCGCCTTCACAGACTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGATGGATTTCTCCTGGAAATGTTAATACTAAAT ACAATGAAAACTTCAAGGGCAGGGCCACACTGACTGCAGACCTATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGATGGATATTCCCTGTATTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0246] WBP3311_2.306.4-VL Amino acid sequence (SEQ ID NO: 350): DIVMSQSPSSLTVSAGEKVTMSC KSSQSLLNSRTRKNYLA WYQQKPGQSPKLLIY WASTRQS GVPDRFTGSGSGTAFTLTISGVQAEDLAVYFCTQ SHTLRT FGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 351): GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAGCCAGGGCAGTCTCCTAAACTACTAATCTACTGG GCATCCACTAGGCAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGCTTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGCACGCAATCTCATACTCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0247] While CDRs are known to contribute to antigen binding, it is known that six, though not all, CDRs are essential and irreplaceable. In other words, it is possible to substitute, alter, or modify one or more CDRs in the anti-CD3 binding moiety derived from WBP3311_2.306.4 while substantially maintaining specific binding affinity to CD3.

[0248] In certain embodiments, the anti-CD3 binding moiety provided herein includes one heavy chain CDR3 sequence of the anti-CD3 antibody WBP3311_2.306.4. In certain embodiments, the anti-CD3 binding moiety provided herein includes a heavy chain CDR3 containing SEQ ID NO: 344. The heavy chain CDR3 region is thought to be centrally located at the antigen-binding site and therefore produce the greatest contact with the antigen and provide the greatest free energy for the antibody's affinity to the antigen. Furthermore, the heavy chain CDR3 may be the overwhelmingly most diverse CDR in terms of length, amino acid composition, and higher-order structure due to multiple diversification mechanisms (Tonegawa S., Nature. 302:575-81 (1983)). The diversity of heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM., Immunity. 13:37-45 (2000)) as well as the desired antigen-binding affinity (Schier R et al., J Mol Biol. 263:551-67 (1996)).

[0249] In certain embodiments, the anti-CD3 binding moieties provided herein include a suitable framework region (FR) sequence, insofar as the anti-CD3 binding moiety can specifically bind to CD3. The CDR sequences provided in Table A are obtained from mouse antibodies, but they can be grafted onto any suitable FR sequence of any suitable species, such as mouse, human, rat, or rabbit, using preferred methods known in the art, such as recombinant technology.

[0250] In certain embodiments, the anti-CD3 binding moiety provided herein is humanized.

[0251] In certain embodiments, the humanized antigen-binding moieties provided herein consist substantially entirely of human sequences, with the exception of non-human CDR sequences. In some embodiments, the variable region FR and, if present, the constant region are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived from different human immunoglobulin genes; for example, the FR sequences may be derived from one human antibody and the constant region from another human antibody. In some embodiments, the humanized antigen-binding moieties include human FR1-4.

[0252] The sequences of the heavy chain and light chain variable regions of the anti-CD3 humanized antibody WBP3311_2.306.4-z1 are provided below.

[0253] WBP3311_2.306.4-z1-VH Amino acid sequence (SEQ ID NO: 352): QVQLVQSGAEVKKPGSSVKVSCKAS GFAFTDYYIH WVRQAPGQGLEWMG WISPGNVNTKYNENFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR DGYSLYYFDY WGQGTLVTVSS Nucleic acid sequence (SEQ ID NO: 353): CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTGAAGAAGCCTGGGTCTAGTGTCAAGGTGTCATGCAAGGCTAGCGGGTTCGCCTTTACTGACTACTACATCCACTGGGTGCGGCAGGCTCCCGGACAAGGGTTGGAGTGGATGGGATGGATCTCCCCAGGCAATGTCAACACAAAGT ACAACGAGAACTTCAAAGGCCGCGTCACCATTACCGCCGACAAGAGCACCTCCACAGCCTACATGGAGCTGTCCAGCCTCAGAAGCGAGGACACTGCCGTCTACTACTGTGCCAGGATGGGTACTCCCTGTATTACTTTGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC

[0254] WBP3311_2.306.4-z1-VL Amino acid sequence (SEQ ID NO: 354): DIVMTQSPDSLAVSLGERATINC KSSQSLLNSRTRKNYLA WYQQKPGQPPKLLIY WASTRQS GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC TQSHTLRT FGGGTKVEIK Nucleic acid sequence (SEQ ID NO: 355): GATATCGTGATGACCCAGAGCCCAGACTCCCTTGCTGTCTCCCTCGGCGAAAGAGCAACCATCAACTGCAAGAGCTCCCAAAGCCTGCTGAACTCCAGGACCAGGAAGAATTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTCATCTACTGG GCCTCCACCCGGCAGTCTGGGGTGCCCGATCGGTTTAGTGGATCTGGGAGCGGACAGACTTCACATTGACAATTAGCTCACTGCAGGCCGAGGACGTGGCCGTCTACTACTGTACTCAGAGCCACACTCTCCGCACATTCGGCGGAGGGACTAAAGTGGAGATTAAG

[0255] b) Anti-CD19 antibody

[0256] In a particular embodiment, the first antigen-binding moiety or the second antigen-binding moiety is an anti-CD19 binding moiety derived from an anti-CD19 antibody, comprising one, two, or three heavy chain CDR sequences selected from the group consisting of SEQ ID NOs: 356-359 and / or one, two, or three light chain CDR sequences selected from SEQ ID NOs: 360-362.

[0257] These CDR sequences are derived from the antibodies shown in Table B below. The CDR sequences of these anti-CD19 antibodies are provided below.

[0258] [Table 8]

[0259] The sequences of the heavy chain and κ-light chain variable regions of the WBP7011_4.155.8 antibody are provided below.

[0260] WBP7011-4.155.8-VH Amino acid sequence (SEQ ID NO: 363): EIQLQQSGPELVKPGASVKVSCKAS GYAFTSYNMY WVKQSHGKSLEWIG YIDPYNGDTTYNQKFKG KATLTVDKSSSTAYMHLNSLTSEDSAVYYCLT TAYAMDY WGQGTSVTVSS Nucleic acid sequence (SEQ ID NO: 364): GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAGGCTTCTGGTTATGCATTCACTAGCTACAACATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTACAATGGTGATACT ACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGCATCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTCTCACTACGGCCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0261] WBP7011-4.155.8-VL Amino acid sequence (SEQ ID NO: 365): QIVLTQSPAIMSASLGEEITLTC SASSTVNYMH WYQQKSGTSPKLLIY STSN LAS GVPSRFSGSGSGTFYSLTIRSVEAEDAADYYC HQWSSYPYT FGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 366): CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGGAGATCACCCTAACCTGCAGTGCCAGCTCGACTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTGATTTATAGCACATCCAACCTG GCTTCTGGAGTCCCTTCTCGCTTCAGTGGCAGTGGGTCTGGGACCTTTTATTCTCTCACAATCAGAAGTGTGGAGGCTGAAGATGCTGCCGATTATTACTGCCATCAGTGGAGTAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0262] In certain embodiments, the anti-CD19 binding moiety disclosed herein includes a heavy chain CDR3 sequence of the anti-CD19 antibody WBP7011_4.155.8 or W7011-4.155.8-z1-P15. In certain embodiments, the anti-CD19 binding moiety provided herein includes a heavy chain CDR3 sequence including SEQ ID NO: 358. The heavy chain CDR3 region is thought to be centrally located at the antigen-binding site and therefore produce the greatest contact with the antigen and provide the greatest free energy for the antibody's affinity to the antigen. Furthermore, the heavy chain CDR3 may be the overwhelmingly most diverse CDR in terms of length, amino acid composition, and higher-order structure due to multiple diversification mechanisms (Tonegawa S., Nature. 302:575-81 (1983)). The diversity of heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM., Immunity. 13:37-45 (2000)) as well as the desired antigen-binding affinity (Schier R et al., J Mol Biol. 263:551-67 (1996)).

[0263] In certain embodiments, the anti-CD19 antibody disclosed herein is humanized. The sequences of the heavy chain and light chain variable regions of the humanized anti-CD19 antibody W7011-4.155.8-z1-P15 are provided below.

[0264] W7011-4.155.8-z1-P15-VH Amino acid sequence (SEQ ID NO: 367): QMQLVQSGPEVKKPGTSVKVSCKAS GYAFTSYNMY WVRQARGQRLEWIG YIDPYNADTTYNQKFKG RVTITRDMSTSTAYMELSSLRSEDTAVYYCLT TAYAMDY WGQGTLVTVSS Nucleic acid sequence (SEQ ID NO: 368): CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTGAAGAAGCCCGGGACATCCGTCAAGGTCTCATGTAAGGCTAGCGGGTACGCATTCACTTCCTACAACATGTACTGGGTGCGCAGGCCAGAGGACAGAGGTTGGAGTGGATCGGCTACATCGACCCATACAACGCCGATACT ACCTACAATCAGAAGTTTAAAGGGCGGTGACCATTACCCGGGATATGTCCACCTCCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACAGCCGTGTACTACTGCCTGACAACAGCCTATGCCATGGACTATTGGGGCCAGGGCACACTTGTGACTGTGAGCAGT

[0265] W7011-4.155.8-z1-P15-VL Amino acid sequence (SEQ ID NO: 369): DIQLTQSPSFLSASVGDRVTITC SASSTVNYMH WYQQKPGKAPKLLIY STSN LAS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC HQWSSYPYT FGQGTKLEIK Nucleic acid sequence (SEQ ID NO: 370): GACATCCAGCTCACCCAATCCCCTTCTTTCCTCCGCAAGTGTCGGAGATAGGGTGACTATCACCTGCTAGCTTCTTCAACCGTGAACTACATGCATTGGTACCAGCAGAAGCCCGGGAAAGCCCCAAAGCTGCTGATCTACAGCACCTCCAATCTG GCCAGTGGAGTGCCAAGCCGGTTTAGCGGGAGCGGCTCCGGCACTGAATTCACTTTGACAATTAGCAGCCTTCAGCCTGAGGACTTTGCCACATATTACTGTCACCAGTGGTCCAGCTACCCCTACACATTCGGGCAGGGCACAAAGCTGGAGATTAAG

[0266] (Bispecific polypeptide complex)

[0267] In one embodiment, the present disclosure provides a bispecific polypeptide complex. As used herein, the term “bispecific” means that there are two antigen-binding sites, each capable of specifically binding to a different antigen or to a different epitope on the same antigen. The bispecific polypeptide complex provided herein comprises a first antigen-binding site comprising a first heavy chain variable domain functionally linked to a first TCR constant region (C1) and a first light chain variable domain functionally linked to a second TCR constant region (C2), wherein C1 and C2 can form a dimer containing at least one non-natural stable interchain linkage between C1 and C2. The bispecific polypeptide complex provided herein further comprises a second antigen-binding site, but the second antigen-binding site does not contain a sequence derived from the TCR constant region.

[0268] In certain embodiments, the present disclosure provides a bispecific polypeptide complex comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein: The first antigen-binding site is: A first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from the N-terminus to the C-terminus of the first T cell receptor (TCR) constant region (C1); and A second polypeptide comprising the first light chain variable domain (VL) of the first antibody, functionally linked from the N-terminus to the C-terminus of the second TCR constant region (C2); Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and The first antibody has the first antigen specificity, The second antigen-binding portion has a second antigen specificity that is different from the first antigen specificity, and The first and second antigen-binding moieties are less prone to mispairing than other cases where both the first and second antigen-binding moieties are natural Fab counterparts.

[0269] The bispecific polypeptide complexes provided herein show significantly less tendency to have mispaired heavy and light chain variable domains. While we do not wish to tie this to any theory, it is conceivable that the stabilized TCR constant regions in the primary antigen-binding moiety can specifically associate with each other, thereby contributing to the highly specific pairing of intended VH1 and VL1, while preventing undesirable mispairing with variable regions that do not provide other intended antigen-binding sites for VH1 or VL1.

[0270] WuXiBody format bispecific polypeptide complexes have a longer in vivo half-life compared to bispecific polypeptide complexes of other formats, and are also relatively easy to manufacture.

[0271] In certain embodiments, the second antigen-binding moiety of the bispecific polypeptide complex provided herein comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2) of a second antibody. In certain embodiments, at least one of VH2 and VL2 is functionally linked to the antibody constant region, or both VH2 and VL2 are functionally linked to the antibody heavy chain and light chain constant regions, respectively. In certain embodiments, the second antigen-binding moiety further comprises an antibody constant CH1 domain functionally linked to VH2 and an antibody light chain constant domain functionally linked to VL2. For example, the second antigen-binding moiety comprises Fab.

[0272] When the first, second, third, and fourth variable domains (e.g., VH1, VH2, VL1, and VL2) are expressed in a single cell, it is highly desirable that VH1 specifically pairs with VL1 and VH2 specifically pairs with VL2, resulting in a bispecific protein product with precise antigen-binding specificity. However, in existing technologies such as hybrids (or quadromas), random pairing of VH1, VH2, VL1, and VL2 occurs, resulting in the generation of up to 10 different species, of which only one is a functionally bispecific antigen-binding molecule. This not only reduces the yield but also complicates the purification of the target product.

[0273] The bispecific polypeptide complex provided herein is exceptional in that the variable domain is less prone to mispairing than other complexes where both the first and second antigen-binding moieties are natural Fab counterparts. In illustrative examples, the first antigen-binding domain contains VH1-C1 paired with VL1-C2, and the second antigen-binding domain contains VH2-CH1 paired with VL2-CL. Surprisingly, C1 and C2 preferentially associate with each other and have a low tendency to associate with CL or CH1, thereby preventing and significantly reducing the formation of undesirable pairs such as C1-CH, C1-CL, C2-CH, and C2-CL. As a result of the specific association of C1-C2, VH1 specifically pairs with VL1, thereby providing the first antigen-binding site, and CH1 specifically pairs with CL, thereby enabling the specific pairing of VH2-VL2, providing the second antigen-binding site. Therefore, the first and second antigen-binding moieties have a low tendency for mismatch, and mispair formation between, for example, VH1-VL2, VH2-VL1, VH1-VH2, and VL1-VL2 is significantly reduced when both the first and second antigen-binding moieties are in the form of, for example, VH1-CH1, VL1-CL, VH2-CH1, and VL2-CL, compared to when they are in the natural Fab counterpart.

[0274] In certain embodiments, the bispecific polypeptide complexes provided herein, when expressed from cells, have significantly lower mispair formation products (e.g., at least 1, 2, 3, 4, 5 or more, fewer mispair formation products) and / or significantly higher product yields (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more, higher yields) than reference molecules expressed under comparable conditions, wherein the reference molecule is identical to that of the bispecific polypeptide complex, except that it has native CH1 instead of C1 and native CL instead of C2.

[0275] In certain embodiments, the first and / or second antigen-binding sites are polyvalent, such as divalent, trivalent, or tetravalent. As used herein, the term “valence” refers to the presence of a specific number of antigen-binding sites in a given molecule. Thus, the terms “divalent,” “tetravalent,” and “hexavalent” mean the presence of two, four, and six binding sites in an antigen-binding molecule, respectively. A divalent molecule can be monospecific if both of its binding sites specifically bind to the same antigen or the same epitope. Similarly, a trivalent molecule can be bispecific if, for example, two binding sites are monospecific to a first antigen (or epitope) and a third binding site is specific to a second antigen (or epitope). In certain embodiments, the first and / or second antigen-binding sites in the bispecific polypeptide complex provided herein can be divalent, trivalent, or tetravalent, having at least two binding sites specific to the same antigen or epitope. In certain embodiments, this provides stronger binding to the antigen or epitope than a monovalent counterpart. In certain embodiments, the first-valence and second-valence binding sites in the bivalent antigen-binding moiety are either structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences, even if they have the same specificity).

[0276] In certain embodiments, the first and / or second antigen-binding moieties are polyvalent and include two or more antigen-binding sites that are functionally linked together, with or without spacers.

[0277] In certain embodiments, the first and / or second antigen-binding moieties include one or more Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, and scFv fragments, as well as other fragments described in the aforementioned papers by Spiess et al. (2015) and Brinkmann et al. (2017), or combinations thereof, which are linked with or without spacers in heavy and / or light chains, and at least one form is capable of binding to a second antibody.

[0278] In certain embodiments, the second antigen-binding moiety comprises two or more Fabs of the second antibody. The two Fabs may be functionally linked to one another; for example, the first Fab may be covalently bound to the second Fab via a heavy chain, with or without a spacer in between.

[0279] In certain embodiments, the first antigen-binding moiety further comprises a first dimer-forming domain, and the second antigen-binding moiety further comprises a second dimer-forming domain. As used herein, the term “dimer-forming domain” refers to peptide domains that are capable of associating with each other to form a dimer, or, in some examples, capable of spontaneous dimerization of two peptides.

[0280] In certain embodiments, a first dimerizing domain may associate with a second dimerizing domain. This association can occur via any suitable interaction, linkage, or bond, for example, through connectors, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, or hydrophobic-hydrophilic interactions, or combinations thereof. Exemplary dimerizing domains include, but are not limited to, antibody hinge regions, antibody CH2 domains, antibody CH3 domains, and other suitable protein monomers capable of dimerizing and associating with each other. The hinge regions, CH2 and / or CH3 domains may originate from any antibody isotype, such as IgG1, IgG2, and IgG4.

[0281] In certain embodiments, the first and / or second dimerizing domains include at least a portion of the antibody hinge region. In certain embodiments, the first and / or second dimerizing domains may further include an antibody CH2 domain and / or an antibody CH3 domain. In certain embodiments, the first and / or second dimerizing domains include at least a portion of the hinge-Fc region, i.e., the hinge-CH2-CH3 domain. In certain embodiments, the first dimerizing domain may be functionally linked to the C-terminus of the first TCR constant region. In certain embodiments, the second dimerizing domain is functionally linked to the C-terminus of the antibody CH1 constant region of the second antigen-binding moiety.

[0282] In a particular embodiment, the first dimer-forming domain is functionally linked (with or without a spacer) to the first TCR constant region (C1) by a third connecting domain.

[0283] When the Fv region of an immunoglobulin is aligned with the TCR immunoglobulin-like domain, the antibody hinge N-terminus and the TCR hinge N-terminus are also aligned. An example is shown in Table 7 below, where the antibody hinge N-terminus (SEQ ID NO: 278 or 279) is aligned with the TCR beta-hinge N-terminus (SEQ ID NO: 280).

