Variant CH3 domains engineered for preferential CH3 heterodimerization, multispecific antibodies comprising same, and methods for making same

Abstract

Variant CH3 domain polypeptides are provided that preferentially form CH3-CH3 heterodimers over CH3-CH3 homodimers. Such variant CH3 domains can be used to promote desired Fc pairings and thus provide efficient development of bispecific and multispecific antibodies and different types of Fc fusions. Methods for producing bispecific antibodies using such variant CH3 domains and methods for producing libraries that include such variant CH3 domains are also provided.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 136,120, filed January 11, 2021, entitled "CH3 DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL CH3 HETERODIMERIZATION AND MULTI-SPECIFIC ANTIBODIES COMPRISING THE SAME," the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to variant CH3 domains that associate with other variant CH3 domains to promote Fc heterodimerization through preferential pairing, as well as polypeptides, molecules, and multispecific antibodies or antigen-binding antibody fragments, and compositions comprising any of the foregoing. The present invention further relates to polynucleotides encoding one or more such variant CH3 domains, polypeptides, molecules, multispecific antibodies or antigen-binding antibody fragments, and compositions and libraries comprising any of the foregoing. The present invention further relates to methods for generating libraries comprising variant CH3 domains and methods for using these libraries to identify variant CH3 domains that associate with other variant CH3 domains to promote Fc heterodimerization. The present invention further relates to methods for screening for variant CH3 domain combinations (sets) that promote Fc heterodimerization, methods for producing heteromeric molecules such as multispecific antibodies or antigen-binding antibody fragments comprising the variant CH3 domain sets, and methods for producing heteromeric molecules such as multispecific antibodies and antigen-binding antibody fragments in which Fc heterodimerization is promoted using the variant CH3 domain sets. [Background technology]

[0003] Efforts are ongoing to develop antibody therapeutics with two or more antigen-binding specificities, such as bispecific antibodies. Bispecific antibodies can be used to block multiple surface receptors associated with cancer, autoimmune diseases, inflammation, or other diseases and conditions. Bispecific antibodies can also be used to place targets in close proximity, regulate protein complex formation, or drive cell-cell contact. The production of bispecific antibodies was first reported in the early 1960s (Nisonoff et al., Arch Biochem Biophys 1961 93(2):460-462), and the first monoclonal bispecific antibodies were generated using hybridoma technology in the 1980s (Milstein et al., Nature 1983 305(5934):537-540). Interest in bispecific antibodies has grown significantly over the past decade due to their therapeutic potential, and bispecific antibodies are now used in outpatient clinics; for example, blinatumomab and emicizumab are approved for the treatment of certain cancers (see Sedykh et al., Drug Des Devel Ther 12:195-208 (2018) and Labrijn et al. Nature Reviews Drug Discovery 18:585-608 (2019) for recent reviews of methods for producing bispecific antibodies and the characteristics of bispecific antibodies approved for pharmaceutical use).

[0004] While bispecific antibodies offer significant advantages over monospecific antibodies, their widespread commercial application is hindered by the lack of efficient / low-cost production methods, their lack of stability, and their lack of a long half-life in humans. Bispecific antibodies can be formed by coexpressing two different heavy chains and two different light chains. However, because heavy chains bind to light chains in a relatively promiscuous manner, coexpression of two heavy chains and two light chains can result in a mixture of 16 possible combinations, representing 10 different antibodies, of which only one corresponds to the desired bispecific antibody (if complete promiscuity exists, the maximum yield in the mixture is 12.5%). Even if a first heavy chain pair with a first specificity and a second heavy chain pair with a second specificity different from the first are produced separately and then mixed for heavy chain-heavy chain pairing, only three possible combinations are possible, of which only one corresponds to the desired bispecific antibody (if complete promiscuity exists, the maximum yield in the mixture is 50%). This mispairing (also called the linkage problem) poses a major challenge in producing bispecific antibodies, and various techniques have been developed to address this issue.

[0005] One strategy used to mitigate heavy chain-heavy chain mispairing is to design bispecific antibodies with a common heavy chain, i.e., two identical heavy chains and two different light chains (see, e.g., Fischer et al., Nature Commun. 6:6113 (2015)). This eliminates the need to eliminate mispairing in antibody products. However, this strategy requires the identification of two antibodies with different specificities but the same heavy chain, i.e., differing only in the light chain, which is difficult and tends to impair the specificity of each binding arm, significantly reducing diversity (see, e.g., Wang et al., MABS 10(8):1226-1235 (2018)). Leucine zippers (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)) are also used.

[0006] Another strategy is to modify the antibody constant region to reduce the occurrence of chain-heavy chain mispairing. Many engineering efforts have been made in the CH3 domain to promote CH3 heterodimerization. Such techniques include, for example, "W-SAV" substitutions (see, e.g., Atwell S. et al., J Mol Biol. 1997 Jul 4;270(1):26-35 and US 5,731,168 (Genentech)); "HA-TF" substitutions (see, e.g., Moore G. et al., mAbs 2011 Nov-Dec;3(6):546-557 and US 10,472,427 (Xencor)); "VYAV-VLLW" substitutions (see, e.g., Von Kreudenstein TS et al., MAbs 2013;5:646-54 and US 9,499,634 (Zymeworks)); "7.8.60" design and "20.8.34" design (see, e.g., Larver-Fay A. et al., Structure. 2016 April 5;24(4):641-651 and US 10,774,156 (University of North Carolina at Chapel Hill and Eli Lilly); electrostatic complementarity through charge exchange substitutions such as "DD-KK" (see, e.g., Gunasekaran K. et al., J Biol Chem 2010;285:19637-46 and US 8,592,562 (Amgen)); and "knob-into-hole" ("KiH") engineering such as "EW-RVT" substitutions (see, e.g., Choi HJ. et al., Mol Cancer Ther. 2013 Dec;12(12):2748-59 and US 9951145B2 (Ajou University)).Further examples of CH3 modifications include US 10,597,464 (Genmab), US 16 / 482,137 (Centrymed), US 9,562,109 (Zymeworks), US 15 / 409,456 (Zymeworks), US 9,624,291 (Ramot at Tel Aviv University), PCT / EP2019 / 083638 (Morphosys), US 9,605,084 (Xencor), US 16 / 062,405 (Alphamab), US 15 / 997,222 (Janssen), US 14 / 989,648 (Zymeworks), US 13 / 892,198 (Zymeworks), US 15 / 586,686 (Hoffmann La Roche), US9,308,258 (Amgen), US9,200,060 (Amgen), US15 / 554,022 (Laboratoire Francais), US9,574,010 (Zymeworks), PCT / US2019 / 023382 (Dana-Farber Cancer Institute), US13 / 814657 (MedImmune), US11 / 228,026 (Xencor), PCT / US2017 / 045139 (Merrimack), US16 / 244,378 (Hoffmann La Roche), and those described in Brinkmann U. et al., MAbs. 2017 Feb-Mar;9(2):182-212 (review).

[0007] Although these CH3 modifications increase the propensity to form CH3 heterodimers, improvements are still needed, especially given the general interest in developing improved multispecific antibodies for use in human therapy. Summary of the Invention

[0008] It is an object of the present invention to provide engineered variant CH3 domains that preferentially form CH3-CH3 heterodimers over CH3-CH3 homodimers. The variant CH3 domains disclosed herein can be incorporated into polypeptides, molecules, or multispecific antibodies or antigen-binding antibody fragments, for example, where heterodimerization is desired.

[0009] In one aspect, provided herein is a variant immunoglobulin heavy chain constant region 3 (CH3) domain polypeptide or a heavy chain polypeptide comprising such a first variant CH3 domain polypeptide. The variant CH3 domain or heavy chain can comprise at least one amino acid substitution (e.g., compared to a parent sequence, e.g., a wild-type sequence, such as SEQ ID NO: 1).

[0010] In some embodiments, such a variant CH3 domain or heavy chain polypeptide may preferentially form a CH3-CH3 heterodimer with another CH3 domain or heavy chain containing a CH3 domain over a CH3-CH3 homodimer with another CH3 domain or heavy chain containing a CH3 domain.

[0011] In some embodiments, a variant CH3 domain polypeptide (which may be referred to as a "first variant CH3 domain polypeptide") is provided that may comprise one or more amino acid substitutions that may comprise or consist of an amino acid substitution at one or more of the following amino acid positions according to EU numbering: 364, 366, 368, 370, 399, 400, 405, 407, and 409. In some embodiments, heavy chain polypeptides comprising such CH3 domain polypeptides may also be provided.