[0284] The third junction domain of the bispecific polypeptide complex provided herein can be selected to include an appropriate length of the antibody hinge N-terminus (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) and an appropriate length of the TCR hinge N-terminus (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues). As used herein, the term "hinge N-terminus" refers to most of the N-terminal fragment of the hinge region. For example, the junction domain may be selected to have all, most, or part of the sequences derived from the antibody hinge N-terminus or the TCR hinge N-terminus, or it may contain more residues derived from the antibody hinge N-terminus than from the TCR hinge N-terminus, or vice versa.

[0285] In certain embodiments, the third connecting domain of the polypeptide complex provided herein has a total length equivalent to the length of the antibody hinge N-terminus or the length of the TCR hinge N-terminus.

[0286] Similarly, an appropriate third connecting domain can be determined based on structure. For example, when superimposing the three-dimensional structures of an antibody and a TCR and determining the length or characteristics of the sequence derived from the N-terminus of the antibody or TCR hinge, the overlap between the N-terminus of the antibody hinge and the N-terminus of the TCR hinge in the superimposed structure is determined and taken into consideration.

[0287] In certain embodiments, the third connecting domain includes a spacer between the N-terminus of the antibody hinge and the N-terminus of the TCR hinge. Any suitable spacer sequence or length can be used, as long as it does not adversely affect antigen binding or polypeptide complex stability.

[0288] Examples of sequences for the antibody hinge N-terminus, TCR hinge N-terminus, and third connection domain are provided in Tables 7, 8, 9, and 10 below.

[0289] In certain embodiments, C1 comprises the manipulated CBeta, and the first dimerizing domain comprises the hinge and Fc of IgG1 or IgG4. Table 7 shows examples of useful connection domain designs for TCR CBeta fused to the antibody hinge. The antibody hinge N-terminus is aligned with the TCR Beta hinge N-terminus. Examples of connection domain designs are also provided in aligned form (see, e.g., SEQ ID NO: 152 or 153). In such embodiments, a third connection domain is included in SEQ ID NO: 53 or 54 (which comprises the third connection domain and the hinge C-terminus). [Table 9]

[0290] In certain embodiments, C1 comprises the manipulated CAlpha or CPre-Alpha, and the first dimerizing domain comprises the hinge and Fc of IgG1 or IgG4. Table 8 shows examples of useful connection domain designs for TCR CAlpha or CPre-Alpha fused to the antibody hinge. The antibody hinge N-terminus is aligned with the TCR Alpha or CPre-Alpha hinge N-terminus. In such embodiments, a third connection domain is contained in Sequence IDs: 134, 135, 140, or 141 (which includes the third connection domain and hinge C-terminus).

[0291] [Table 10]

[0292] In certain embodiments, C1 comprises the manipulated CGamma, and the first dimerizing domain comprises the hinge and Fc of IgG1 or IgG4. Table 9 shows examples of useful connection domain designs for TCR CGamma fused to the antibody hinge. The antibody hinge N-terminus is aligned with the TCR Gamma hinge N-terminus. Examples of connection domain designs are also provided in aligned form (see, e.g., SEQ ID NO: 165 or 166). In such embodiments, a third connection domain is contained in SEQ ID NO: 121 or 122 (which includes the third connection domain and the hinge C-terminus).

[0293] [Table 11]

[0294] In certain embodiments, C1 comprises an engineered CDelta, and the first dimerizing domain comprises the hinge and Fc of IgG1 or IgG4. Table 10 shows examples of useful connection domain designs for TCR CDelta fused to an antibody hinge. The antibody hinge N-terminus is aligned with the TCR Delta hinge N-terminus. Examples of connection domain designs are also provided in aligned form. In such embodiments, a third connection domain is contained in Sequence ID No. 127 or 128 (which includes the third connection domain and the hinge C-terminus).

[0295] [Table 12]

[0296] In certain embodiments, the first dimer-forming domain is functionally linked to the C-terminus of the manipulated TCR constant region and together forms a chimeric constant region. In other words, this chimeric constant region includes the first dimer-forming domain functionally linked to the manipulated TCR constant region.

[0297] In certain embodiments, the chimeric constant region comprises an engineered CBeta bound to a first hinge-Fc region derived from IgG1, IgG2, or IgG4. Examples of sequences of such chimeric constant regions are provided in Tables 11, 12, 13, and 14.

[0298] In certain embodiments, the chimeric constant region includes an engineered CAlpha bonded to a first hinge derived from IgG1, IgG2, or IgG4. Examples of sequences of such chimeric constant regions are provided in Tables 11, 12, and 13.

[0299] In certain embodiments, the chimeric constant region contains a third connecting domain comprising or including sequence numbers 134, 135, 140, or 141, and includes an engineered CPre-Alpha bound to a first hinge derived from IgG1, IgG2, or IgG4. Examples of sequences of such chimeric constant regions are provided in Tables 15 and 16.

[0300] In certain embodiments, the chimeric constant region comprises an engineered CGamma bonded to a first hinge derived from IgG1, IgG2, or IgG4. Examples of sequences of such chimeric constant regions are provided in Tables 17 and 18.

[0301] In certain embodiments, the chimeric constant region includes an engineered CDelta bound to a first hinge derived from IgG1, IgG2, or IgG4. Examples of sequences of such chimeric constant regions are provided in Tables 17 and 18.

[0302] In certain embodiments, the chimeric constant region further comprises a first antibody CH2 domain and / or a first antibody CH3 domain. For example, the chimeric constant region further comprises a first antibody CH2-CH3 domain conjugated to the C-terminus of a third connecting domain. Examples of sequences of such chimeric constant regions are provided in Table 19.

[0303] In a particular embodiment, the first chimeric constant region and the second TCR constant domain are sequence numbers: 177 / 176, 179 / 178, 184 / 183, 185 / 183, 180 / 176, 181 / 178, 182 / 178, 184 / 186, 185 / 186, 188 / 187, 196 / 187, 190 / 189, 192 / 191, 192 / 193, 195 / 194, 198 / 197, 200 / 199, 202 / 201, 203 / 201 This includes pairs of sequences selected from the group consisting of 203 / 204, 205 / 204, 206 / 204, 208 / 207, 208 / 209, 211 / 210, 213 / 212, 213 / 151, 214 / 212, 214 / 151, 234 / 233, 232 / 231, 216 / 215, 218 / 217, 220 / 219, 222 / 221, 224 / 223, 226 / 225, 227 / 223, 229 / 228, 229 / 230, 236 / 235, and 238 / 237.

[0304] These pairs of chimeric constant regions and second TCR constant domains are useful in that they can be manipulated to fuse with a desired antibody variable region to provide the polypeptide complex disclosed herein. For example, the antibody heavy chain variable region is fused with the chimeric constant region (including C1) to form the first polypeptide chain of the polypeptide complex provided herein; and similarly, the antibody light chain variable region is fused with the second TCR constant domain (including C2) to form the second polypeptide chain of the polypeptide complex provided herein.

[0305] These pairs of chimeric constant regions and second TCR constant domains can be used as a platform for constructing the first antigen-binding moiety of the bispecific polypeptide complex provided herein. For example, the variable region of the first antibody can be fused at the N-terminus of the platform sequence (e.g., VH to the chimeric constant domain and VL to the TCR constant domain). The second antigen-binding moiety can then be designed and constructed to associate with the bispecific polypeptide complex provided herein, thereby constructing the bispecific polypeptide complex.

[0306] In certain embodiments, the second dimerizing domain includes a hinge region. The hinge region may be derived from an antibody such as IgG1, IgG2, or IgG4. In certain embodiments, the second dimerizing domain may optionally further include an antibody CH2 domain and / or an antibody CH3 domain, such as a hinge-Fc region. The hinge region may be bound to the antibody heavy chain of the second antigen-binding site (e.g., Fab).

[0307] In a bispecific polypeptide complex, the first and second dimerizing domains are capable of associating with a dimer. In certain embodiments, the first and second dimerizing domains are different and associate in a manner that inhibits homodimerization and / or promotes heterodimerization. For example, the first and second dimerizing domains may be selected so as not to be identical and to preferentially form heterodimers among themselves rather than homodimers among them. In certain embodiments, the first and second dimerizing domains are capable of associating with a heterodimer via knob-into-hole formation, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased mobility.

[0308] In certain embodiments, the first and second dimerizing domains each contain CH2 and / or CH3 domains mutated to enable knob-into-hole formation. The knob can be obtained in the first CH2 / CH3 polypeptide by replacing a smaller amino acid residue with a larger one, and the hole can be obtained by replacing a larger residue with a smaller one. For details on the mutation sites for knob-into-hole formation, please refer to the aforementioned papers by Ridgway et al. (1996), Spiess et al. (2015), and Brinkmann et al. (2017).

[0309] In certain embodiments, the first and second dimer-forming domains include a first CH3 domain of the IgG1 isotype containing S139C and T151W substitutions (SEQ ID NO: 295, knob), and a second CH3 domain of the IgG1 isotype containing Y134C, T151S, L153A, and Y192V substitutions (SEQ ID NO: 296, hole). In another embodiment, the first and second dimer-forming domains include a first CH3 domain of the IgG4 isotype containing S136C and T148W substitutions (SEQ ID NO: 298, knob), and a second CH3 domain of the IgG4 isotype containing Y131C, T148S, L150A, and Y189V substitutions (SEQ ID NO: 299, hole). The sequences and numbering of wild-type Fc IgG1 (SEQ ID NO: 294) and Fc IgG4 (SEQ ID NO: 297) are shown in Figures 20A–20D. As noted earlier, when XnY refers to the Fc region (e.g., the CH3 domain of the Fc region), the amino acid residue numbering is based on the numbering shown in Figures 20A-20D.

[0310] In certain embodiments, the first and second dimer-forming domains further include a first hinge region and a second hinge region. For example, substitutional charge pairs can be introduced into the hinge regions of IgG1 and IgG2 to promote heterodimer formation. For further details, see the aforementioned paper by Brinkmann et al. (2017).

[0311] Dual Specificity Format

[0312] The bispecific polypeptide complexes provided herein may be any preferred bispecific format known in the art. In certain embodiments, the bispecific polypeptide complex is based on a reference bispecific antibody format. As used herein with respect to a bispecific format, “based on” means that the bispecific polypeptide complex provided herein takes the same bispecific format as a reference bispecific antibody, except that one antigen-binding moiety is modified to include VH functionally linked to C1 and VL functionally linked to C2, where C1 and C2 are associated by at least one non-natural interchain linkage as previously defined. Examples of reference bispecific antibody formats known in the art include, but are not limited to, (i) bispecific antibodies with a symmetric Fc, (ii) bispecific antibodies with an asymmetric Fc, (iii) conventional antibodies with an additional antigen-binding moiety, (iv) bispecific antibody fragments, (v) conventional antibody fragments with an additional antigen-binding moiety, and (vi) bispecific antibodies with human albumin or human albumin-binding peptides added.

[0313] BsIgG is monovalent for each antigen and can be produced by the simultaneous expression of two light chains and two heavy chains in a single host cell. Additional IgG is engineered to form bispecific IgG by adding an additional antigen-binding unit to either the amino or carboxyl terminus of either the light chain or heavy chain. The additional antigen-binding unit can be a single-domain antibody (non-paired VL or VH) such as DVD-Ig, a paired antibody variable domain (e.g., Fv or scFv), or an engineered protein scaffold. Any antigen-binding unit in BsIgG, particularly the paired VH-CH1 / VL-CL, can be modified to replace CH1 with C1 and CL with C2 as disclosed herein to form the bispecific polypeptide complex provided herein.

[0314] A bispecific antibody fragment is an antigen-binding fragment derived from an antibody but lacking some or all of the antibody's constant domain. Examples of such bispecific antibody fragments include, for example, single-domain antibodies, Fv, Fab, and diabodies. To form the bispecific polypeptide complex provided herein, the antigen-binding site in the bispecific antibody fragment (e.g., particularly paired VH-CH1 / VL-CL) can be modified to include the polypeptide complex disclosed herein (e.g., VH-C1 / CL-C2).

[0315] In certain embodiments, the bispecific polypeptide complexes provided herein are based on “whole” antibody formats such as whole IgG or IgG-like molecules, as well as smaller recombinant formats such as tandem single-chain variable region molecules (taFv), diabodies (Db), single-chain diabodies (scDb), and various other derivatives thereof (compared to bispecific antibody formats as described by Byrne H. et al., (2013) Trends Biotech, 31 (11): 621-632). Examples of bispecific antibodies are based on formats including, but not limited to, quadromas, chemically coupled Fabs (antigen-binding fragments), and BiTEs (bispecific T cell engagers).

[0316] In certain embodiments, the bispecific polypeptide complexes provided herein are based on a bispecific format selected from the following: triomabs; hybrid hybridomas (quadromas); multispecific antikalin platforms (Pieris); diabodies; single-chain diabodies; tandem single-chain Fv fragments; TandAb, trispecific Ab (Affimed); Darts (dual affinity retargeting; Macrogenics); bispecific Xmab (Xencor); bispecific T cell engagers (Bite; Amgen; 55kDa); Tr iplebodies; tribody (Fab-scFv) fusion protein (CreativeBiolabs) multifunctional recombinant antibody derivatives; duobody platform (Genmab); dock-and-lock platform; knob-into-hole (KIH) platform; humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-Ig (dual variable domain immunoglobulin) (Abbvie); kappa-lambdabody; TBTI (tetravalent bispecific tandem Ig); and CrossMab.

[0317] In certain embodiments, the bispecific polypeptide complexes provided herein are based on bispecificity formats selected from bispecificity IgG-like antibodies (BsIgG), including CrossMab; DAF (two-in-one); DAF (four-in-one); DutaMab; DT-IgG; knob-in-hole common LC; knob-in-hole aggregate; charged pair; Fab-arm exchange; SEEDbody; triomab; LUZ-Y; Fcab; kappa-lambda-body; and orthogonal Fab. For a detailed description of bispecificity antibody formats, see Spiess C., Zhai Q., ​​and Carter PJ (2015) Molecular Immunology 67: 95-106, which is incorporated herein by reference in its entirety.

[0318] In certain embodiments, the bispecific polypeptide complexes provided herein are based on a bispecific format selected from antibodies with added IgG having an additional antigen-binding moiety, including DVD-IgG;IgG(H)-scFv;scFv-(H)IgG;IgG(L)-scFv;scFV-(L)IgG;IgG(L,H)-Fv;IgG(H)-V;V(H)-IgG;IgG(L)-V;V(L)-IgG;KIH IgG-scFab;2scFv-IgG;IgG-2scFv;scFv4-Ig;scFv4-Ig;Zybody; and DVI-IgG (Four-in-One) (see same literature).

[0319] In certain embodiments, the bispecific polypeptide complexes provided herein are based on a format selected from bispecific antibody fragments, including nanobody; nanobody-HAS; BiTE; diabody; DART; TandAb; scdiabody; sc-diabody-CH3; diabody-CH3; triplebody; miniantibody; minibody; TriBi minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F(ab')2; F(ab')2-scFv2; scFv-KIH; Fab-scFv-Fc; tetravalent HCAb; scdiabody-Fc; diabody-Fc; tandem scFv-Fc; and intrabody (see the same literature).

[0320] In certain embodiments, the bispecific polypeptide complexes provided herein are based on bispecific formats such as Doc and Lock;ImmTAC;HSAbody;scDiabody-HAS; and tandem scFv-toxin (see the same literature).

[0321] In certain embodiments, the bispecific polypeptide complexes provided herein are based on a format selected from bispecific antibody conjugates, including IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2 (see the same literature).

[0322] In certain embodiments, the first antigen-binding moiety and the second binding moiety can associate with an Ig-like structure. The Ig-like structure resembles a natural antibody having a Y-shaped construct with two arms for antigen binding and one stem for association and stabilization. The similarity to a natural antibody can offer various advantages, such as good in vivo pharmacokinetics, desired immunological response, and stability. An Ig-like structure comprising the first antigen-binding moiety provided herein associated with the second antigen-binding moiety provided herein is known to have thermal stability comparable to that of Ig (e.g., IgG). In certain embodiments, the Ig-like structure provided herein is at least 70%, 80%, 90%, 95%, or 100% of natural IgG.

[0323] In a particular embodiment, the bispecific polypeptide complex has four polypeptide chains: i) VH1-C1-hinge-CH2-CH3; ii) VL1-C2; iii) VH2-CH1-hinge-CH2-CH3; and iv) VL2-CL, where C1 and C2 are capable of forming a dimer containing at least one non-natural interchain bond, and two hinge regions and / or two CH3 domains are capable of forming one or more interchain bonds that can facilitate dimerization.

[0324] Antigen specificity of bispecific complexes

[0325] The bispecific complex provided herein has two antigen specificities. The first and second antigen-binding moieties are directed toward the first and second antigen specificities, respectively.

[0326] The first and second antigen specificities may be identical; in other words, the first and second antigen-binding sites may bind to the same antigen molecule, or to the same epitope of the same antigen molecule.

[0327] Alternatively, the first and second antigen specificities may be different. For example, the first and second antigen-binding moieties may bind to different antigens. Such a bispecific polypeptide complex is useful, for example, in bringing two different antigens into close proximity, thereby promoting their interaction (e.g., bringing immunological cells into close proximity to tumor antigens or pathogen antigens, thereby promoting recognition or elimination of such antigens by the immune system). Another example is that the first and second antigen-binding moieties may bind to different (and optionally non-overlapping) epitopes of a single antigen. This is useful in enhancing the recognition of or binding to target antigens, particularly mutable antigens (e.g., viral antigens).

[0328] In some embodiments, one of the antigen-specific portions of the bispecific complex provided herein is directed to a T cell-specific receptor molecule and / or a natural killer cell (NK cell)-specific receptor molecule. In some embodiments, one of the first and second antigen-binding portions is capable of specifically binding to CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, or CD95, while the other is capable of specifically binding to a tumor-associated antigen.

[0329] In certain embodiments, one of the antigen-specificities of the bispecific complex provided herein is directed toward CD3. In certain embodiments, the first antigen-binding moiety of the bispecific complex is capable of specifically binding to CD3. In certain embodiments, the second antigen-binding moiety of the bispecific complex is capable of specifically binding to CD3.

[0330] In certain embodiments, the antigen-binding moieties of the bispecific complex include VH1 and VL1 derived from both anti-CD3 antibodies. In certain embodiments, the first polypeptide and the second polypeptide are sequence numbers: 2 / 1, 3 / 4 / , 5 / 1, 6 / 3, 7 / 3, 9 / 8, 10 / 8, 9 / 11, 10 / 11, 13 / 12, 15 / 14, 17 / 16, 17 / 18, 20 / 19, 21 / 12, 28 / 3, 29 / 3, 30 / 12, 31 / 12, 65 / 64, 67 / 66, 69 / 68, 70 / 68, 70 / 71, 72 / 71, 73 / 71, 75 / 74, 75 / 76, 78 / 77, 86 / 85, 90 / 89, 91 / Provided herein are polypeptide complexes or bispecific polypeptide complexes comprising a pair of sequences selected from the group consisting of 92 / , 94 / 93, 96 / 95, 98 / 97, 99 / 95, 101 / 100, 101 / 102, 106 / 105, 108 / 107, 110 / 109, 112 / 111, 137 / 136, 138 / 136, 137 / 139, and 138 / 139, wherein the variable region (T3) of the anti-CD3 antibody is fused to the constant region of the TCR, as shown in Table 20.

[0331] In some embodiments, one antigen specificity of the bispecific complex provided herein is directed to T cell-specific receptor molecules and / or natural killer cell (NK cell)-specific receptor molecules, and the other antigen specificity is directed to tumor-associated surface antigens. In certain embodiments, the first antigen-binding moiety of the bispecific complex can specifically bind to T cell-specific receptor molecules and / or natural killer cell (NK cell)-specific receptor molecules (such as CD3), and the second antigen-binding moiety can specifically bind to tumor-associated antigens (such as CD19), or vice versa.

[0332] In certain embodiments, the bispecific polypeptide complex comprises a 4-sequence combination selected from the group consisting of SEQ ID NOs: 22 / 12 / 24 / 23 (E17, IgG1), 25 / 12 / 26 / 23 (E17, IgG4), and 25 / 12 / 27 / 23 (F16), as shown in Example 8 and Table 20, where the first antigen-binding moiety binds to CD3 and the second antigen-binding moiety binds to CD19. The E17 design is a bispecific bivalent antibody, and the F16 design is a bispecific trivalent antigen-binding complex with two repeat sequences of the anti-CD19 antibody Fab.

[0333] In certain embodiments, the bispecific polypeptide complex comprises a first antigen-binding moiety that binds to CTLA-4 and a second antigen-binding moiety that binds to PD-1, or vice versa.

[0334] In a particular embodiment, the bispecific polypeptide complex comprises four polypeptide chains, including: i) VH1 functionally linked to a first chimeric constant region; ii) VL1 functionally linked to a second chimeric constant region; iii) VH2 functionally linked to a normal antibody heavy chain constant region; and iv) VL2 functionally linked to a normal antibody light chain constant region. In a particular embodiment, the first chimeric constant region may each contain C1-hinge-CH2-CH3 as previously defined. In a particular embodiment, the second chimeric constant region may contain C2 as previously defined. In a particular embodiment, the normal antibody heavy chain constant region may each contain CH1-hinge-CH2-CH3 as previously defined. In a particular embodiment, the normal antibody light chain constant region may contain CL as previously defined.

[0335] The following construct names are used interchangeably in this disclosure: E17-Design_2-QQQQ and W3438-T3U4.E17-1.uIgG4.SP; F16-Design-2-QQQQ and W3438-T3U4.F16-1.uIgG4.SP; U6T5.G25.IgG4 and W3248-U6T5.G25-1.uIgG4.SP; and U6T1.G25R.IgG4 and W3248-U6T1.G25R-1.uIgG4.SP.

[0336] Preparation method

[0337] This disclosure provides polypeptide complexes and isolated nucleic acids or polynucleotides encoding bispecific polypeptide complexes as provided herein.

[0338] As used herein, the terms “nucleic acid” or “polynucleotide” refer to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof. Unless otherwise specified, the term encompasses polynucleotides including known analogs of native nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular polynucleotide sequence also implicitly encompasses its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitution can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed-base and / or deoxyinosine residue (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0339] The nucleic acids or polynucleotides encoding polypeptide complexes and bispecific polypeptide complexes provided herein can be constructed using recombinant techniques. For this purpose, the DNA encoding the antigen-binding portion (such as the CDR or variable region) of the parent antibody is isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody). Similarly, the DNA encoding the TCR constant region can also be obtained. As an example, a polynucleotide sequence encoding the variable domain (VH) and a polynucleotide sequence encoding the first TCR constant region (C1) are obtained and functionally ligated to enable transcription and expression in host cells to construct the first polypeptide. Similarly, the polynucleotide sequence encoding VL is functionally ligated to the polynucleotide sequence encoding C1 so that a second polypeptide can be expressed in host cells. If necessary, polynucleotide sequences encoding one or more spacers can also be functionally ligated to other encoding sequences to enable expression of the desired product.

[0340] The encoding polynucleotide sequence can be further functionally ligated to one or more regulatory sequences in the expression vector, and the expression or generation of the first and second polypeptides can be made feasible and under appropriate control.

[0341] The encoding polynucleotide sequences(s) can be inserted into a vector for further cloning (DNA amplification) or expression using recombination techniques known in the art. In another embodiment, the polypeptide complexes and bispecific polypeptide complexes provided herein may be prepared by homologous recombination known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters (e.g., SV40, CMV, EF-1α), and transcription termination sequences.

[0342] As used herein, the term “vector” refers to a vehicle into which a protein-coding polynucleotide is functionally inserted to result in the expression of that protein. Typically, this construct also includes appropriate regulatory sequences. For example, the polynucleotide molecule may include regulatory sequences located in the 5'-flanking region of a nucleotide sequence encoding a guide RNA and / or a nucleotide sequence encoding a position-specified polypeptide modification, functionally linked to the coding sequence in a manner that enables the expression of a desired transcript / gene in a host cell. Vectors may be used to transform, transduce, or translocate host cells to result in the expression of a gene element held within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain various elements for controlling expression, including a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, a vector may contain an origin of replication. A vector may also contain, non-limitingly, substances that assist its entry into cells, including viral particles, liposomes, or protein coatings.

[0343] In some embodiments, the vector system includes strains such as mammals, bacteria, and yeast, and includes, but is not limited to, plasmids such as pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, and pACT2.2, as well as vectors available from other laboratories and commercially. Suitable vectors may include plasmids or viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0344] Vectors comprising polynucleotide sequences provided herein can be introduced into host cells for cloning or gene expression. As used herein, "host cell" refers to the cell into which the exogenous polynucleotide and / or vector is introduced.

[0345] Suitable host cells for cloning or expressing DNA in vectors as described herein are prokaryotic cells, yeast, or higher eukaryotic cells, as previously described. Suitable prokaryotic cells for this purpose include eubacteria such as Gram-negative or Gram-positive bacteria, such as the Enterobacteriaceae family including Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella tiphymulius), Serratia (e.g., Serratia marcescens), and Sigella, as well as Bacillus (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces.

[0346] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for the cloning or expression of vectors encoding polypeptide complexes and bispecific polypeptide complexes. Saccharomyces cerevisiae, or common baker's yeast, are among the most commonly used lower eukaryotic host microorganisms. However, the following numerous other genera, species, and strains are generally available and useful here: Schizosaccharomyces pombe; Kluiveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. dorosophilarum (ATCC 36,906), K. thermotolerance, and K. marxianus; Yarrowia (EP 402,226); Pichia pastris (EP 183,070); Candida; Trichoderma lesia (EP 244,234); Neurospora crassa crassa); genus Schwaniomyces, e.g., Schwaniomyces occidentalis; and filamentous fungi, e.g., Neurospora, Penicillium, Trypocladium, and Aspergillus hosts, such as A. nigerans and A. niger.

[0347] The host cells suitable for the expression of glycosylated polypeptide complexes and bispecific polypeptide complexes provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants derived from hosts, as well as corresponding insect host cells in tolerable states, have been identified, such as Spodoptera fulgiperda (caterpillar), Aedes aedipuchi (mosquito), Aedes arbopictus (mosquito), Drosophila melanogaster (fruit fly), and Bonmix mori. Various virus strains for transfusion are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bonmix mori NPV, and such viruses are used as viruses herein in accordance with the present invention, particularly as viruses for transfusion of Spodoptera fulgiperda cells. Plant cell cultures of cotton, corn, potatoes, soybeans, petunias, tomatoes, and tobacco can also be used as hosts.

[0348] However, the most interesting aspect is vertebrate cells, and the proliferation of vertebrate cells in tissue culture is becoming a conventional procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), e.g., Expi293; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); Dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocytes (Hep G2, HB 8065); Mouse breast cancer cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatocellular carcinoma line (Hep G2).

[0349] Host cells transformed by the aforementioned expression vector or cloning vector can be cultured in a standard nutrient medium appropriately modified for promoter induction, transformant selection, or cloning vector amplification.

[0350] For the preparation of polypeptide complexes and bispecific polypeptide complexes provided herein, host cells transformed with expression vectors may be cultured in various media. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Re. 30,985 may be used as a culture medium for host cells. These optional media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin®), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with host cells selected for expression and will be obvious to those skilled in the art.

[0351] In one embodiment, the Disclosure provides a method for expressing the polypeptide complex and the bispecific polypeptide complex provided herein, which comprises culturing the host cells provided herein under conditions in which the polypeptide complex or the bispecific polypeptide complex is expressed.

[0352] In certain embodiments, the Disclosure provides a method for constructing a polypeptide complex provided herein, which comprises: a) introducing into a host cell: a first polynucleotide encoding a first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from N-terminus to C-terminus to a first TCR constant region (C1); and a second polynucleotide encoding a second polypeptide comprising a first light chain variable domain (VL) of a first antibody functionally linked from N-terminus to C-terminus to a second TCR constant region (C2): wherein C1 and C2 are capable of forming a dimer comprising at least one non-natural interchain linkage between C1 and C2, the non-natural interchain linkage being capable of stabilizing the dimer of C1 and C2, and the first antibody having first antigen specificity; and b) expressing the polypeptide complex in a host cell.

[0353] In certain embodiments, the Disclosure provides a method for constructing a bispecific polypeptide complex provided herein, which involves: a) delivering to a host cell: a first polynucleotide encoding a first polypeptide containing a first heavy chain variable domain (VH) of a first antibody functionally linked from the N-terminus to the C-terminus of a first TCR constant region (C1); a second polynucleotide encoding a second polypeptide containing a first light chain variable domain (VL) of a first antibody functionally linked from the N-terminus to the C-terminus of a second TCR constant region (C2); and one or more additional polynucleotides encoding a second antigen-binding moiety. a) a step of introducing: where C1 and C2 are capable of forming a dimer comprising at least one non-natural interchain linkage between a first mutated residue in C1 and a second mutated residue in C2, the non-natural interchain linkage is capable of stabilizing the dimer of C1 and C2, the first antigen-binding moiety and the second antigen-binding moiety have reduced mispairing than in another case where the first antigen-binding moiety is a natural Fab counterpart, and the first antibody has first antigen specificity and the second antibody has second antigen specificity; b) a step of expressing a bispecific polypeptide complex in host cells.

[0354] In certain embodiments, the method further includes isolating the polypeptide complex.

[0355] When recombinant technology is used, the polypeptide complexes and bispecific polypeptide complexes provided herein may be generated intracellularly, in the perimembranous lumen, or secreted directly into the culture medium. If the product is generated intracellularly, the first step is to remove any particulate debris, either from the host cell or lysed fragments, by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992), describe a procedure for isolating antibodies secreted into the perimembranous lumen of Escherichia coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If the product is secreted into the culture medium, the supernatant from such an expression system is generally concentrated first using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit protein degradation, a protease inhibitor such as PMSF may be included in one of the aforementioned steps, and an antibiotic may be included to prevent the accidental growth of contaminants.

[0356] Polypeptide complexes and bispecific polypeptide complexes provided herein, prepared from cells, can be purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0357] If the polypeptide complex or bispecific polypeptide complex provided herein contains an immunoglobulin Fc domain, protein A can be used as an affinity ligand depending on the type and isotype of the Fc domain present in the polypeptide complex. Protein A can be used to purify polypeptide complexes based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is bound is most frequently agarose, but other matrices are also available. Mechanically stable matrices such as controlled porous glass (CPG) or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than those achievable with agarose.

[0358] If the polypeptide complex or bispecific polypeptide complex provided herein contains a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE®, chromatography on anion or cation exchange resins (e.g., polyaspartate columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0359] Following any one or more preliminary purification steps, the mixture containing the polypeptide complex of interest and impurities is subjected to low-pH hydrophobic interaction chromatography using an elution buffer with a pH of approximately 2.5 to 4.5, preferably performed with a low salt concentration (e.g., about 0 to 0.25 M salt).

[0360] In certain embodiments, the bispecific polypeptide complexes provided herein can be readily purified in high yield using conventional methods. One advantage of the bispecific polypeptide complexes is the significant reduction in mispairing between the variable domain sequences of the heavy and light chains. This reduces the generation of undesirable byproducts and allows for the acquisition of high-purity products in high yield using a relatively simple purification process.

[0361] derivative

[0362] In certain embodiments, a polypeptide complex or a bispecific polypeptide complex can be used as the basis for conjugation with a desired conjugate.

[0363] Various conjugates are intended to be linked to polypeptide complexes or bispecific polypeptide complexes provided herein (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be linked to polypeptide complexes or bispecific polypeptide complexes by means of covalent bonding, affinity bonding, intercalation, coordination bonding, complex formation, association, blending, or addition, among other methods.

[0364] In certain embodiments, the polypeptide complexes or bispecific polypeptide complexes provided herein may be manipulated to include a specific site outside the epitope-binding portion used to bind to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent linkage to the conjugate.

[0365] In certain embodiments, the polypeptide complex or bispecific polypeptide complex may be linked to a conjugate indirectly, or indirectly, for example, through another conjugate or through a linker.

[0366] For example, polypeptide complexes having reactive residues such as cysteine ​​or bispecific polypeptide complexes may have their reactive groups linked to thiol-reactive substances such as maleimide, iodoacetamide, or pyridyl disulfide, or to other thiol-reactive conjugation partners (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means, (1990) Bioconjugate Chem. 1:2; Hermanson, G., Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0367] In another example, the polypeptide complex or bispecific polypeptide complex is conjugated to biotin, and then indirectly conjugated to a second conjugation which is conjugated to avidin. In yet another example, the polypeptide complex or bispecific polypeptide complex may be linked to a linker which further links to the conjugate. Examples of linkers include bifunctional coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suherate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and his-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents are N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), which provide disulfide linkages.

[0368] This conjugate may be a detectable label, a pharmacokinetic modification, a purified portion, or a cytotoxic portion. Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferases, glucoamylase, lysozyme, sugar oxidase, or β-D-galactosidase), and radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I,35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32The conjugate may contain P, other lanthanides, luminescent labels, a chromophore moiety, digoxigenin, biotin / avidin, a DNA molecule for detection, or gold. In certain embodiments, the conjugate may be a pharmacokinetic modification moiety, such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and ethylene glycol / propylene glycol copolymers. In certain embodiments, the conjugate may be a purification moiety, such as magnetic beads. The "cytotoxic moiety" may be any substance that is harmful to cells or that damages or kills cells. Examples of cytotoxic components include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and their analogues, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), and alkylating agents (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine). This includes, but is not limited to, chloretamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and mitotic inhibitors (e.g., vincristine and vinblastine).

[0369] Methods for conjugating conjugates to proteins such as antibodies, immunoglobulins, or fragments thereof can be found, for example, in U.S. Patents 5,208,020; 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, the entirety of which is incorporated herein by reference.

[0370] Pharmaceutical composition

[0371] This disclosure also provides pharmaceutical compositions containing polypeptide complexes or bispecific polypeptide complexes provided herein, and pharmaceutically acceptable carriers.

[0372] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient(s), and / or salt are generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0373] "Pharmacovigilant carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that has acceptable bioactivity and is non-toxic to the target.Pharmacovigilant carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmacovigilant liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, metal ion sequestering agents or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0374] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in the pharmaceutical compositions provided herein reduces the oxidation of polypeptide complexes or bispecific polypeptide complexes. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving protein stability and maximizing half-life. Accordingly, in certain embodiments, compositions containing the polypeptide complexes or bispecific polypeptide complexes disclosed herein and one or more antioxidants, such as methionine, are provided.

[0375] For further illustration, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or glucose-added lactate Ringer's injection; non-aqueous vehicles such as plant-derived non-volatile oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents at bacteriostatic or fungiostatic concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); metal ion sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antimicrobial agent used as a carrier may be added to the pharmaceutical composition in a re-administered container containing phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline solution, glucose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or substances such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0376] This pharmaceutical composition may be in the form of a solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, and magnesium carbonate.

[0377] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in a conventional form, such as a solution, suspension, or emulsion, or in a solid form suitable for preparing a solution, suspension, or emulsion. The injectable preparation may include sterile and / or nonpyrogenic solutions ready for injection, sterile dry soluble products such as lyophilized powders that are directly combined with a solvent immediately before use, sterile suspensions ready for injection, sterile dry insoluble products that are directly combined with a vehicle immediately before use, and sterile and / or nonpyrogenic emulsions. These solutions may be aqueous or non-aqueous.

[0378] In certain embodiments, the parenteral preparation in unit doses is packaged in ampoules, vials, or needle syringes. All preparations for parenteral administration must be sterile and nonpyrogenic, as is known and practiced in the art.

[0379] In certain embodiments, sterile lyophilized powders are prepared by dissolving the polypeptide complexes or bispecific polypeptide complexes disclosed herein in a suitable solvent. The solvent may contain excipients to improve stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients used include, but are not limited to, water, glucose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable substances. In one embodiment, the solvent may include a buffer such as citrate, sodium phosphate, or potassium, or other such buffers known to those skilled in the art, at a nearly neutral pH. Subsequent sterile filtration of this solution, followed by lyophilization under standard conditions known to those skilled in the art, provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or repeated dose of the polypeptide complexes, bispecific polypeptide complexes, or compositions thereof provided herein. Vials that are slightly overfilled (e.g., about 10%) with the amount required for a single dose or set of doses are acceptable to facilitate accurate sample collection and accurate medication administration. Lyophilized powders can be stored under appropriate conditions, such as about 4°C to room temperature.