[0012] Optionally, such a variant CH3 domain or heavy chain polypeptide preferentially forms a heterodimer (a heterodimer of the CH3 domain) with a second variant CH3 domain polypeptide or a heavy chain polypeptide comprising the second variant CH3 domain polypeptide. In some cases, the second variant CH3 domain polypeptide (a) may differ from the first variant CH3 domain polypeptide by at least one amino acid, and (b) may include an amino acid substitution at one or more of the following positions according to EU numbering: 364, 366, 368, 370, 399, 400, 405, 407, and 409. Further optionally, (i) the first CH3 domain further includes the amino acid substitution S354C, and the second CH3 domain further includes the amino acid substitution Y349C. Alternatively, (ii) the first CH3 domain further includes the amino acid substitution Y349C, and the second CH3 domain further includes the amino acid substitution S354C. When present, the combination of the S354C and Y349C substitutions may allow for a disulfide bond between the first and second variant CH3 domains, which may further promote CH3-CH3 heterodimerization.

[0013] In some embodiments, further optionally, (i) if the amino acid substitution in the first variant CH3 domain consists of T366Y, then the amino acid substitution in the second variant CH3 domain does not consist of Y407T; (ii) if the amino acid substitution in the first variant CH3 domain consists of Y407T, then the amino acid substitution in the second variant CH3 domain does not consist of T366Y; and (iii) if the amino acid substitution in the first variant CH3 domain consists of T366W, then the amino acid substitution in the second variant CH3 domain does not consist of T366Y. (iv) if the amino acid substitutions in the first variant CH3 domain consist of T366S, L368A, and Y407V, the amino acid substitutions in the second variant CH3 domain do not consist of T366W; (v) if the amino acid substitutions in the first variant CH3 domain consist of S354C and T366W, the amino acid substitutions in the second variant CH3 domain do not consist of Y349C, T366S, L368A, and Y407V; and (vi) if the amino acid substitutions in the first variant CH3 domain consist of Y3 (vii) if the amino acid substitutions in the first variant CH3 domain consist of S364H and F405A, the amino acid substitutions in the second variant CH3 domain do not consist of Y349T and T394F; and (viii) if the amino acid substitutions in the first variant CH3 domain consist of Y349T and T394F, the amino acid substitutions in the second variant CH3 domain do not consist of S364H and T394F. and F405A; (ix) if the amino acid substitutions in the first variant CH3 domain consist of T350V, L351Y, F405A, and Y407V, then the amino acid substitutions in the second variant CH3 domain do not consist of T350V, T366L, K392L, and T394W; (x) if the amino acid substitutions in the first variant CH3 domain consist of T350V, T366L, K392L, and T394W, then the amino acid substitutions in the second variant CH3 domain do not consist of T350V, L351Y, F405A, and Y407V;(xi) if the amino acid substitutions in the first variant CH3 domain consist of K392D and K409D, the amino acid substitutions in the second variant CH3 domain do not consist of E356K and D399K; (xii) if the amino acid substitutions in the first variant CH3 domain consist of E356K and D399K, the amino acid substitutions in the second variant CH3 domain do not consist of K392D and K409D; (xiii) if the amino acid substitutions in the first variant CH3 domain consist of D221E, P228E, and L368E, the amino acid substitutions in the second variant CH3 domain do not consist of D221R, P228R, and K409R; and the first CH3 domain and the second CH3 domain are human IgG1 (xiv) if the amino acid substitutions in the first variant CH3 domain consist of D221R, P228R, and K409R, then the amino acid substitutions in the second variant CH3 domain do not consist of D221E, P228E, and L368E; and the first CH3 domain and the second CH3 domain are derived from a human IgG1 CH3 domain; and (xv) if the amino acid substitutions in the first variant CH3 domain consist of C223E, P228E, and L368E, then the amino acid substitutions in the second variant CH3 domain do not consist of C223R, E225R, P228R, and K409R; and the first CH3 domain and the second CH3 domain are derived from a human IgG2 (xvi) if the amino acid substitutions in the first variant CH3 domain consist of C223R, E225R, P228R, and K409R, the amino acid substitutions in the second variant CH3 domain do not consist of C223E, P228E, and L368E; the first CH3 domain and the second CH3 domain are derived from human IgG2 CH3 domains; (xvii) if the amino acid substitutions in the first variant CH3 domain consist of K360E and K409W, the amino acid substitutions in the second variant CH3 domain do not consist of Q347R, D399V, and F405T; (xviii) if the amino acid substitutions in the first variant CH3 domain consist of Q347R, D399V, and F405T, the amino acid substitutions in the second variant CH3 domain do not consist of K360E and K409W;(xix) if the amino acid substitutions in the first variant CH3 domain consist of K360E, K409W, and Y349C, then the amino acid substitutions in the second variant CH3 domain do not consist of Q347R, D399V, F405T, and S354C; (xx) if the amino acid substitutions in the first variant CH3 domain consist of Q347R, D399V, F405T, and S354C, then the amino acid substitutions in the second variant CH3 domain do not consist of K360E, K409W, and Y349C; and (xxi) if the amino acid substitutions in the first variant CH3 domain consist of (xxii) if the amino acid substitutions in the first variant CH3 domain consist of 351D, consist of 349E, consist of 349D, consist of 368E, consist of 368D, consist of 349E and 355E, consist of 349E and 355D, consist of 349D and 355E, or consist of 349D and 355D, (xxiii) if the amino acid substitution in the first variant CH3 domain consists of F405L, the amino acid substitution in the second variant CH3 domain does not consist of K409R; (xxiv) if the amino acid substitution in the first variant CH3 domain consists of K409R, the second variant the amino acid substitutions in the CH3 domain do not consist of F405L; (xxv) if the amino acid substitutions in the first variant CH3 domain consist of K360D, D399M, and Y407A, the amino acid substitutions in the second variant CH3 domain do not consist of E345R, Q347R, T366V, and K409V; (xxvi) if the amino acid substitutions in the first variant CH3 domain consist of E345R, Q347R, T366V, and K409V, the amino acid substitutions in the second variant CH3 domain do not consist of K360D, D399M, and Y407A;(xxvii) if the amino acid substitutions in the first variant CH3 domain consist of Y349S, K370Y, T366M, and K409V, then the amino acid substitutions in the second variant CH3 domain do not consist of E356G, E357D, S364Q, and Y407A; and (xxviii) if the amino acid substitutions in the first variant CH3 domain consist of E356G, E357D, S364Q, and Y407A, then the amino acid substitutions in the second variant CH3 domain do not consist of Y349S, K370Y, T366M, and K409V. In each of the foregoing, the substitution positions are according to EU numbering.