[0380] Reconstitution of lyophilized powders with sterile water for injection provides formulations for parenteral administration. In one embodiment, a suitable carrier of sterile and / or nonpyrogenic water or other liquid is added to the lyophilized powder for reconstitution. The exact amount is determined by the chosen therapy being performed and can be determined empirically.

[0381] Treatment method

[0382] Also provided are therapeutic methods comprising administering a therapeutically effective amount of the polypeptide complex or bispecific polypeptide complex provided herein to a subject in need thereof, thereby treating or preventing a condition or disorder. In certain embodiments, the subject is determined to have a disorder or condition that is likely to respond to the polypeptide complex or bispecific polypeptide complex provided herein.

[0383] As used herein, “to treat” or “to cure” a condition includes preventing or alleviating a condition, delaying the onset or rate of development of a condition, reducing the risk of development of a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, causing complete or partial regression of a condition, curing a condition, or any combination thereof.

[0384] The therapeutically effective dose of polypeptide complexes and bispecific polypeptide complexes provided herein will depend on various factors known in the art, such as body weight, age, medical history, current medications, the subject's health status, and the possibility of cross-reactivity, allergies, hypersensitivity, and adverse side effects, as well as the route of administration and the stage of disease development. The dose may be increased or decreased proportionally by a person skilled in the art (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0385] In certain embodiments, the polypeptide complex or bispecific polypeptide complex provided herein may be administered in therapeutically effective doses of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain embodiments, the polypeptide complex or bispecific polypeptide complex provided herein is administered in doses of approximately 50 mg / kg or less, and in certain embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dose may vary throughout the course of treatment. For example, in certain embodiments, the initial dose may be higher than subsequent doses. In certain embodiments, the dose may fluctuate throughout the course of treatment depending on the subject's response.

[0386] The medication regimen may be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0387] Polypeptide complexes or bispecific polypeptide complexes provided herein may be administered by any route known in the art, such as parenteral routes (e.g., intravenous, intramuscular, or intradermal injection, including subcutaneous, intraperitoneal, or intravenous infusion) or non-parenteral routes (e.g., oral, intranasal, intraocular, sublingual, intrarectal, or topical).

[0388] In certain embodiments, the conditions or disorders treated by the polypeptide complex or bispecific polypeptide complex provided herein are cancer or cancerous conditions, autoimmune diseases, infections and parasitic diseases, cardiovascular diseases, neurological disorders, neuropsychiatric conditions, trauma, inflammation, or coagulation disorders.

[0389] As used herein, “cancer” or “cancerous condition” means any medical condition mediated by the growth, proliferation, or metastasis of neoplastic or malignant cells, and includes both solid tumors and non-solid tumors such as leukemia. As used herein, “tumor” means a solid mass of neoplastic and / or malignant cells.

[0390] With respect to cancer, “to treat” or “to cure” means inhibiting or slowing the growth, proliferation, or metastasis of neoplastic or malignant cells, preventing or delaying the development of growth, proliferation, or metastasis of neoplastic or malignant cells, or any combination thereof. With respect to tumor, “to treat” or “to cure” includes eradicating all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the development of the tumor, or any combination thereof.

[0391] For example, with respect to the use of polypeptide complexes or bispecific polypeptide complexes of this disclosure for the treatment of cancer, a therapeutically effective dose is a dose or concentration of the polypeptide complex capable of eradicating all or part of the tumor, inhibiting or slowing tumor growth, inhibiting the growth or proliferation of cells mediating the cancerous condition, inhibiting tumor cell metastasis, improving any symptoms or markers associated with the tumor or cancerous condition, preventing or delaying the development of the tumor or cancerous condition, or a combination thereof.

[0392] In certain embodiments, the conditions and disorders include tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin lymphoma (CHL) and primary mediastinal cancer. These include B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and EBV-negative PTLD, as well as EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary exudative lymphoma, Hodgkin lymphoma, etc., as well as neoplasms of the central nervous system (CNS), such as primary CNS lymphoma, spinal cord tumors, and brainstem gliomas.

[0393] In certain embodiments, conditions and disorders include CD19-related diseases or conditions such as B-cell lymphoma, optionally Hodgkin lymphoma or non-Hodgkin lymphoma, where non-Hodgkin lymphoma includes diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), or Valdenström macroglobulinemia (WM).

[0394] In certain embodiments, conditions and disorders include hyperproliferative conditions or infections that can be treated by modulating the immune response with CTLA-4 and / or PD-1. Examples of hyperproliferative conditions include, but are not limited to, solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions.

[0395] This polypeptide complex or bispecific polypeptide complex may be administered alone or in combination with one or more additional therapeutic means or substances.

[0396] In certain embodiments, when used for the treatment of cancer, tumors, or proliferative disorders, the polypeptide complexes or bispecific polypeptide complexes provided herein may be administered in combination with chemotherapy, radiotherapy, surgery for cancer treatment (e.g., tumor resection), one or more antiemetics or other treatments for the treatment of complications arising from chemotherapy, or any other therapeutic agents for use in the treatment of cancer or related medical disorders. As used herein, “administered in combination” includes being administered simultaneously as part of the same pharmaceutical composition, simultaneously as separate compositions, or as separate compositions at different time points. A composition administered before or after another substance is considered to be administered “in combination” with the substance, as the term is used herein, even if the composition and the second substance are administered via different routes. Where possible, additional therapeutic agents administered in combination with the polypeptide complexes or bispecific polypeptide complexes provided herein shall be administered according to the schedule listed in the package insert for the additional therapeutic agent, or according to the Physicians' Desk Reference, 70th edition (2016) or protocols well known in the art.

[0397] In certain embodiments, the therapeutic agent can induce or boost an immune response against cancer. For example, tumor vaccines can be used to induce an immune response against a specific tumor or cancer. Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon-α, -β, and -γ; colony-stimulating factors such as macrophage-CSF, granulocyte-macrophage-CSF, and granulocyte-CSF; interleukins such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; and tumor necrosis factors such as TNF-α and TNF-β. Substances that inactivate immunosuppressive targets, such as TGF-β beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, can also be used. Another group of substances includes those that activate the immune response against tumor or cancer cells, such as those that enhance T cell activation (e.g., agonists of T cell costimulatory molecules such as CTLA-4, ICOS, and OX-40), and those that enhance dendritic cell function and antigen presentation.

[0398] kit

[0399] This disclosure further provides kits comprising polypeptide complexes or bispecific polypeptide complexes provided herein. In some embodiments, the kits are useful for detecting, capturing, or concentrating one or more targets of interest in a biological sample. The biological sample may include cells or tissues.

[0400] In some embodiments, the kit comprises a polypeptide complex or bispecific polypeptide complex provided herein conjugated with a detectable label. In certain other embodiments, the kit comprises an unlabeled polypeptide complex or bispecific polypeptide complex provided herein, and further comprises a labeled secondary antibody capable of conjugating to the unlabeled polypeptide complex or bispecific polypeptide complex provided herein. The kit may further include instructions for use and packaging for separating each component in the kit.

[0401] In certain embodiments, the polypeptide complexes or bispecific polypeptide complexes provided herein are associated with a substrate or apparatus. Useful substrates or apparatus may be, for example, magnetic beads, microtiter plates, or test specimens. They may be useful for binding assays (such as ELISA), immunograph assays, or for capturing or concentrating target molecules in biological samples.

[0402] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below fall, in whole or in part, within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive ability and without departing from the spirit of the invention. It will be understood that many modifications can be made in the procedures described herein, while still remaining within the boundaries of the invention. It is the inventors' intention that such modifications fall within the scope of the invention.

[0403] [Table 13]

[0404] [Table 14]

[0405] Table 15

[0406] Table 16

[0407] Table 17

[0408] Table 18-1 Table 18-2

[0409] Table 19

[0410] Table 20-1 Table 20-2

[0411] Table 21-1 Table 21-2 Table 21-3 Table 21-4 Table 21-5 Table 21-6 Table 21-7 Table 21-8 Table 21-9 Table 21-10 Table 22-1 Table 22-2 Table 22-3 Table 22-4 Table 22-5 Table 22-6 Table 22-7 Table 22-8 Table 22-9 Table 22-10 Table 22-11 [Table 22-12] [Table 22-13] [Table 22-14] [Table 22-15] [Table 22-16] [Table 22-17] [Table 22-18] [Table 22-19] [Table 22-20] [Table 22-21] [Table 22-22] [Table 22-23] [Table 22-24] [Examples]

[0412] (Example 1: Design and manipulation of antibody and TCR chimeric protein) (TCR sequence)

[0413] TCRs are heterodimeric proteins constructed from double strands. Approximately 95% of human T cells have TCRs consisting of alpha and beta chains, while the remaining 5% have TCRs consisting of gamma and delta chains. The constant region of the human alpha chain contains only one gene, TRAC. The constant region of the human beta chain has two subclasses, the genes TRBC1 and TRBC2. In the Protein Databank (PDB), the number of crystal structures of TRBC1 is relatively larger than that of TRBC2, and therefore the TRBC1 sequence was selected as the main backbone for designing the polypeptide complex ("WuXiBody") disclosed herein. The typical amino acid sequence of TRBC1 can be found in PDB structure 4L4T.

[0414] (Interchain disulfide bonds in TCRs)

[0415] The inventors used the TCR crystal structure to derive their WuXiBody design. Unlike the native TCR anchored to the membrane on the T cell surface, soluble TCR molecules are less stable, but their 3D structure is very similar to that of antibody Fab. In fact, the instability of TCRs under the soluble conditions used is a major obstacle to elucidating their crystal structure (Wang, 2014, op. cit.). The inventors adopted a strategy of introducing a pair of Cys mutations into the TCR constant region and found that this significantly improved chain aggregation and enhanced expression.

[0416] The effect of interchain disulfide bonds on antibody expression

[0417] To determine whether disulfide bonds play a role in maintaining the WuXiBody structure, constructs of the chimeric antibody with and / or without disulfide bonds in the TCR constant region were expressed. The SDS-PAGE results of the expressed WuXiBodies are shown in Figures 15-17. All expressed WuXiBodies were whole IgG-like constructs with two identical arms. Expression of constructs with and without cysteine ​​mutations between CBeta S56 and CAlpha T49, between CBeta S16 or A18 and CPre-Alpha S11, between CGamma Q57 and CDelta V50, and between CGamma A19 and CDelta E88 was tested.

[0418] Expression of a construct lacking a disulfide bond in CBeta / CAlpha (SEQ ID NO: 32 / 42) showed that this construct could not maintain the antibody structure (see Figure 15B). Expression of constructs lacking disulfide bonds in CBeta / CPre-Alpha and CGamma / CDelta was also tested and yielded similar results. In contrast, constructs containing mutated cysteine ​​residues were able to form interchain disulfide bonds, which allowed them to maintain an Ig-like structure (see Figure 15A).

[0419] WuXibody Chimera Domain Design

[0420] Cysteine ​​pair mutations in the constant region of the TCR (numbered according to the sequences in Figures 19A-19E) were introduced into different construct designs of TCR chimeric antibodies, which are shown in Table 21.

[0421] [Table 23]

[0422] For paired Cys mutations in the TCR alpha-beta constant region, the T49C-S56C disulfide bond was used in all designs.

[0423] The connections linking the antibody variable domain and the TCR constant domain, their relative fusion orientation, and the connections linking Fc were all carefully fine-tuned to create a stable and functional WuXiBody. Since the TCR structure is very similar to the antibody Fab, the inventors superimposed an antibody Fv homology model onto the TCR variable region (PDB 4L4T, Figure 2B). The superimposed structure indicates that antibody Fv is structurally equivalent to the TCR constant domain. Based on this structural alignment and corresponding sequences, all relevant operational parameters were designed as exemplified below.

[0424] (Domain orientation)

[0425] Since both the VH-CBeta / VL-CAlpha and crossed VH-CAlpha / VL-CBeta fusion orientations allow for precise assembly of chimeric proteins, the inventors designed and tested both orientations. The sequence homology of VH-VL is close to that of TCR VBeta-VAlpha. The inventors named the VH-CBeta / VL-CAlpha formula the "normal orientation" and VH-CAlpha / VL-CBeta the "crossed orientation".

[0426] (First and second connection domains)

[0427] The inventors aligned the sequences of antibodies and TCRs based on structural alignment and acknowledged that the connections defined in the germline sequences do not always correspond to what is indicated on the structure. For example, based on sequence definition, the connection between VH and CH1 should begin to the right of the last two "SS" residues in the VH region. However, structurally, these two residues are already part of the connection. The inventors defined their connections based on structure rather than sequence.

[0428] Tables 1 and 2 of this disclosure show structure-based sequence alignments for two tested orientations. Predicting which domains are compatible with which ligation domains is challenging, so the inventors checked how the antibody and TCR ligations overlapped on the superimposed structure and estimated possible configurations using each one individually. These ligation domain designs are listed in Tables 1 and 2.

[0429] (Third connection domain)

[0430] Using a strategy similar to that described earlier, the human IgG1 and IgG4 hinges were aligned with the proximal region of the TCR membrane (i.e., the TCR hinge), and their overlap at the structural level was further checked. Tables 7 and 8 of this disclosure list the designs of the third connecting domain.

[0431] (FG loop and DE loop)

[0432] Alignment of the TCR constant region structure with the antibody structure revealed that the FG and DE loops of the TCR beta chain are longer than the corresponding regions in antibody CH1. Figures 3A-3B show the difference in the constant regions between the T cell beta chain and the antibody heavy chain. To test how these two loops disrupt the structure when CH1 is replaced by TCR beta, constructs with and without these two loops were designed.

[0433] Based on the considerations mentioned above, a total of nine constructs were designed using combinations of these parameters, as listed in Tables 22 and 23.

[0434] [Table 24]

[0435] [Table 25]

[0436] (Example 2: Preparation and characterization of monospecific TCR / antibody chimeras) Before fusing the TCR constant domains into bispecific antibody constructs, the feasibility of introducing them into typical monospecific IgG was first evaluated. A laboratory-developed anti-CD3 antibody, designated T3, was selected, and this proof-of-concept (POC) study was performed. The constant domains CH1 and CL of T3 IgG were replaced with the corresponding TCR constant regions (CAlpha and CBeta). All nine different strategies listed in Table 22 (see above) were applied, and all constructs were expressed in the Expi293 system.

[0437] Table 24 lists the expression levels of the designed proteins in the collected supernatant quantified by Q-ELISA. In general, most "normal orientation" designs showed better expression than "cross-orientation" formats, and most "TCR connection" designs showed better expression than "antibody connection." Two "normal orientation" constructs, Design_5 and Design-6, showed comparable expression to Design_2. Of the two "cross-orientation" constructs, Design_7 and 8 showed better expression than Design_3 and 4. Low expression was observed in the extra-long FG loop within the TCR CBeta, suggesting that this FG loop may cause significant steric crash with the fused antibody VL domain.

[0438] [Table 26]

[0439] These results differed completely from what Wu et al. observed with their similar antibody-TCR chimeric design (Wu et al., 2015, op. cit.): their “cross-orientation” design resulted in low expression; their “normal orientation” design did not even show expression.

[0440] To confirm whether the expressed proteins had accurate folding and retained their original function, the inventors tested their binding on CD3-positive Jurkat cells. FACS binding of all samples was performed at three different concentrations: 5.0, 0.4, and 0.032 nm. The original wild-type antibody T3 was used as a positive control. Dose-dependent CD3 binding data are listed in columns 3-5 of Table 24. Designs 2, 6, and 6a showed the best binding ability, comparable to the native antibody T3. Interestingly, all three constructs were, inadvertently, the three best expression formats in mammalian cells. This strong correlation suggests that the level of expression or binding stems from the same molecular origin, namely the compatibility between the antibody variable domain and the TCR constant domain, which necessitates careful design of components such as the connecting domain and interchain disulfide bonds.

[0441] Based on these expression levels and binding activity, Design_2 was selected as the final format to proceed with.

[0442] (Example 3: Deglycosylation) N-glycosylation sites of post-translational modifications (PTMs) on antibodies are causing protein heterogeneity and are beginning to pose a challenge in the development phase. Therefore, we attempted to remove N-glycosylation sites from the constant region of the TCR. A total of four N-glycosylation sites were found in the constant region of the TCR. One is on CBeta (N69, see SEQ ID NO: 244), and the other three are on CAlpha (N34, N68, and N79, see SEQ ID NO: 241). Expression data from this disclosure suggest that these sites, particularly the site on CAlpha, were indeed severely glycosylated when this molecule was expressed in mammalian cells.

[0443] All glycosylation sites on Design_2 were removed by substituting four Asn residues with Gln or Ala (referred to as Design_2-QQQQ or -AAAA, see Table 25). While this strategy is very common in protein manipulation, Gln / Ala mutations have been reported to affect the expression level of TCR / antibody chimeric proteins (Wu et al., 2015, op. cit.). To mitigate this risk, residues derived from Pre-TCR (N68S on CAlpha) and macaque TCR (N79 on CAlpha, N69E on CBeta) (referred to as Design_2-QSKE and -ASKE) were also used at the corresponding positions (see Table 25). In addition, the presence of an atypical glycosylation site (N61) on CAlpha has also been reported (Wollscheid et al., Nature Biotechnology, 27(4), 378-386 (2009), "Mass Spectrometry Identification and Relative Determination of N-Linked Cell Surface Glycoproteins", Wollsheid B., Baush-Fluck D., Henderson C., O'Brien R., Bubel M., Schiess R., Aebersold R., Watts JD, Nat. Biotechnol. 27:378-386(2009) [PubMed][Europe PMC]). Consequently, this residue was also mutated in Gln (referred to as Design_2-QQQQQ, see Table 25). All mutants were expressed in Expi293 for further testing.