[0014] In some embodiments, a first variant CH3 domain polypeptide or a heavy chain polypeptide comprising the first variant CH3 domain polypeptide may comprise an amino acid substitution at one or more of the following amino acid positions according to EU numbering: 364, 366, 400, 407, and 409, and may optionally preferentially form a heterodimer with a second variant CH3 domain polypeptide or a heavy chain polypeptide comprising the second CH3 domain polypeptide that comprises one or more of the following amino acid positions according to EU numbering: 366, 368, 370, 399, 405, and 407.

[0015] In some embodiments, a first variant CH3 domain polypeptide or a heavy chain polypeptide comprising the first variant CH3 domain polypeptide may comprise an amino acid substitution at one or more of the following amino acid positions: 366, 368, 370, 399, 405, and 407, and may optionally preferentially form a heterodimer with a second variant CH3 domain polypeptide or a heavy chain polypeptide comprising the second CH3 domain polypeptide comprising one or more of the following amino acid positions: 364, 366, 400, 407, and 409. In some embodiments, a first variant CH3 domain polypeptide may comprise an amino acid substitution only at (I) one or more of the following amino acid positions: 364, 366, 400, 407, and 409 according to EU numbering, or (II) one or more of the following amino acid positions: 366, 368, 370, 399, 405, and 407 according to EU numbering.

[0016] In some embodiments, the amino acid substitutions in the first variant CH3 domain polypeptide are at: (i) 366, (ii) 368, (iii) 407, (iv) 366 and 407, (v) 366 and 368, (vi) 366 and 409, (vii) 368 and 370, (viii) 368 and 407, (ix) 399 and 405, (x) 400 and 409, (xi) 364, 366, and 409, (xii) 364, 407, and 409, (xiii) 366, 368, and 370, (xiv) 366, 368, and 407, (xv) 366, 399, and 405, xvi) 366th, 400th, and 409th, (xvii) 366th, 407th, and 409th, (xviii) 368th, 400th, and 409th, (xix) 399th, 405th, and 407th, (xx) 400th, 407th, and 409th, (xxi) 366th, 399th, 405th, and 407th, (xxii (xxiii) 366, 400, 407, and 409; (xxiv) 366, 368, 399, 405, and 407; or (xxv) 366, 368, 400, 407, and 409. In each of the foregoing, the substitution positions are according to EU numbering.

[0017] In some embodiments, the first variant CH3 domain polypeptide has one or more of the following: (i) the amino acid substitution in the first variant CH3 domain comprises or consists of an amino acid substitution at position 366, and optionally, the amino acid substitution in the second variant CH3 domain comprises or consists of an amino acid substitution at position 407, or at positions 366 and 407; (ii) the amino acid substitution in the first variant CH3 domain comprises or consists of an amino acid substitution at position 368, and optionally, the amino acid substitution in the second variant CH3 domain comprises or consists of an amino acid substitution at position 369; (iii) the amino acid substitution in the first variant CH3 domain comprises or consists of an amino acid substitution at position 407, and optionally, the amino acid substitution in the second variant CH3 domain comprises or consists of an amino acid substitution at position 366; (iv) the amino acid substitution in the first variant CH3 domain comprises or consists of an amino acid substitution at positions 366 and 407, and optionally, the amino acid substitution in the second variant CH3 domain comprises or consists of an amino acid substitution at position 366. (v) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366 and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 368, or 368 and 370; (vi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368 and 370, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366 and 409. (vii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 399 and 405, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 400 and 409; (viii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 400 and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 399 and 405.(ix) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 368, and 407; (x) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 407, and 409, and optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 368, and 370. (xi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 368, and 370, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 407, and 409; (xii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain (xiii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366 and 368, and optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 407, and 409; (xiv) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 407, and 409, and optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 407, and 409. (xv) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368 and 407, and optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 366, and 409; (xvi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally,(xvii) the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 368 and 407; (xvii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 400, 407, and 409, or at positions 366, 400, 407, and 409, or at positions 366, 368, 400, 407, and 409; i) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 400, 407, and 409, or at positions 366, 400, 407, and 409, or at positions 366, 368, 400, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, and 405; (xix) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 400, and 409. optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407; (xx) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407; (xxi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409; (xxii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, 405,and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 368, 400, and 409; (xxiii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366 and 368, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 407, and 409; (xxiv) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368, 400, and 409; (xxv) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368 and 407, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 366, and 409; (xxvi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366 and 368; (xxvii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 400, 407, and 409; (xxviii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 400, 407, and 409, or 366, 368, 400, 407, and 409, or 366, 368, 400, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, and 405;(xxix) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407; (xxx) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407, and optionally the amino acid substitutions in the second variant CH3 domain (xxxi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368, 400, and 409, and optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409; or (xxxii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409, and optionally, the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 368, 400, and 409. In each of the foregoing, the substitution positions are according to EU numbering.

[0018] In some embodiments, a first variant CH3 domain can include a substitution (to cysteine) at amino acid position 349 according to EU numbering that allows for a CH3 disulfide bond and can form a disulfide bond with another variant CH3 domain that includes an amino acid substitution (to cysteine) at amino acid position 354 according to EU numbering.

[0019] In some embodiments, a first variant CH3 domain can include an amino acid substitution (to cysteine) at amino acid position 354 according to EU numbering that allows for a CH3 disulfide bond and can form a disulfide bond with another variant CH3 domain that includes an amino acid substitution (to cysteine) at amino acid position 349 according to EU numbering.