[0444] [Table 27]

[0445] [Table 28]

[0446] Expression levels in the supernatant were estimated by Q-ELISA in Table 25. Interestingly, our single deglycosylation design slightly reduced expression levels. Simple mutations with Gln or Ala had no negative effect on the non-reducing gel (Figure 4) and a 150 kd band was observed. On the reducing gel (Figure 4), a 25 kd band was observed. Both indicate successful removal of glycosylation on the light and heavy chains. Mutant proteins with N-glycan removal from the TCR constant region were tested for CD3 binding. Figure 5 shows CD3 + The study showed different mutant protein binding to Jurkat cells. The mutant protein curve shifted only slightly to the right compared to the wild-type antibody T3, which is attributed to the detection of an antibody more sensitive to human IgG than the chimera. There was no change in maximum binding. Overall, most deglycosylation designs did not show any significant differences in either expression or binding. Design_2-QQQQ was selected as the design for further testing.

[0447] In a similar study conducted by Wu et al. (Wu et al., 2015, op. cit.), they performed a deglycosylation mutation on their "cross-orientation" format because their "normal orientation" format was not expressed.

[0448] (Example 4: Design of a WuXiBody based on TCR pre-alpha / beta) Pre-T cell antigen receptors (pre-TCRs) expressed by immature thymocytes play a central role in early T cell development. Pre-TCRs possess a typical beta chain, but their sequence and structure are entirely different from those of the typical alpha chain; they lack a special pre-alpha chain, which only includes a usable constant region. Since the constant region of the typical TCR is compatible with the antibody variable region, pre-TCRs (see PDB 3OF6, SEQ ID NO: 246) were also expected to aid in the design of chimeric proteins. The antibody designs are shown in Table 27.

[0449] [Table 29]

[0450] As listed in Table 28, a total of 10 chimeric constructs were designed by combining these parameters.

[0451] [Table 30]

[0452] [Table 31-1] [Table 31-2]

[0453] The experience gained in Examples 1-3 suggested that connection domains with "normal orientation" and more TCR residues are better suited for producing good chimeric proteins. Therefore, the same strategy was adopted to design the light and heavy chain connection domains shown in Tables 3 and 4. Unlike typical alpha chains, the TCR pre-alpha chain contains only one glycosylation site (N50), which was mutated to a Gln residue (see Sequence ID: 247). The entire heavy chain with a beta constant region is the same as that of Design_2 in Table 22, and includes an N-glycosylation site (N69) substituted with a Gln residue (see Sequence ID: 244).

[0454] The third connecting domain in normal orientation was designed identically to that shown in Table 7, and the third connecting domain in cross-orientation was designed identically to that shown in Table 8 (TCR alpha / beta-based chimeric antibody).

[0455] The pre-TCR lacked a native interchain disulfide bond on the third connecting domain. Similar to the manipulations performed on the typical TCR, the inventors logically introduced disulfide bonds at the beta- and pre-alpha interfaces within the constant region to improve the stability of this chimeric protein (see Table 11). All interface residues on the pre-TCR crystal structure (PDB 3OF6) were examined to obtain a list of interchain pairs whose CAlpha and CBeta carbon atoms were within 7 Å and 5 Å, respectively (see Table 11). Each defined pair was then substituted with a Cys residue, and this mutant protein was expressed in Expi293 cells.

[0456] (Example 5: Design of a TCR gamma / delta-based chimeric antibody) While TCRs constructed from gamma and delta chains are not very common, the heterodimer properties of this protein can also help in the design of novel chimeric formats. Following the same strategies and procedures validated in Example 1, we designed a novel chimera by using the constant region of a delta-gamma TCR to replace the corresponding region of an antibody. The structure of the delta-gamma TCR (see PDB 4LFH, SEQ ID NOs: 249 and 252) was used to facilitate structure-guided sequence alignment between the antibody and the TCR.

[0457] Tables 5 and 6 list the connection domains designed for "normal orientation" and "cross-orientation," respectively. The corresponding IgG1 and IgG4 connection domains for different orientations are shown in Tables 9 and 10. The structure of the delta-gamma TCR is more similar to the antibody than the alpha-beta TCR. No additional FG and DE loops were designed. All N-glycosylation sites (N65 on the gamma, and N16 and N79 on the delta, see SEQ ID NO: 250) were removed by Gln(Q) substitution. The contact interface disulfide bond was designed based on the same strategy introduced in Example 4.

[0458] [Table 32]

[0459] As listed in Table 31, a total of 13 chimeric constructs were designed using combinations of these parameters.

[0460] [Table 33]

[0461] [Table 34-1] [Table 34-2]

[0462] (Example 6: Antibody heavy-light chain mispairing test) One challenge in producing IgG-like bispecific antibodies is the uncontrolled mispairing of light and heavy chains. The inventors evaluated whether TCR beta and alpha-substituted CH1 and CL domains could aggregate with normal IgG heavy and light chains when co-expressed in a single host cell.

[0463] In addition to the anti-CD3 antibody T3, the inventors developed a monoclonal antibody U4 that targets the B lymphocyte antigen CD19. To check the likelihood of mispairing of the light and heavy chains of the two native antibodies, the light-heavy chain pairs of T3 and U4 were intentionally switched (T3_light-U4_heavy, T3_heavy-U4_light) and co-expressed in Expi293 cells. The same test using TCR-modified T3 was also performed as a parallel comparison. Figures 6A-6B show SDS-PAGE data for proteins in both IgG1 and IgG4. For the switched pairs using native antibodies, the 150kd band in unreduced PAGE and the 50kd and 23kd bands in reduced PAGE clearly identified aggregates of mispaired IgG proteins. However, after the introduction of TCR-modified T3, the 150kd band was no longer observed in the gel, indicating that none of the non-cognitive pairs could aggregate into the antibody-like molecule. These data confirm that the TCR-modified Fab designed by the inventors can effectively prevent mispairing of non-cognitive chains.

[0464] (Example 7: Preparation and characterization of Fab-TCR chimeras) To confirm that bispecific antibodies can be designed using TCR-modified antibody Fab, Fab fragments cleaved at two positions were constructed. Figure 8 shows that TCR-modified T3 Fab by N-glycan removal was successfully expressed and purified (T3-Fab-Design_2.his1 (SEQ ID NO: 30 / 12) and T3-Fab-Design_2.his2 (SEQ ID NO: 31 / 12)). Their CD3 binding ability was also confirmed. + The results were evaluated in Jurkat cells and compared with the monovalent form of wild-type T3. Figure 9 shows that chimeric Fab and monovalent T3 have qualitatively similar binding behaviors. These discrepancies may arise from differences in protein detection methods using His and Fc tags.

[0465] (Example 8: Preparation and characterization of TCR-based knob-into-hole bispecific antibodies) After successfully fusing the TCR constant domain to the monospecific antibody T3 and confirming that the novel format effectively prevents chain mispairing with antibody U4, the inventors proceeded to construct a bispecific format.

[0466] TCR-modified T3 and wild-type U4, both possessing a "knob-into-hole" mutation utilizing the Fc CH3 domain, were co-expressed from Expi293 cells. The "knob-into-hole" mutations were generated in the IgG1 isotype at S139C and T151W (SEQ ID NO: 295, knob) in the CH3 domain of T3, and at Y134C, T151S, L153A, and Y192V (SEQ ID NO: 296, hole) in the CH3 domain of U4. Alternatively, the knob-into-hole mutations were generated in the IgG4 isotype at S136C and T148W (SEQ ID NO: 298, knob) in the CH3 domain of T3, and at Y131C, T148S, L150A, and Y189V (SEQ ID NO: 299, hole) in the CH3 domain of U4. Figures 7A-7B show the SDS-PAGE data of the proteins produced in IgG1 and IgG4 after purification. The yields after the first step of protein A purification reached 125 mg / L and 173.7 mg / L for IgG1 and IgG4, respectively. The precise molecular weights, i.e., the approximately 150 kd band on the non-reducing gel, as well as the approximately 50 and 25 kd bands on the reducing gel, were all clearly observed. The purified samples were further validated by SEC-HPLC. The purity of IgG1 and IgG4 reached 98.63% and 100%, respectively. This data indicates that both IgG-like molecules, IgG1 and IgG4, were well expressed and aggregated. These novel TCR-related bispecificity formats were named "E17-Design_2-QQQQ" (Sequence IDs: 22 / 12 / 24 / 23 for IgG1 and 25 / 12 / 26 / 23 for IgG4).

[0467] The expected molecular weights were observed for the designed bispecific antibodies, but it was necessary to verify whether each arm maintained its initial binding ability to its individual cognitive antigens. Since E17-Design_2 is a monovalent conjugate for each target, the inventors also constructed monovalent forms of the natural T3 and natural U4 for parallel comparison. Figures 10A and 10B show CD3, respectively. + Jurkat cells and CD19 + The results of FACS binding of designed bispecific antibodies against Ramos cells are shown. The TCR-modified T3 arm showed moderate binding loss compared to wild-type T3, while IgG4 performed better than IgG1 and was closer to the native protein. Binding of the U4 arm was not reduced by the nearby engineered T3 arm. This was similar to the binding to the original U4 antibody in the monovalent form. Interestingly, however, IgG1 performed better than IgG4 in this case. The reason why isotype is important in maintaining monovalent binding is not clear. Factors such as the stability of the TCR constant region, the selection of the third junction domain design, or the interaction between the two Fab arms may produce the observed phenomenon.

[0468] The monovalent binding of the TCR-modified bispecific format to CD3 and CD19 was reduced for both compared to their bivalent parent antibodies. CD3-mediated T cell activation is known to be extremely sensitive. Strong stimulation of T cells can cause side effects. Therefore, relatively weak CD3 binding was probably acceptable and even desirable for safety reasons. However, weak CD19 binding directly affects the bispecific antibody's ability to kill B cells, consequently reducing its efficacy. To confirm the importance of CD19 binding and to test the universality of our chimeric design, we manufactured another bispecific construct in IgG4, referred to as "F16-Design_2-QQQQ," in which the designed T3 arm was still monovalent, but the U4 arm was bivalent.

[0469] This novel construct was expressed and purified, and its binding experiments were performed directly. Figures 11A-11B show its FACS binding data compared to the previously designed E17 and two parental antibodies, T3 and U4. Interestingly, F16-Design_2-QQQQ showed improved CD3- and CD19-binding (sequence numbers 25 / 12 / 27 / 23 in the order of HC / LC(anti-CD3) / HC / LC(anti-CD19)). Its CD19-binding (sequence numbers 27 / 23) was equivalent to that of the wild-type antibody U4. This data confirms that our chimeric design for T3 can be applied to different bispecificity formats.

[0470] (Example 9: In vitro assay of bispecific antibody-directed tumor cell death) An in vitro functional assay was performed to check the activity of the designed bispecific format in T cell-engaged death of malignant B cells. The E17 construct was tested first. Parental monospecific antibodies T3 and U4 were used as negative controls. Figure 12 shows the dose-dependent cell death function of this E17 bispecific format. E17-IgG4(EC 50 =57pM) is E17-IgG1 (EC 50 It was more potent than E17 (EC17 = 624 pM). To enhance this cell death activity, the F16 format, which has two CD19-binding sites, was also compared with E17. As shown in Figure 13, E17 (EC17 = 624 pM) was more potent. 50 Compared to F16 (EC = 17.7 pM), 50 The efficacy of the solution (=5.5 pM) was improved threefold. This data confirmed that CD19 binding affects the cell death effect. An unrelated human IgG4 antibody was used as a negative control.

[0471] (Example 10: Mass Spectrometry Characterization) To confirm that the produced bispecific antibody had an accurate aggregate, the inventors characterized the molecule E17-Design_2-QQQQ by mass spectrometry. The theoretical molecular weight differences between the two heavy chains and the two light chains were approximately 4000 Da and 500 Da, respectively. Figure 14A shows the protein spectrum under non-reducing conditions. The peak at 148180 Da was the expected molecular weight of the accurately aggregated bispecific antibody. No other peaks were observed indicating a "knob-into-hole" mutation in the Fc region, or that the inventors' TCR-substituted CH1 / CL region worked to properly pair the desired quadruple chain. It is worth noting that the non-reducing mass spectrum cannot help distinguish the accurately aggregated bispecific antibody from IgG in which both light chains were mispaired. However, Example 4 points out that the mispaired heavy and light chains were neither expressed nor aggregated, which does not rule out the possibility of mispairing of both heavy and light chain pairs.

[0472] Under non-reducing conditions (see Figure 14A), a peak was observed at 149128 Da, which is approximately 947 Da higher than the calculated molecular weight. Mass spectrometry was also performed using the protein under reducing conditions. Figure 14B shows the presence of a peak 948 Da away from the VL-CAlpha chimeric light chain, which indicates an O-glycan modification (GlcNAc+Hex+2*NeuAc) on the light chain.

[0473] (Example 11: Thermal stability test) The inventors further used differential scanning fluorescence (DSF) to determine the protein melting temperature T m The thermal stability of the designed bispecific antibodies was tested and compared in both IgG1 and IgG4 by measuring [specific parameters]. Natural monospecific T3 and TCR-modified T3 (Design_2 and Design_2-QQQQ) were used as controls.

[0474] Table 33 shows the measured T of the new constructs. on Value, T mThe values ​​were listed. In general, all molecules showed reasonable thermal stability. The IgG1-like molecule was more stable than the IgG4-like molecule. m The value was 74°C. The TCR antibody chimeric protein was relatively low at approximately 60°C. m This suggests that TCR CBeta-CAlpha has less resistance to high temperatures compared to the normal antibody CH1-CL. This is consistent with what was reported in the study by Wu et al. (Wu et al., 2015, cited above), and also suggests that the CAlpha domain is less stable than CBeta (Toughiri et al., mAbs, 862 (July), 1276-1285 (2016)).

[0475] Mutations that remove N-glycosylation in the TCR constant region did not affect the thermal stability of this chimeric protein. Our bispecific antibody E17-Design_2-QQQQ exhibited similar T characteristics to those of Design_2-QQQQ. m , and T3 lower than natural T3 m It had.

[0476] [Table 35]

[0477] (Example 12: Materials and Methods) (Antibody T3 Fv homology modeling)

[0478] The antibody Fv structural model was constructed using the software Discovery Studio (BIOVIA) based on its Fv amino acid sequence. Both the light chain and heavy chain sequences were first annotated with Kabat numbering to identify the three CDRs and the framework of each chain. Next, each segment (either the CDR or the framework) was searched for in the antibody database using BLAST against all antibody structures in the PDB. Where high resolution and low B factor were present, the structure of the best-matching sequence was used to construct a homology model. Then, all modeled segments were assembled to construct the light chain and heavy chain structural models. The relative orientation between these two modeled chains was estimated by measuring the angle of the antibody structure with the most similar overall sequence. All molecular visualization and analysis work was performed using PyMOL software (Schrodinger).

[0479] (Vector construction)

[0480] The VL, VH, Ck, and CH1 genes were amplified by PCR from existing laboratory DNA templates. The CAlpha and CBeta genes were synthesized by Genewiz. Native or chimeric light chain genes were inserted into linear vectors containing the CMV promoter and kappa signal peptide. DNA fragments of VH-CH1 or VH-CBeta were inserted into linear vectors containing the human IgG4 / IgG1 constant region CH2-CH3. These vectors also contained the CMV promoter and human antibody heavy chain signal peptide. Plasmid ligation, transformation, and DNA preparation were performed using standard molecular biology protocols. Site-directed mutagenesis was performed by PCR amplification using mutagenic primers, followed by DpnI digestion of the template DNA.

[0481] (Protein expression)

[0482] The heavy and light chain vectors were simultaneously transfused into Expi293 cells (Thermofisher Scientific). The ratio of the different vectors for simultaneous transfusion was adjusted according to the expected structure of the antibody, and the initial expression results were shown on SDS-PAGE. Briefly, 40 μg of plasmid and 108 μl of expifectamine were transfused into 1.2 × 10⁶ cells in a volume of 40 ml. 8 Individual cells were used for translocation. Enhancer 1 and Enhancer 2 were added 20 hours after translocation. Translocated cells were cultured in an elliptical shaker rotating at 120 rpm at 8% CO2 and 37°C. Five days after translocation, the supernatant was collected by centrifugation and cell fragments were removed by 0.22 μm filtering.

[0483] (Facial expression detection by SDS-PAGE)

[0484] The supernatant collected on day 5 was mixed with NuPAGE LDS sample buffer (4×), NuPAGE sample reducing agent (10×), and H2O. The reduced sample was heated at 75°C and then loaded onto a gel. The gel was electrophoresed for 35 minutes at a constant pressure of 200V. Next, the gel was stained with SimplyBlue® SafeStain (Invitrogen, LC6065) and microfaved for 5 minutes. Destaining was performed by incubation with water and microwaved for 7 minutes. Images of the gel were taken using Universal Hood III (Bio-Rad).

[0485] (purification)

[0486] Protein A chromatography purification

[0487] MabSelect® SuRe® (MSS) Protein A resin was obtained from GE Healthcare and packed into a glass column (BioRad). Purification by Protein A chromatography was performed at room temperature with a flow rate of 0.2 ml / min using a peristaltic pump as the power source. After loading the sample, 100 mM glycine (pH 3.5) in 10 column volumes (CV) was used for elution, and different fractions were collected. The protein concentration in the different fractions was measured using NanoDrop® 2000 (Thermo Fisher Scientific). This protein purity was detected by SDS-PAGE and SEC-HPLC.

[0488] Ion exchange chromatography (IEC)

[0489] IEC chromatography experiments were performed using a 1 ml Hi-trap SP HP column from GE Healthcare Life Sciences, equipped with an AKTA Pure system (GE Healthcare). The programmed method was set up as follows: column washing with 10 CV of Wash Buffer A (10 mM NaH2PO4, pH 6.0); application of the sample using the sample inlet; equilibration of the column with 10 CV of Wash Buffer A (10 mM NaH2PO4, pH 6.0); elution of the column with Wash Buffer A and Wash Buffer B (10 mM NaH2PO4, 1 M NaCl, pH 6.0). Gradient elution conditions were applied as a linear step of 50 CV with 30% Wash Buffer B, a linear step of 5 CV with 100% Wash Buffer B, and a step of filling with 10 CV with 100% Wash Buffer B. Fractions were collected in 0.5 ml portions per tube according to the UV absorbance value (collection threshold set to 5 mAU).

[0490] Size exclusion chromatography (SEC)

[0491] This chromatography experiment was performed using a Superdex® 200 Increase 10 / 300 GL column and an AKTA system from GE Healthcare Life Sciences. The experiment was conducted using PBS (137 mM NaCl, 2.68 mM KCl, 1.76 mM KH2PO4, 10 mM Na2HPO4, pH 7.0) at a flow rate of 0.5 ml / min. Fractions were collected in 0.5 ml portions using an automated collection program (collected values ​​were set to 5 mAU UV absorbance).