[0020] In certain embodiments, the first variant CH3 domain polypeptide may comprise one or more of the following amino acid substitutions: Y349C, S354C, S364D, S364L, T366Q, T366R, T366S, T366V, T366W, L368A, L368F, L368S, L368I, K370G, K370Y, D399Q, S400T, F405L, Y407V, Y407G, K409R, K409L, and / or K409G, where in each of the foregoing, the substitution positions are according to EU numbering. In certain embodiments, the first variant CH3 domain polypeptide comprises one of the following sets of amino acid substitutions: (i) T366V, (ii) T366W, (iii) L368F, (iv) Y407V, (v) T366V and L368F, (vi) T366S and Y407G, (vii) T366Q and K409R, (viii) T366R_K409G, (ix) L368F and K370G, (x) L3 68I and Y407G, (xi) S400T and K409L, (xii) D399Q and F405L, (xiii) S364D, Y407V, and K409G, (xiv) S364L, T366W, and K409G, (xv) T366V, L368S, and K370Y, (xvi) T366S, L368I, and Y407G, (xvii) T366V, D399Q, and F405L, (xviii) T3 66W, D399Q, and F405L, (xix) T366V, S400T, and K409L, (xx) T366W, S400T, and K409L, (xxi) T366Q, Y407V, and K409R, (xxii) L368F, S400T, and K409L, (xxiii) D399Q, Y407V, and F405L, (xxiv) S400T, Y407V, and K409L, (xxv) T366S , D399Q, F405L, and Y407G, (xxvi) T366Q, D399Q, F405L, and K409R, (xxvii) T366S, S400T, Y407G, and K409L, (xxviii) T366S, L368A, D399Q, Y407V, and F405L, or (xxix) T366S, L368A, S400T, Y407V, and K409L. Optionally, the first variant CH3 domain polypeptide may further comprise Y349C or S354C.In each of the foregoing, the substitution positions follow EU numbering.

[0021] In certain embodiments, the first variant CH3 domain polypeptide comprises: (i) the amino acid substitution in the first variant CH3 domain comprises or consists of T366V, and optionally the amino acid substitution in the second variant CH3 domain comprises or consists of Y407V; (ii) the amino acid substitution in the first variant CH3 domain comprises or consists of T366W, and optionally the amino acid substitution in the second variant CH3 domain comprises or consists of T366S and Y407G; (iii) the first variant CH3 domain comprises or consists of T366S and Y407G; (iv) the amino acid substitution in the first variant CH3 domain comprises or consists of L368F, and optionally the amino acid substitution in the second variant CH3 domain comprises or consists of T366Q and K409R; (iv) the amino acid substitution in the first variant CH3 domain comprises or consists of Y407V, and optionally the amino acid substitution in the second variant CH3 domain comprises or consists of T366V; (v) the amino acid substitution in the first variant CH3 domain comprises or consists of T366S and Y407G. (vi) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366Q and K409R, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of L368F; (vii) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366R and K409G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of L368F. (viii) the amino acid substitutions in the first variant CH3 domain comprise or consist of L368F and K370G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366R and K409G; (ix) the amino acid substitutions in the first variant CH3 domain comprise or consist of S400T and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of D399Q and F405L;(x) the amino acid substitutions in the first variant CH3 domain comprise or consist of D399Q and F405L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of S400T and K409L; (xi) the amino acid substitutions in the first variant CH3 domain comprise or consist of S364D, Y407V, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366V, L368S, and K370Y; (xii) in the first variant CH3 domain (xiii) the amino acid substitutions in the first variant CH3 domain comprise or consist of S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366S, L368I, and Y407G; (xiv) the amino acid substitutions in the first variant CH3 domain comprise or consist of S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366S, L368I, and Y407G; (xv) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366S, L368A, Y407V, D399Q, and F405L; (xvi) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of S364L, T366W, and K409G. (xvii) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366S, Y407G, D399Q, and F405L; (xvii) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366S, Y407G, D399Q, and F405L;(xviii) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366S, Y407G, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366W, S400T, and K409L; (xix) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366W, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain comprise T366S, L368A, Y407V, S40 (xx) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366S, L368A, Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366W, D399Q, and F405L; (xxi) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366W, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366W, D399Q, and F405L. (xxii) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366S, Y407G, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366W, D399Q, and F405L; (xxiii) the amino acid substitutions in the first variant CH3 domain comprise or consist of Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of Y407V, S400T, and K409L. (xxiv) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of Y407V, S400T, and K409L; (xxv) the amino acid substitutions in the first variant CH3 domain comprise or consist of Y407V, D399Q, and F405L, and optionally(xxvi) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of Y407V, D399Q, and F405L; (xxvii) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366Q, K409R, D399Q, and F405L. (xxviii) the amino acid substitutions in the first variant CH3 domain comprise or consist of L368F, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366Q, K409R, D399Q, and F405L; (xxix) the first variant CH3 domain comprise or consist of L368F, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366Q, K409R, D399Q, and F405L. (xxx) the amino acid substitutions in the first variant CH3 domain comprise or consist of Y407V, T366Q, and K409R, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of T366V and L368F; (xxxi) the amino acid substitutions in the first variant CH3 domain comprise or consist of T366V and L368F, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of Y407V, T366Q, and K409R. (xxxii) the amino acid substitutions in the first variant CH3 domain comprise or consist of S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of L368I and Y407G, or (xxxii) the amino acid substitutions in the first variant CH3 domain comprise or consist of L368I and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of S364L, T366W, and K409G. In each of the foregoing, the substitution positions are according to EU numbering.

[0022] In certain embodiments, the first variant CH3 domain polypeptide may further comprise Y349C according to EU numbering, and optionally, the second variant CH3 domain polypeptide may further comprise S354C according to EU numbering.

[0023] In certain embodiments, the first variant CH3 domain polypeptide may comprise S354C according to EU numbering, and optionally, the second variant CH3 domain polypeptide may further comprise Y349C according to EU numbering.

[0024] In some preferred embodiments, the first variant CH3 domain polypeptide comprises: (1) the amino acid substitution in the first variant CH3 domain consists of T366W, and optionally, the amino acid substitution in the second variant CH3 domain consists of T366S and Y407G; (2) the amino acid substitution in the first variant CH3 domain consists of T366S and Y407G, and optionally, the amino acid substitution in the second variant CH3 domain consists of T366W; and (3) the amino acid substitution in the first variant CH3 domain consists of S364L, T366W, and K. (3) the amino acid substitutions in the first variant CH3 domain consist of T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain consist of S364L, T366W, and K409G; (4) the amino acid substitutions in the first variant CH3 domain consist of T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain consist of S364L, T366W, and K409G; (5) the amino acid substitutions in the first variant CH3 domain consist of S354C and T366W, and optionally the amino acid substitutions in the second variant CH3 domain consist of Y349C, T (6) the amino acid substitutions in the first variant CH3 domain consist of S354C, T366S, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain consist of Y349C and T366W; (7) the amino acid substitutions in the first variant CH3 domain consist of S354C, S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain consist of Y349C, T366S, L368I, and Y407G; (8) the amino acid substitutions in the first variant CH3 domain consist of S354C, S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain consist of Y349C, T366S, L368I, and Y407G; (9) the amino acid substitutions in the first variant CH3 domain consist of S354C, T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain consist of Y349C, S364L, T366W, and K409G; (10) the amino acid substitutions in the first variant CH3 domain consist of Y349C, T366S, and Y407G;Optionally, the amino acid substitutions in the second variant CH3 domain consist of S354C, S354C, and T366W; (11) the amino acid substitutions in the first variant CH3 domain consist of Y349C, S364L, T366W, and K409G; optionally, the amino acid substitutions in the second variant CH3 domain consist of S354C, T366S, L368I, and Y407G; (12) the amino acid substitutions in the first variant CH3 domain consist of Y349C, T366S, L368I, and Y407G; optionally, the amino acid substitutions in the second variant CH3 domain consist of S354C, S364L, T366W, and K409G. In each of the foregoing, the substitution positions are according to EU numbering.

[0025] In further embodiments, the first variant CH3 domain polypeptide may comprise an amino acid sequence according to SEQ ID NO: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, or 161, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, or 162, respectively.

[0026] In further embodiments, the first variant CH3 domain polypeptide may comprise an amino acid sequence according to SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, or 162, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, or 161, respectively.

[0027] In further embodiments, the first variant CH3 domain polypeptide may comprise an amino acid sequence according to SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, or 163, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, or 164, respectively.

[0028] In further embodiments, the first variant CH3 domain polypeptide may comprise an amino acid sequence according to SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, or 164, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, or 163, respectively.

[0029] In further embodiments, the first variant CH3 domain polypeptide may comprise an amino acid sequence according to SEQ ID NO: 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, or 165, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 154, or 164, respectively.