[0492] Ni Sepharose® Excel Chromatography Purification

[0493] For the purification of 6×His-tagged proteins using Ni Sepharose® Excel chromatography, Ni Sepharose® Excel resin was purchased from GE Healthcare. This resin was packed into a glass column (BioRad). After washing the column with 10 column volumes (CV) of ddH2O to remove the resin storage buffer, it was used for the purification of 6×His-tagged proteins. Briefly, the Ni column purification was performed at room temperature using a peristaltic pump at a flow rate of 0.2 ml / min. After loading the sample, 10 CV of PBS (50 mM phosphate, 150 mM NaCl, pH 7.0) was used for washing, followed by removal of weakly bound proteins with 5 CV of elution buffer 1 (50 mM phosphate, 150 mM NaCl, 20 mM imidazole, pH 7.0). The bound protein was eluted using 10CV Elution Buffer 2 (50 mM phosphate, 150 mM NaCl, 500 mM imidazole, pH 7.0). After elution, the collected protein was measured using NanoDrop® 2000 (Thermo Fisher Scientific). The purity of the eluted protein was detected by SDS-PAGE and SEC-HPLC. The column was regenerated using 10CV ddH2O and 10CV Stripping Buffer (50 mM Tris, 500 mM NaCl, 50 mM EDTA, pH 7.4) for sanitation, using 10CV 6M guanidine hydrochloride (pH 7.4) and 10CV 0.1M nickel sulfate. The regenerated column was filled with 20% ethanol and stored at 4°C.

[0494] Size exclusion - High-performance liquid chromatography (SEC-HPLC)

[0495] The purity of the samples was analyzed using a TSK-GEL G3000SWXL column (7.8 mm × 300 mm) from Tosoh Bioscience and an Agilent 1200 HPLC system (Agilent Technologies). The column was equilibrated with phosphate buffer (50 mM sodium phosphate, 150 mM NaCl, pH 7.0) at a flow rate of 1.0 ml / min. After filtering and injecting 50 μl of protein sample, UV absorbance was monitored at 280 nm. Purity was estimated by integrating this chromatogram.

[0496] Measurement of antibody concentration by ELISA

[0497] ELISA plates were coated with 200 ng / ml of (Fab)2 derived from goat anti-human IgG-Fc in coating buffer (200 mM Na2CO3 / NaHCO3, pH 9.2). After incubation overnight at 4°C, the plates were washed once with PBS buffer using a deep-well washer (Biotek ELx405). The plates were then blocked with 2% BSA in PBS buffer and incubated at room temperature for 1 hour. The plates were washed three times with washing buffer, and the positive control antibody and diluted samples were added. After incubation for 2 hours, the plates were washed six times with 300 μl of washing buffer, and biotinylated goat anti-human Ig-Fc (Bethyl, 100 μl / well, 1:5000 dilution in 2% BSA) was added as the detection antibody. After incubation and washing, SA-HRP (Invitrogen, diluted 1:8000 in 2% BSA) was added. The plate was then incubated at room temperature for another hour. The plate was washed six times with 300 μl / well washing buffer. The substrate TMB was added and allowed to develop color for 10 minutes. A stop solution (2M HCl, 100 μl / well) was added to stop further color development, and the absorbance was measured at 450 nm using a plate reader (Molecular Device SpectraMax® M5e).

[0498] Target-binding assay

[0499] The binding ability of the designed molecules is, for each, CD3 + Jurkat cell line and CD19 + The evaluation was performed using Ramos cell lines. Both cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI1640 medium (Invitrogen, catalog number 22400105) supplemented with 10% fetal bovine serum (FBS, Corning, catalog number 35-076-CV).

[0500] 10 per well 5 Aliquots of individual cells were collected, washed with 1% bovine serum albumin (BSA, BovoGen-BSAS), and subsequently incubated in a 96-well round-bottom plate (Corning, catalog no. 3799) with serially diluted test antibodies at 4°C for 1 hour. After two washes with 1% BSA, the plate was further incubated with PE-conjugated goat anti-human IgG Fc antibody (Jackson Immuno Research Laboratories, catalog no. 109-115-098) at 4°C for 30 minutes. After two more washes, the cells were analyzed by flow cytometry using a FACSCanto II cytometer (BD Biosciences), and the relevant fluorescence intensity was quantified using FlowJo software. EC50 values ​​were obtained using Prism software (GraphPad Software) with a 4-parameter nonlinear regression analysis.

[0501] (Bispecific antibody - targeted tumor cell death)

[0502] To obtain human T cells, peripheral blood mononuclear cells (PBMCs) derived from healthy donors were newly isolated from heparinized venous blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare-17-1440-03). These cells were cultured for 6 days in RPMI1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin solution (Sciencell, catalog number: 0503), 50 units / mL human IL-2 ligand protein, and 10 ng / mL OKT3 antibody (EBioscience, catalog number 16-0037-85). The resulting CD8 cells were then cultured. + For T cell enrichment, PBMCs were passed through an EasySep (Stemcell, catalog number: 19053) column. CD8 from the negative selection column. + T cells were used as effector cells.

[0503] In cytotoxicity assays, CD19 is used as the target cell. + Raji cells were pre-labeled with 20 nM Cell Trace pharmacy (Invitrogen, catalog number C34564) at 37°C for 30 minutes. The cell pellet was then washed twice with phenol-free RPMI1640 medium (Invitrogen, catalog number 11835030) supplemented with 10% FBS. Pharmacy-stained Raji B cells (20,000 cells / well) were isolated in 96-well round-bottom plates (Corning, catalog number 3799) using CD8. +T cells (target cell:effector cell ratio 1:5) and serially diluted bispecific antibodies were incubated at 37°C for 4 hours. After incubation, 3 μM propidium iodide (PI, Invitrogen, catalog no. P3566) was added and thoroughly mixed to confirm cell death. After 15 minutes, the cells were analyzed by flow cytometry using a FACSCanto II cytometer. Bispecific antibody-mediated cytotoxicity can be defined as the percentage of PI-positive target cells among pharmed-positive target cells. The EC50 of T cell-engaged cytotoxicity was determined using Prism software (GraphPad Software).

[0504] (Mass spectrometry characterization)

[0505] This protein was diluted to 0.4 mg / mL and deglycosylated by incubation at 37°C for at least 4 hours with 1 μL of PNGase F (Glyko, GKE-5006D) in 100 μL of 20 mM Tris buffer (pH 8.0) (protein-to-enzyme ratio 40:1). Aliquots of the deglycosylated bispecific antibody were partially reduced at room temperature for 15 minutes by adding 2 μL of 1 M DTT to a final concentration of 20 mM. 2 μg of each sample was injected at 0.4 mL / min onto an Acquity UPLC BEH300 C4 column (2.1 × 100 mm, 1.7 μm). Mobile phase A was 0.1% formic acid (FA) in HPLC-grade water. Mobile phase B was 0.1% FA in acetonitrile. For both non-reducing and reducing conditions, an effective elution gradient from 24%B to 34%B was used from 3.0 min to 15.0 min. After separation by RP UPLC, the mass of bispecific proteins under both non-reducing and reducing conditions was detected by Waters Xevo G2 Q-TOF. The MS signals were deconvolved using BiophamaLynx 1.3 software. Theoretical mass-average molecular weights of the light and heavy chain components were determined using the GPMaw program (v.6.00).

[0506] (Thermal stability test using DSF)

[0507] The DSF assay was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems). Briefly, 19 μL of antibody solution was mixed with 1 μL of 62.5 × SYPRO orange solution (Invitrogen) and added to a 96-well plate (Biosystems). This plate was heated at a rate of 2°C / min from 26°C to 95°C, and the resulting fluorescence data was collected. The negative derivative of the fluorescence change with respect to different temperatures was calculated, and the maximum value was taken as the melting temperature T. h It was defined as follows: If a protein has multiple unfolding transitions, the first two T h It was reported that T h1 and T h2 It was named that. T h1 The formal melting temperature T is always used to facilitate comparisons between different proteins. m It is interpreted as: Data collection and T h The calculation was performed automatically by the operating software. Once the software reported plots of negative derivatives at different temperatures, the points in the curve plot that begin to decrease from the pre-transition baseline were plotted at the starting temperature T. on It can be roughly estimated as follows.

[0508] (Example 13: O-glycan identification) The mass spectrometry data revealed an O-glycan on the TCR-modified T3 light chain. Unlike N-glycosylation sites, which can be positioned based on amino acid sequence patterns, O-glycosylation sites are difficult to predict from the sequence. This T3 TCR-chimeric light chain consisted of the V region of the T3 parent antibody and the constant region of the TCR alpha chain. Both regions can potentially contain an O-glycosylation site. Mass spectrometry was performed again on the original T3 monoclonal antibody, and it was confirmed that this parent antibody did not contain an O-glycan, indicating that the O-glycan is located within the TCR alpha constant region.

[0509] It is known that O-glycosylation occurs almost exclusively at Se or Thr residues, and that 21 Ser / Thr residues exist within the TCR alpha constant region (shown in bold in the sequence below). To pinpoint the exact location of the O-glycosylation sites, Ala scanning was performed to replace each individual Ser / Thr with Ala, constructing 21 TCR-modified, single-specific T3 molecules. Latent O-glycans on each mutant were released from the protein, labeled with 2-aminobenzoic acid, and quantified by HPLC combined with a fluorescence detector. Loss of the O-glycan signal can indicate the location of the O-glycosylation site.

[0510] [Table 36]

[0511] To identify and quantify O-glycans, we developed acid hydrolysis and HPLC-based methods. Samples were hydrolyzed with 2M TFA (trifluoroacetic acid) to release O-glycan monosaccharides. GalN (galactosamine), released from GalNAc (N-acetyl-D-galactosamine) and Gal (galactose) in the O-glycans, was labeled with 2-aminobenzoic acid and analyzed by HPLC combined with FLD (fluorescence detector), and quantified using an external calibration curve. Since the released GalN content is a specific monosaccharide for O-glycans, it directly correlated with the amount of O-glycans. These results report the number of moles of GalN per mole of protein, which represents the amount of O-glycans contained in one mole of protein.

[0512] Table 34 shows the quantified O-glycan levels for all mutants. The bispecific molecule E17-Design_2-QQQQ was used as the control protein. This data showed that there were 0.24 mol of O-glycan available on 1 mol of E17-Design_2-QQQQ protein. Since this is a bispecific antibody with only one TCR-modified T3 light chain, the sum of the O-glycan levels of the two chains should be twice that, i.e., approximately 0.48 mol / mol. Of all 21 mutants, most retained the predicted amount of O-glycan. Samples #3, #8, #10, and #20 showed slight signal depletion. Sample #19 showed clear O-glycan loss. Its signal was even lower than that of the control protein. Therefore, position S91 was identified as the major O-glycosylation site. S19, S36, S41, and S94 were identified as possible O-glycosylation sites.

[0513] [Table 37]

[0514] (Example 14: Binding to Fcγ receptor, C1q, and FcRn) (method)

[0515] (Fcγ receptor binding affinity via SPR)

[0516] Antibody binding affinity to FcγRs was detected using Biacore T200 (or Biacore 8K). Each receptor was captured on a CM5 sensor chip (GE) immobilized with anti-his antibody. Different concentrations of antibody were injected onto the sensor chip at a flow rate of 30 uL / min, followed by a 60-second association phase and a 60-second dissociation phase. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0517] The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgram. This experimental data was fitted using a 1:1 model (for FcγRI) or a steady-state model (for other receptors) employing Langmuir analysis. The molar concentration of the antibody was calculated using a molecular weight of 150 kDa.

[0518] (C1q binding by ELISA)

[0519] An ELISA plate (Nunc) was coated overnight at 4°C with a 3 μg / mL antibody sample. After blocking and washing, C1q was gradient diluted starting from 600 μg / mL and incubated at room temperature for 2 hours. The plate was then washed and subsequently incubated with sheep anti-human C1q Ab-HRP for 1 hour. After washing, a TMB substrate was added, and the interaction was stopped with 2 M HCl. Absorbance at 450 nm was measured using a microplate reader (molecular device).

[0520] (FcRn binding affinity by SPR)

[0521] Antibody binding affinity to FcRn was detected using Biacore T200 (or Biacore 8K). Each antibody was immobilized on a CM5 sensor chip (GE). Different concentrations of FcRn in delivery buffer (50 mM Na2HPO4 / NaH2PO4, 150 mM NaCl, 0.05% Tween 20, pH 6.0) were injected onto the sensor chip at a flow rate of 30 μL / min, followed by a 60-second association phase and then a 60-second dissociation phase. After each binding cycle, the chip was regenerated with 1×PBS (pH 7.4).

[0522] The sensorgrams of the blank surface and buffer channel were subtracted from the sensorgram of the test sample. This experimental data was fitted using a steady-state model. The molar concentration of FcRn was calculated using a molecular weight of 45 kDa.

[0523] (result)

[0524] Since all the IgG1 mentioned earlier are IgG1 with LALA mutations, the binding activity of E17-Design_2-QQQQ to FcγRI, FcγRIIa(H167), FcγRIIa(R167), FcγRIIb, FcγRIIIa(F176), FcγRIIIa(V176), and FcγRIIIb in both IgG4 and wild-type IgG1 (T3U4.E17-2.(2).uIgG1 (knob-into-hole wild-type IgG1)) was investigated by SPR.

[0525] [Table 38]

[0526] Affinity is summarized in Tables 35 (IgG4) and 36 (wild-type IgG1). Both molecules showed typical human IgG4 and wild-type IgG1 binding affinity to all Fcγ receptors.

[0527] [Table 39]

[0528] [Table 40]

[0529] The binding activity of the antibodies to C1Q was investigated by ELISA (Figures 21A-21B). E17-Design_2-QQQQ in IgG4 did not show a binding signal in ELISA (Figure 21A), while E17-Design_2-QQQQ in wild-type IgG1 and control human IgG1 antibodies showed a normal binding signal (Figure 21B).

[0530] (Example 15: Symmetrical formats G19, G19R, G25, G25R) Due to safety concerns, antibody therapeutic targets such as CD3×CD19 benefit from bispecific antibodies with monovalent CD3 binding. Considering this, we designed and successfully fabricated asymmetric bispecific formats E17 and F16 by integrating WuXiBody Fab and using knob-into-hole technology. However, some bispecific targets, such as CTLA-4×PD-1, benefit from symmetric formats, which allow for the aggregation of two different antibodies while retaining their original valencies (i.e., a total of tetravalents), achieving the desired synergistic effect. The core unit of WuXiBody is the chimeric Fab, which can be readily incorporated into both asymmetric and symmetric formats to ensure accurate pairing of cognitive light-heavy chains. We designed four WuXiBody-based symmetric formats: G19, G19R, G25, and G25R.

[0531] Figure 22 provides a schematic description of the four symmetry formats. In G19 and G25, two WuXiBody chimeric Fabs were grafted onto the c-terminus and n-terminus of the normal antibody, respectively. The difference between G19 and G19R, or between G25 and G25R, is the inverted positions of the normal and chimeric Fabs in each individual format. The heavy chain portions of the two Fabs and IgG-Fc were integrated into a single chain, while the two light chains were free to fold and assemble independently. When the three vectors were simultaneously transfused into host cells, the heavy chain-heavy chain association was expected to function like a normal antibody during the expression process, while each light chain was expected to self-assemble with its own cognitive partner on the heavy chain.

[0532] We designed a bispecific CTLA-4 × PD-1 antibody in a symmetrical WuXiBody format. A novel anti-PD-1 antibody, W3055_1.153.7 (referred to as U6), and a commercially available anti-CTLA-4 antibody, ipilimumab (referred to as T1), were used to connect to this novel format. The IgG4 isotype was selected to ensure depletion of ADCC and CDC activity against the molecule. Since both U6 and T1 can be placed at the top or bottom of this format (referred to as U6T1 and T1U6, respectively), a single format G19 was used first to investigate both cases.

[0533] The relevant sequences of the test WuXiBody are provided below:

[0534] [Table 41-1] [Table 41-2] [Table 41-3] [Table 41-4] [Table 41-5] [Table 41-6]

[0535] Both U6T1 and T1U6 constructs were successfully expressed in the Expi293 system, and the expressed proteins achieved a purity of approximately 90% after one-step purification by protein A chromatography. Figures 23A-23B show the SDS-PAGE and SEC-HPLC characteristics of the purified proteins. To verify their binding ability, cell-based binding assays to both PD-1 and CTLA-4 targets were subsequently performed. Figures 24A-24B show that both U6 and T1 exhibited reduced binding when positioned at the bottom of the format. Considering that the function of PD-1 is relatively more important than that of CTLA-4 (CTLA-4 antibodies are known to have more severe side effects), the PD-1 binding side was positioned at the top to test how to optimize CTLA-4 binding when positioned at the bottom, as well as to maximize U6 binding (i.e., U6T1 rather than T1U6).

[0536] The other three WuXiBody formats, G19R, G25, and G25R (shown in Figure 22), were also investigated. In addition, the benchmark antibody AK-104 (Akeso Biopharma), a PD-1 / CTLA-4 bispecific antibody used in clinical trials, was obtained and used as a control for direct comparison.

[0537] Due to the importance of PD-1 function, U6 was retained on the apex side of all formats to maximize PD-1 binding, while T1 was retained on the basal side and showed moderate CTLA-4 binding. All constructed molecules were well expressed in Expi293, and purity >90% was easily achieved after one-step purification by protein A chromatography. Figures 25A-25B show the purified proteins characterized by SDS-PAGE and SEC-HPLC.

[0538] Next, cell-based binding assays were performed to both PD-1 and CTLA-4 to check the binding ability of all newly constructed molecules. Figures 26A-26B show a comparison of binding curves between the designed constructs and the benchmark antibody. The data showed that all proteins had PD-1 binding very similar to that of the benchmark antibody. In addition, CTLA-4 binding was significantly improved in the G25 and G25R formats, achieving performance equivalent to the benchmark antibody (≤2x). However, the G19R format still did not work well. Perhaps G19 and G19R share the same problem of hindering effective binding of T1.

[0539] The functions of these molecules were further characterized by verification of their competitive ability against the two target ligands, PD-L1 and CD80. Figures 27A-27B confirm that these molecules performed comparably to the benchmark in competition with PD-L1. On the CTLA-4 side, format G25R showed similar ability to the benchmark in competition with CD80. The other two formats yielded considerably worse results. The difference between G25 and G25R lies in the position of the TCR constant region. Conversion of T1 to the WuXiBody format appeared to enhance T1 activity, although T1 remained below U6. This provides a good example demonstrating that functional leads can be effectively screened by scanning a limited number of WuXiBody-derived formats.

[0540] Therefore, we obtained a functional PD-1 / CTLA-4 bispecific antibody similar to the benchmark antibody. The WuXiBody format is highly universal, meaning that any novel antibody can fit into these formats and exhibit its function. If a good parent antibody is available, it can be used to create a molecule superior to the benchmark antibody.

[0541] To prove this concept, another anti-CTLA-4 antibody, W3162_1.154.8-z35 (referred to as T5), which has a much stronger affinity than ipilimumab, was developed and executed in all four formats G19, G19R, G25, and G25R shown in Figure 22. Again, all novel constructs were well expressed in Expi293 cells and easily purified by one-step protein A chromatography. The purity of these proteins is shown in Figures 28A-28B.

[0542] All U6T5 molecules, the previously identified U6T1.G25R molecule, and the benchmark antibody were bound and compared. The results are listed in Figures 29A-29B. The PD-1 side retained the initial binding behavior observed earlier, because the PD-1 antibody was not replaced in any of these formats. However, regarding the CTLA-4 side, all U6T5 constructs (even in G19 and G19R formats) showed significantly better binding than U6T1.G25R and the benchmark molecule. U6T5.G25 was the most potent of all these novel proteins, showing a 1.6× improvement in EC compared to the benchmark antibody. 50 It also exhibited a >3× improved peak value. This molecule was further characterized in ELISA dual-binding assays and FACS competitive assays. Figure 30 demonstrates that these molecules effectively dual-binding to both targets simultaneously. Data in Figures 31A-31B confirm that U6T5.G25 has significantly improved competitiveness with CD80 compared to CTLA-4. This demonstrates that the WuXiBody format is sufficiently flexible to manipulate different parent antibodies. When this molecule is incorporated (plugged) into the WuXiBody format, the superior features of the parent antibody are well preserved and reflected.

[0543] We characterized the thermal stability of molecules covering all four of these symmetry formats. Most of these molecules exhibited a melting temperature of approximately 60°C (see Table 37), which is consistent with the asymmetric formats described earlier.

[0544] [Table 42]

[0545] (Example 16: Light-heavy switched chimera fabric) A total of 111 possible WuXiBody-based formats were successfully designed. In addition to the previously shown E17, F16, G19, G19R, G25, and G25R, several novel formats with light-heavy crossover TCR-chimeric Fabs were also designed. These were designated G26, G26R, and G27, as shown in Figure 32. In this study, antibody pairs U6 and T4 were used, where T4 was the anti-CTLA-4 antibody WBP3162-1.146.19-z12. The T4U6 pair was developed for formats G27 and G26R. Figures 33A-33B show purified proteins characterized by SDS-PAGE and SEC-HPLC. Both proteins were expressed, and T4U6.G27.IgG4 had low purity, while T4U6.G26R.IgG4 had accurate molecular weight and high purity. The binding ability of the latter molecule was characterized in FACS binding. Figures 34A-34B show that PD-1 binding, being located on the bottom side, was affected, while CTLA binding, being located on the top side of the format, was fully recovered.

[0546] U6T4 pairs were tested in format G26. Both expression and purification processes worked well, as shown in Figures 35A-35B. Both ELISA and FACS binding were performed, and the data are shown in Figures 36A-36D. These data demonstrated that light-heavy cross-linked chimeric Fabs can still function well. The melting temperature of this molecule was approximately 63.4°C, as shown in Table 38.

[0547] [Table 43]

[0548] (Example 17: Bispecific anti-CD13×CD19 WuXiBody) background

[0549] (targeted biology)

[0550] Human CD19 is a type I transmembrane protein belonging to the immunoglobulin superfamily (Carter et al., Curr Dir Autoimmun, 2004, 7:4-32). It is expressed on most B cells but is not detected on plasma cells, stem cells, or normal myeloid cells (Tedder, Nat Rev Rheumatol, 2009, 5(10):572-577). CD19 plays a crucial role in establishing the endogenous B cell signaling threshold by regulating both B cell receptor (BCR)-dependent and BCR-independent signaling (Wang et al., Experimental Hematology & Oncology, 2012, 1:36). CD19 has a broader expression range than CD20. The pattern of CD19 expression is maintained in B-cell malignancy, which allows for targeting of tumor indicators of early B cells, such as all subtypes of B-cell lymphoma from slowly progressive to aggressive, as well as early B-cell tumors such as acute lymphoblastic leukemia (ALL), which converts to B-cell chronic lymphocytic leukemia and acute non-T lymphoblastic leukemia and cannot be targeted by rituximab. Several CD19 monoclonal antibodies have been investigated for lymphoma therapy (U.S. Patent Application Publication 20140072587 A1, U.S. Patent No. 8,242,252 B2, and U.S. Patent No. 8,097,703 B2).

[0551] The CD3 T cell coreceptor is a protein complex composed of four distinct chains: a CD3 gamma chain, a CD3 delta chain, and two CD3 epsilon chains. These four chains associate with the molecule known as the T cell receptor (TCR) and the zeta chain to produce an activation signal in T lymphocytes. The TCR, zeta chain, and CD3 molecules constitute the TCR complex, where the TCR subunit recognizes and binds to antigens, and the CD3 subunit transposes and transports antigenic stimuli into signaling pathways, ultimately regulating T cell activity. The CD3 protein is present on virtually all T cells. The CD3-TCR complex modulates T cell function in both innate and adoptive immune responses, as well as cellular and humoral immune functions. These include broad-spectrum cytotoxic effects, such as the elimination of pathogenic organisms and the regulation of tumor growth. Mouse monoclonal antibodies specific to human CD3, such as OKT3 (Kung et al., Science, 1979, 206: 347-9), were first-generation CD3 antibodies developed for therapeutic purposes. While OKT3 possesses potent immunosuppressive efficacy, its clinical use was hindered by serious side effects linked to its immunogenic and promitotic capabilities (Chatenoud, Nature Reviews, 2003, 3:123-132). OKT3 induces an anti-globulin response, promoting rapid clearance and neutralization (Chatenoud et al., Eur. J. Immunol., 1982, 137:830-8). Furthermore, OKT3 induced T...

Claims

1. It is a polypeptide complex: A first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from the N-terminus to the C-terminus of a first T cell receptor (TCR) constant region (C1); and A second polypeptide comprising a first light chain variable domain (VL) of a first antibody functionally linked from the N-terminus to the C-terminus of a second TCR constant region (C2); Here, C1 and C2 can form a dimer containing at least one unnatural interchain bond between C1 and C2, and the unnatural interchain bond can stabilize the dimer, and The first antibody is a polypeptide complex having the first antigen specificity.

2. The polypeptide complex according to claim 1, wherein the non-natural interchain bond is formed between a first mutated residue in C1 and a second mutated residue in C2.

3. The polypeptide complex according to claim 2, wherein at least one of the first and second mutated residues is a cysteine ​​residue.

4. The polypeptide complex according to any one of claims 1 to 3, wherein the non-natural interchain bond is a disulfide bond.

5. The polypeptide complex according to any one of claims 1 to 4, wherein the first mutated residue is contained within the contact interface of C1, and / or the second mutated residue is contained within the contact interface of C2.

6. The polypeptide complex according to any one of claims 1 to 5, wherein at least one native cysteine ​​residue is absent or present in C1 and / or C2.

7. The polypeptide complex according to any one of claims 1 to 6, wherein at least one native N-glycosylation site is either absent or present within C1 and / or C2.

8. The polypeptide complex according to any one of claims 1 to 7, wherein the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unnatural interchain bonds, and optionally at least one unnatural interchain bond is a disulfide bond.

9. a) Whether C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) Whether C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) Whether C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an manipulated CGamma and C2 includes an manipulated CDelta; or f) C1 includes an operated CDelta and C2 includes an operated CGamma: A polypeptide complex according to any one of claims 1 to 8.

10. The first VH is functionally connected to C1 in the first connection domain, and The polypeptide complex according to any one of claims 1 to 9, wherein the first VL is functionally linked to C2 by the second connecting domain.

11. The polypeptide complex according to claim 10, wherein the first and / or second connecting domains comprise a suitable length of C-terminal fragment of antibody V / C linkage (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) and a suitable length of N-terminal fragment of TCR V / C linkage (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues).

12. The manipulated CBeta contains mutated cysteine ​​residues within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86 and 122-127; and / or The polypeptide complex according to claim 9, wherein the manipulated CaPha contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 6-29, 37-67, and 86-95.

13. The polypeptide complex according to claim 9, wherein the manipulated CBeta comprises a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from S56C, S16C, F13C, V12C, E14C, F13C, L62C, D58C, S76C, and R78C, and / or the manipulated CAlpha comprises a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C, and S62C.

14. The manipulated CBeta and manipulated CAlpha are S56C in CBeta and T49C in Calpha, S16C in CBeta and Y11C in Calpha, F13C in CBeta and L13C in Calpha, S16C in CBeta and L13C in Calpha, V12C in CBeta and S16C in Calpha, E14C in CBeta and S16C in Calpha, F13C in CBeta and V23C in Calpha, L62C in CBeta and Y44C in Calpha, C The polypeptide complex according to claim 13, comprising a pair of mutated cysteine ​​residues that substitute for a pair of amino acid residues selected from the group consisting of D58C in Beta and T46C in Calpha, S76C in CBeta and T46C in Calpha, S56C in CBeta and L51C in Calpha, S56C in CBeta and S62C in Calpha, and R78C in CBeta and S62C in Calpha, wherein the pair of cysteine ​​residues is capable of forming a non-natural interchain disulfide bond.

15. The polypeptide complex according to any one of claims 12 to 14, wherein a native cysteine ​​residue is absent or present at position C74 of the manipulated CBeta.

16. The polypeptide complex according to any one of claims 12 to 15, wherein the at least one native glycosylation site is absent or present in the manipulated CBeta and / or manipulated CaAlpha.

17. The polypeptide complex according to claim 16, wherein the native glycosylation site in the manipulated CBeta is N69, and / or the number of native glycosylation sites in the manipulated CaAlpha is selected from N34, N68, N79, and any combination thereof.

18. The polypeptide complex according to any one of claims 12 to 17, wherein the manipulated CBeta lacks or retains an FG loop and / or a DE loop that includes amino acid residues 101-117 of the natural CBeta.

19. The polypeptide complex according to any one of claims 12 to 18, wherein the manipulated CaPha comprises SEQ ID NO: 43-48 and / or the manipulated CBeta comprises SEQ ID NO: 33-41.

20. The polypeptide complex according to any one of claims 10 to 19, wherein C1 comprises manipulated CBeta, and C2 comprises manipulated CAlpha; and the first connecting domain comprises or includes sequence number 49 or 50, and / or the second connecting domain comprises or includes sequence number 51 or 52.

21. The polypeptide complex according to any one of claims 12 to 20, wherein C1 comprises manipulated CAlpha, and C2 comprises manipulated CBeta; and the first connecting domain comprises or is sequence number 129 or 130, and / or the second connecting domain comprises or is sequence number 49 or 50.

22. The manipulated CBeta contains mutated cysteine ​​residues within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86 and 122-127; and / or The polypeptide complex according to claim 9, wherein the manipulated CPre-Alpha contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 7-19, 26-34, 56-75, and 103-106.

23. The polypeptide complex according to claim 9, wherein the manipulated CBeta comprises a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from S16C, A18C, E19C, F13C, A11C, S56C, and S76C, and / or the manipulated CPre-Alpha comprises a mutated cysteine ​​residue that substitutes with an amino acid residue at a position selected from S11C, A13C, I16C, S62C, T65C, and Y59.

24. The manipulated CBeta and manipulated CPre-Alpha contain S16C in CBeta and S11C in CPre-Alpha, A18C in CBeta and S11C in CPre-Alpha, E19C in CBeta and S11C in CPre-Alpha, F13C in CBeta and A13C in CPre-Alpha, S16C in CBeta and A13C in CPre-Alpha, A11C in CBeta and CPre-Alpha The polypeptide complex according to claim 23, comprising a pair of mutated cysteine ​​residues that substitute for a pair of amino acid residues selected from the group consisting of I16C, S56C in CBeta and S62C in CPre-alpha, S56C in CBeta and T65C in CPre-alpha, and S76C in CBeta and Y59C in CPre-alpha, wherein the pair of mutated cysteine ​​residues is capable of forming a non-natural interchain disulfide bond.

25. The polypeptide complex according to any one of claims 22 to 24, wherein the at least one native glycosylation site is absent or present in the manipulated CBeta and / or manipulated CPre-Alpha.

26. The polypeptide complex according to claim 25, wherein the glycosylation site absent or present in the manipulated CBeta is N69, and / or the glycosylation site absent in the manipulated CPre-Alpha is N50.

27. The polypeptide complex according to any one of claims 22 to 26, wherein the manipulated CBeta lacks or retains an FG loop encompassing amino acid residues 101-107 of the natural CBeta and / or a DE loop at a position encompassing amino acid residues 66-71 of the natural CBeta.

28. The polypeptide complex according to any one of claims 22 to 27, wherein the manipulated CPre-Alpha includes sequence numbers: 82-83, 311, 312, 313, 314, 315, 316, 317, or 318, and / or the manipulated CBeta includes sequence numbers: 84, 33-41, 319, 320, 321, 322, 323, or 324.

29. The polypeptide complex according to any one of claims 10-11 and 22-28, wherein C1 comprises manipulated CBeta, and C2 comprises manipulated CPre-Alpha; and the first connecting domain comprises SEQ ID NO: 49 or 50, and / or the second connecting domain comprises SEQ ID NO: 81 or 131.

30. The polypeptide complex according to any one of claims 10-11 and 22-28, wherein C1 comprises manipulated CPre-Alpha, and C2 comprises manipulated CBetha; and the first connecting domain comprises SEQ ID NO: 132 or 133, and / or the second connecting domain comprises SEQ ID NO: 49 or 50.

31. The manipulated CDelta contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 8-26, 43-64, and 84-88; and / or, The polypeptide complex according to claim 9, wherein the manipulated CGamma contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 11-35 and 55-76.

32. The polypeptide complex according to claim 9, wherein the manipulated CGamma comprises a mutated cysteine ​​residue that is substituted with an amino acid residue at a position selected from S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and / or the manipulated CDelta comprises a mutated cysteine ​​residue that is substituted with an amino acid residue at a position selected from F12C, M14C, N16C, D46C, V50C, F87C, and E88C.

33. The polypeptide complex according to claim 32, wherein the manipulated CGAmma and manipulated CDelta contain a pair of mutated cysteine ​​residues that substitute for a pair of amino acid residues selected from the group consisting of Q57C in CGAmma and V50C in CDelta, A19C in CGAmma and E88C in CDelta, S17C in CGAmma and F12C in CDelta, E20C in CGAmma and F12C in CDelta, F14C in CGAmma and M14C in CDelta, T12C in CGAmma and N16C in CDelta, M62C in CGAmma and D46C in CDelta, and A19C in CGAmma and F87C in CDelta, and the pair of cysteine ​​residues introduced therein is capable of forming interchain disulfide bonds.

34. The polypeptide complex according to any one of claims 31 to 33, wherein the at least one native glycosylation site is absent or present in the manipulated CGamma and / or manipulated CDelta.

35. The polypeptide complex according to claim 34, wherein the native glycosylation site in the manipulated CGamma is N65, and / or the number of native glycosylation sites in the manipulated CDelta is one or both of N16 and N79.

36. The polypeptide complex according to any one of claims 31 to 35, wherein the manipulated CGamma includes sequence number 113, or 114, 333, 334, 335, 336, 337, 338, 339, or 340, and / or the manipulated CDelta includes sequence number 115, 116, 325, 326, 327, 328, 329, 330, 331, or 332.

37. The polypeptide complex according to any one of claims 10-11 and 31-36, wherein C1 comprises manipulated CGamma, and C2 comprises manipulated CDelta; and the first connecting domain comprises sequence number 117 or 118, and / or the second connecting domain comprises sequence number 119 or 120.

38. The polypeptide complex according to any one of claims 10-11 and 31-36, wherein C1 comprises manipulated CDelta, and C2 comprises manipulated CGamma; and the first connecting domain comprises sequence number 123 or 124, and / or the second connecting domain comprises sequence number 125 or 126.

39. The polypeptide complex according to any one of claims 1 to 38, wherein the first antigen specificity is directed toward an exogenous antigen, an endogenous antigen, an autoantigen, a neoantigen, a viral antigen, or a tumor antigen.

40. A bispecific polypeptide complex comprising a first antigen-binding moiety associated with a second antigen-binding moiety: The first antigen-binding site is: A first polypeptide comprising a first heavy chain variable domain (VH) of a first antibody functionally linked from the N-terminus to the C-terminus of the first T cell receptor (TCR) constant region (C1), and A second polypeptide comprising a first light chain variable domain (VL) of the first antibody, functionally linked from the N-terminus to the C-terminus of the second TCR constant region (C2): Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and The first antibody has the first antigen specificity, The second antigen-binding portion has a second antigen-specificity that is different from the first antigen-specificity, and The first and second antigen-binding moieties are a bispecific polypeptide complex that is less prone to mispairing than other complexes where both the first and second antigen-binding moieties are natural Fab counterparts.

41. A polypeptide complex according to any one of claims 1 to 39 having first antigen specificity, which is associated with a second antigen-binding moiety having a second antigen specificity different from the first antigen specificity, and comprises a first antigen-binding moiety, and The first and second antigen-binding moieties are a bispecific polypeptide complex that is less prone to mispairing than other complexes where both the first and second antigen-binding moieties are natural Fab counterparts.

42. The bispecific polypeptide complex according to any one of claims 40 to 41, wherein the second antigen-binding portion comprises a heavy chain variable domain and a light chain variable domain of a second antibody having second antigen specificity.

43. The bispecific polypeptide complex according to any one of claims 40 to 42, wherein the second antigen-binding portion comprises Fab.

44. The bispecific polypeptide complex according to any one of claims 40 or 43, wherein the first antigen specificity and the second antigen specificity are directed toward two different antigens or toward two different epitopes on one antigen.

45. The bispecific polypeptide complex according to claim 44, wherein one of the first and second antigen specificities is directed toward a T cell specific receptor molecule and / or a natural killer cell (NK cell) specific receptor molecule, and the other is directed toward a tumor-associated antigen.

46. The bispecific polypeptide complex according to claim 45, wherein one of the first and second antigen specificities is directed toward CD3 and the other is directed toward a tumor-associated antigen.

47. The bispecific polypeptide complex according to claim 46, wherein one of the first and second antigen specificities is directed toward CD3 and the other is directed toward CD19.

48. The bispecific polypeptide complex according to any one of claims 40 to 47, wherein the first antigen-binding portion further comprises a first dimer-forming domain, and the second antigen-binding portion further comprises a second dimer-forming domain, wherein the first and second dimer-forming domains are associated.

49. The bispecific polypeptide complex according to claim 48, wherein the association is via a connector, disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction, or a combination thereof.

50. The bispecific polypeptide complex according to claim 48, wherein the first and / or second dimer-forming domain optionally comprises at least a portion of an antibody hinge region derived from IgG1, IgG2, or IgG4.

51. The bispecific polypeptide complex according to claim 50, wherein the first and / or second dimer-forming domain further comprises an antibody CH2 domain and / or an antibody CH3 domain.

52. The bispecific polypeptide complex according to claim 48, wherein the first dimer-forming domain is functionally linked to the first TCR constant region (C1) by a third connecting domain.

53. a) C1 includes the manipulated CBeta, and the third connection domain is included in sequence number 53 or 54; b) C1 contains the manipulated CAlpha and the third connection domain is included in sequence number: 134, 135, 140, or 141; c) C1 contains the manipulated CPre-Alpha and the third connection domain is included in sequence number: 134, 135, 140, or 141; d) C1 includes the manipulated CGamma and the third connection domain is included in sequence number: 121 or 122; or e) The bispecific polypeptide complex according to claim 52, wherein C1 comprises an engineered CDelta, and the third connecting domain is included in SEQ ID NO: 127 or 128.

54. The bispecific polypeptide complex according to claim 49, wherein the second dimer-forming domain is functionally linked to the heavy chain variable domain of the second antigen-binding portion.

55. The bispecific polypeptide complex according to any one of claims 48 to 54, wherein the first and second dimer-forming domains are associated in a manner that is different and inhibits homodimer formation and / or favors heterodimer formation.

56. The bispecific polypeptide complex according to claim 55, wherein the first and second dimer-forming domains are capable of associating with the heterodimer via knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased mobility.

57. The first antigen-binding portion comprises a first polypeptide containing VH functionally linked to a chimeric constant region, and a second polypeptide containing VL functionally linked to C2, where the chimeric constant region and C2 are sequence numbers: 177 / 176, 179 / 178, 184 / 183, 185 / 183, 180 / 176, 181 / 178, 182 / 178, 184 / 186, 185 / 186, 188 / 187, 196 / 187, 190 / 189, 192 / 191, 192 / 193, 195 / 194, 198 / 197, 200 / 199, 202 / 201, 2 A bispecific polypeptide complex according to any one of claims 40 to 56, comprising a pair of sequences selected from the group consisting of 03 / 201, 203 / 204, 205 / 204, 206 / 204, 208 / 207, 208 / 209, 211 / 210, 213 / 212, 213 / 151, 214 / 212, 214 / 151, 234 / 233, 232 / 231, 216 / 215, 218 / 217, 220 / 219, 222 / 221, 224 / 223, 226 / 225, 227 / 223, 229 / 228, 229 / 230, 236 / 235, and 238 / 237.

58. The aforementioned first antigenicity is directed toward CD3, and the first polypeptide and the second polypeptide are sequence numbers: 2 / 1, 4 / 3, 5 / 1, 6 / 3, 7 / 3, 9 / 8, 10 / 8, 9 / 11, 10 / 11, 13 / 12, 15 / 14, 17 / 16, 17 / 18, 20 / 19, 21 / 12, 65 / 64, 67 / 66, 69 / 68, 70 / 68, 70 / 71, 72 / 71, 73 / 71, 75 / 74, 75 / 76, A bispecific polypeptide complex according to any one of claims 40 to 57, comprising a pair of sequences selected from the group consisting of 78 / 77, 86 / 85, 90 / 89, 91 / 92, 94 / 93, 96 / 95, 98 / 97, 99 / 95, 101 / 100, 101 / 102, 106 / 105, 108 / 107, 110 / 109, 112 / 111, 137 / 136, 138 / 136, 137 / 139, and 138 / 139.

59. The bispecific polypeptide complex according to any one of claims 40 to 58, wherein the first antigen-binding portion is capable of binding to CD3, and the second antigen-binding portion is capable of binding to CD19, and the bispecific polypeptide complex comprises a combination of four polypeptide sequences selected from the group consisting of SEQ ID NOs: 22 / 12 / 24 / 23, 25 / 12 / 26 / 23, and 25 / 12 / 27 / 23.

60. A conjugate comprising a polypeptide complex according to any one of claims 1 to 39, or a bispecific polypeptide complex according to any one of claims 40 to 59, conjugated to a portion.

61. An isolated polynucleotide encoding a polypeptide complex according to any one of claims 1 to 39, or a bispecific polypeptide complex according to any one of claims 40 to 59.

62. An isolated vector comprising the polynucleotide described in claim 61.

63. A host cell comprising an isolated polynucleotide according to claim 61, or an isolated vector according to claim 62.

64. A method for expressing a polypeptide complex according to any one of claims 1 to 39, or a bispecific polypeptide complex according to any one of claims 40 to 59, comprising culturing the host cells according to claim 63 under conditions in which the polypeptide complex or the bispecific polypeptide complex is expressed.

65. A method for producing a polypeptide complex according to any one of claims 1 to 39, or a bispecific polypeptide complex: a) To host cells: A first polynucleotide encoding a first polypeptide containing a first heavy chain variable region (VH) of a first antibody, functionally linked from the N-terminus to the C-terminus of the first TCR constant region (C1), and A step of introducing a second polynucleotide encoding a second polypeptide containing the first light chain variable domain (VL) of a first antibody, which is functionally linked from the N-terminus to the C-terminus of a second TCR constant region (C2), Here, C1 and C2 can form a dimer containing at least one unnatural interchain bond between C1 and C2, and the unnatural interchain bond can stabilize the dimer, and A step in which the first antibody has first antigen specificity; b) A method comprising the step of expressing a polypeptide complex in host cells.

66. a) To the host cell, A step of introducing one or more additional polynucleotides encoding a second antigen-binding portion, Here, the second antigen-binding portion has a second antigen specificity that is different from the first antigen specificity; b) The method according to claim 65, further comprising the step of expressing a bispecific polypeptide complex in host cells.

67. The method according to any one of claims 64 to 66, further comprising the step of isolating the polypeptide complex or the bispecific polypeptide complex.

68. A composition containing a polypeptide complex according to any one of claims 1 to 39, or a bispecific polypeptide complex according to any one of claims 40 to 59.

69. A pharmaceutical composition comprising a polypeptide complex according to any one of claims 1 to 39, or a bispecific polypeptide complex according to any one of claims 40 to 59, and a pharmaceutically acceptable carrier.

70. A method for treating a condition in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of the polypeptide complex according to any one of claims 1 to 39, or the bispecific polypeptide complex according to any one of claims 40 to 59.

71. The method according to claim 70, wherein the condition can be mitigated, eliminated, treated, or prevented when both the first antigen and the second antigen are regulated.

72. 1) The heavy chain variable domain (VH) of the first antibody functionally linked to the constant region (C1) of the first T cell receptor (TCR), and A first antigen-binding moiety comprising: a first antibody light chain variable domain (VL) functionally linked to a second TCR constant region (C2); Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises the manipulated CDelta and C2 comprises the manipulated CGamma: the first antigen-binding moiety, and 2) The VH of the second antibody functionally linked to the CH1 domain of the antibody heavy chain, and A second antigen-binding moiety comprising the VL: of a second antibody functionally linked to the constant (CL) domain of the antibody light chain; Here, the first antigen-binding portion and the second antigen-binding portion are such that the second antigen-binding portion is less prone to mispairing than the other when both the first and second antigen-binding portions are natural Fab counterparts: A polypeptide complex containing [the specified component].

73. The polypeptide complex according to claim 72, further comprising a third antigen-binding moiety including a VH of a third antibody functionally linked to the CH1 domain of an antibody heavy chain, and a VL of a third antibody functionally linked to the CL domain of an antibody light chain, wherein the CH1 of the third antigen-binding moiety is functionally linked to the VH of the second antigen-binding moiety.

74. 1) The heavy chain variable domain (VH) of the first antibody functionally linked to the constant region (C1) of the first T cell receptor (TCR), and A first antigen-binding moiety comprising: a first antibody light chain variable domain (VL) functionally linked to a second TCR constant region (C2); Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises the manipulated CDelta and C2 comprises the manipulated CGamma: the first antigen-binding moiety, and 2) A second antigen-binding moiety comprising VH of a second antibody functionally linked to C1, and VL of a second antibody functionally linked to C2; Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises engineered CDelta and C2 comprises engineered CGamma; second antigen-binding moiety, and 3) The VH of a third antibody functionally linked to the antibody heavy chain CH1 domain, and A third antigen-binding moiety including the VL: of a third antibody functionally linked to the antibody light chain CL domain; and 4) The VH of the fourth antibody functionally linked to the antibody heavy chain CH1 domain, and The fourth antigen-binding moiety, including the VL of the fourth antibody functionally linked to the antibody light chain CL domain: It is a polypeptide complex containing, This polypeptide complex further comprises the first and second antibody CH2 domains and the first and second antibody CH3 domains, Herein, a polypeptide complex in which the VH derived from the first antigen-binding moiety and the VH derived from the second antigen-binding moiety are functionally linked to the first and second antibody CH3 domains, respectively, the CH1 derived from the third antigen-binding moiety and the CH1 derived from the fourth antigen-binding moiety are functionally linked to the first and second antibody CH2 domains, respectively, and the third and fourth antigen-binding moieties are capable of forming a dimer.

75. 1) The heavy chain variable domain (VH) of the first antibody functionally linked to the constant region (C1) of the first T cell receptor (TCR), and A first antigen-binding moiety comprising: a first antibody light chain variable domain (VL) functionally linked to a second TCR constant region (C2); Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises engineered CDelta and C2 comprises engineered CGamma; first antigen-binding moiety, and 2) A second antigen-binding moiety comprising VH of a second antibody functionally linked to C1, and VL of a second antibody functionally linked to C2; Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises engineered CDelta and C2 comprises engineered CGamma; second antigen-binding moiety, and 3) The VH of a third antibody functionally linked to the antibody heavy chain CH1 domain, and A third antigen-binding moiety including the VL: of a third antibody functionally linked to the antibody light chain CL domain; and 4) The VH of the fourth antibody functionally linked to the antibody heavy chain CH1 domain, and The fourth antigen-binding moiety, including the VL of the fourth antibody functionally linked to the antibody light chain CL domain: It is a polypeptide complex containing, This polypeptide complex further comprises the first and second antibody CH2 domains and the first and second antibody CH3 domains, Herein, a polypeptide complex in which C1 derived from the first antigen-binding moiety and C1 derived from the second antigen-binding moiety are functionally linked to the first and second antibody CH2 domains, respectively, VH derived from the third antigen-binding moiety and VH derived from the fourth antigen-binding moiety are functionally linked to the first and second antibody CH3 domains, respectively, and the first antigen-binding moiety and the second antigen-binding moiety are capable of forming a dimer.

76. 1) The heavy chain variable domain (VH) of the first antibody functionally linked to the constant region (C1) of the first T cell receptor (TCR), and A first antigen-binding moiety comprising: a first antibody light chain variable domain (VL) functionally linked to a second TCR constant region (C2); Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises engineered CDelta and C2 comprises engineered CGamma; first antigen-binding moiety; and 2) A second antigen-binding moiety comprising VH of a second antibody functionally linked to C1, and VL of a second antibody functionally linked to C2; Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises engineered CDelta and C2 comprises engineered CGamma; second antigen-binding moiety, and 3) The VH of a third antibody functionally linked to the antibody heavy chain CH1 domain, and A third antigen-binding moiety including the VL: of a third antibody functionally linked to the antibody light chain CL domain; and 4) The VH of the fourth antibody functionally linked to the antibody heavy chain CH1 domain, and The fourth antigen-binding moiety, including the VL of the fourth antibody functionally linked to the antibody light chain CL domain: It is a polypeptide complex containing, This polypeptide complex further comprises the first and second antibody CH2 domains and the first and second antibody CH3 domains, Herein, a polypeptide complex is provided in which the CH1 derived from the third antigen-binding region and the CH1 derived from the fourth antigen-binding region are functionally linked to the first and second antibody CH2 domains, respectively; the C1 derived from the first antigen-binding region is functionally linked to the VH derived from the first antigen-binding region; the C1 derived from the second antigen-binding region is functionally linked to the VH derived from the second antigen-binding region; and the third and fourth antigen-binding regions are capable of forming a dimer.

77. 1) The heavy chain variable domain (VH) of the first antibody functionally linked to the constant region (C1) of the first T cell receptor (TCR), and A first antigen-binding moiety comprising: a first antibody light chain variable domain (VL) functionally linked to a second TCR constant region (C2); Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises engineered CDelta and C2 comprises engineered CGamma; first antigen-binding moiety, and 2) A second antigen-binding moiety comprising VH of a second antibody functionally linked to C1, and VL of a second antibody functionally linked to C2; Here, C1 and C2 are capable of forming a dimer containing at least one unnatural interchain bond between the first mutated residue in C1 and the second mutated residue in C2, and the unnatural interchain bond is capable of stabilizing the dimer, and Here, a) C1 contains manipulated CBeta and C2 contains manipulated CAlpha; b) C1 contains manipulated CAlpha and C2 contains manipulated CBeta; c) C1 contains manipulated CBeta and C2 contains manipulated CPre-Alpha; d) C1 contains manipulated CPre-Alpha and C2 contains manipulated CBeta; e) C1 includes an operated CGamma and C2 includes an operated CDelta; or f) C1 comprises engineered CDelta and C2 comprises engineered CGamma; second antigen-binding moiety, and 3) The VH of a third antibody functionally linked to the antibody heavy chain CH1 domain, and A third antigen-binding moiety including the VL: of a third antibody functionally linked to the antibody light chain CL domain; and 4) The VH of the fourth antibody functionally linked to the antibody heavy chain CH1 domain, and The fourth antigen-binding moiety, including the VL of the fourth antibody functionally linked to the antibody light chain CL domain: It is a polypeptide complex containing, This polypeptide complex further comprises the first and second antibody CH2 domains and optionally the first and second antibody CH3 domains, Herein, a polypeptide complex is provided in which C1 from the first antigen-binding region and C1 from the second antigen-binding region are functionally linked to the first and second antibody CH2 domains, respectively; CH1 from the third antigen-binding region is functionally linked to VH from the first antigen-binding region; CH1 from the fourth antigen-binding region is functionally linked to VH from the second antigen-binding region; and the first antigen-binding region and the second antigen-binding region are capable of forming a dimer.

78. The manipulated CBeta comprises cysteine ​​residues mutated within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86 and 122-127; and / or The polypeptide complex according to any one of claims 72 to 77, wherein the manipulated CaPha contains a mutated cysteine ​​residue within a contact interface selected from the group consisting of amino acid residues 6-29, 37-67, and 86-95.

79. The manipulated CBeta and manipulated CAlpha are S56C in CBeta and T49C in CAlpha, S16C in CBeta and Y11C in CAlpha, F13C in CBeta and L13C in CAlpha, S16C in CBeta and L13C in CAlpha, V12C in CBeta and S16C in CAlpha, E14C in CBeta and S16C in CAlpha, F13C in CBeta and V23C in CAlpha, L62C in CBeta and Y44C in CAlpha, C The polypeptide complex according to claim 78, comprising a pair of mutated cysteine ​​residues that substitute for a pair of amino acid residues selected from the group consisting of D58C in Beta and T46C in CaAlpha, S76C in CBeta and T46C in CaAlpha, S56C in CBeta and L51C in CaAlpha, S56C in CBeta and S62C in CaAlpha, and R78C in CBeta and S62C in CaAlpha, wherein the pair of cysteine ​​residues is capable of forming a non-natural interchain disulfide bond.

80. The polypeptide complex according to claim 79, wherein the manipulated CBeta comprises S56C and the manipulated CAlpha comprises T49C.

81. The polypeptide complex according to any one of claims 72 to 80, wherein the native cysteine ​​residue at position C74 of the manipulated CBeta is absent.

82. The polypeptide complex according to any one of claims 72 to 81, wherein the at least one native glycosylation site is not present in the manipulated CBeta and / or manipulated CaAlpha.

83. The polypeptide complex according to claim 82, wherein the native glycosylation site in the manipulated CBeta is N69, and / or the number of native glycosylation sites in the manipulated CaAlpha is selected from N34, N68, N79, and any combination thereof.

84. An isolated polynucleotide encoding a polypeptide complex according to any one of claims 72 to 83.

85. An isolated vector comprising the polynucleotide described in claim 84.

86. A host cell comprising the isolated polynucleotide according to claim 84 or the isolated vector according to claim 85.

87. A composition containing the polypeptide complex according to any one of claims 72 to 83.

88. A pharmaceutical composition comprising a polypeptide complex according to any one of claims 72 to 83, and a pharmaceutically acceptable carrier.

89. A method for treating a condition in a subject requiring the treatment, comprising administering a therapeutically effective amount of the polypeptide complex described in any one of claims 72 to 83 to the subject.

90. The polypeptide complex according to any one of claims 9 to 21 and 72 to 83, wherein at least one native Ser residue in the manipulated CaAlpha is mutated to reduce O-glycosylation.

91. The polypeptide complex according to any one of claims 40 to 56, wherein C1 or C2 comprises an engineered CaAlpha, and at least one native Ser residue in the engineered CaAlpha is mutated to reduce O-glycosylation.

92. The polypeptide complex according to any one of claims 90 to 91, wherein the mutated amino acid residue is selected from S19, S36, S41, S91, and S94.

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