[0030] In further embodiments, the first variant CH3 domain polypeptide may comprise an amino acid sequence according to SEQ ID NO: 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 154, or 164, and optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, or 165, respectively. In some preferred embodiments, any one of the foregoing first variant CH3 domain polypeptides comprises an amino acid sequence according to SEQ ID NO: 11 or 71, and optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 12 or 72, respectively.

[0031] In some preferred embodiments, the first variant CH3 domain polypeptide of any one of the foregoing comprises an amino acid sequence according to SEQ ID NO: 12 or 72, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 11 or 71, respectively.

[0032] In some preferred embodiments, the first variant CH3 domain polypeptide of any one of the foregoing comprises an amino acid sequence according to SEQ ID NO: 13 or 73, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 14 or 74, respectively.

[0033] In some preferred embodiments, the first variant CH3 domain polypeptide of any one of the foregoing comprises an amino acid sequence according to SEQ ID NO: 14 or 74, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 13 or 73, respectively.

[0034] In some preferred embodiments, any one of the foregoing first variant CH3 domain polypeptides comprises an amino acid sequence according to SEQ ID NO: 15 or 75, and optionally, a second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 16 or 76, respectively; or

[0035] In some preferred embodiments, the first variant CH3 domain polypeptide of any one of the foregoing comprises an amino acid sequence according to SEQ ID NO: 16 or 76, and optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 15 or 75, respectively.

[0036] While various variant CH3 domains are described above, any polypeptide, such as a heavy chain polypeptide, that comprises any of such variant CH3 domains is encompassed by the present invention.

[0037] In one aspect, provided herein is a polypeptide comprising a variant CH3 domain polypeptide or a heavy chain polypeptide comprising the variant CH3 domain polypeptide, wherein the variant CH3 domain in the polypeptide can be any of the first variant CH3 domain or the second variant CH3 domain described above.

[0038] In some of the foregoing embodiments, the polypeptide may be an immunoglobulin polypeptide.

[0039] The polypeptide may further comprise (i) an antigen-binding domain, (ii) an immunoglobulin heavy chain constant region 1 (CH1) domain or a variant CH1 domain, (iii) an immunoglobulin heavy chain constant region 2 (CH2) domain or a variant CH2 domain, and / or (iv) an immunoglobulin light chain constant region (CL) domain.

[0040] Optionally, the polypeptide may satisfy any of the following: (ii) the CH1 domain comprises a wild-type CH1 amino acid sequence or comprises one or more amino acid substitutions or modifications compared to the wild-type CH1 amino acid sequence; (iii) the CH2 domain comprises a wild-type CH2 amino acid sequence or comprises one or more amino acid substitutions or modifications compared to the wild-type CH2 amino acid sequence; and / or (iv) the CL domain comprises a wild-type CL amino acid sequence or comprises one or more amino acid substitutions or modifications compared to the wild-type CL amino acid sequence.

[0041] In some embodiments, where the polypeptide comprises an antigen-binding domain, the antigen-binding domain may comprise an immunoglobulin heavy chain variable region ("VH") domain, an immunoglobulin light chain variable region ("VL") domain, a single-chain fragment variable region ("scFv"), a nanobody, or a combination thereof.

[0042] In some embodiments, the polypeptide comprises, in addition to the first variant CH3 domain, optionally, in N-terminal to C-terminal direction: (i) a VH domain; (ii) a VH domain and a CH1 domain; (iii) a VH domain and a CH2 domain; (iv) a VH domain, a CH1 domain, and a CH2 domain; (v) a VH domain and a CL domain; (vi) a VH domain, a CL domain, and a CH2 domain; (vii) a VL domain; (viii) a VL domain and a CH1 domain; (ix) a VL domain and a CH2 domain, (x) a VL domain, a CH1 domain, and a CH2 domain, (xi) a VL domain and a CL domain, (xii) a VL domain, a CL domain, and a CH2 domain, (xiii) an scFv, (xiv) an scFv domain and a CH1 domain, (xv) an scFv domain and a CH2 domain, (xvi) an scFv, a CH1 domain, and a CH2 domain, (xvii) an scFv domain and a CL domain, or (xviii) an scFv, a CL domain, and a CH2 domain.

[0043] In some embodiments, a polypeptide may satisfy any of the following: (I) comprise a VH domain and be bound to or paired with another polypeptide comprising a VL domain, wherein the VH domain and the VL domain form an antigen-binding site; or (II) comprise a VL domain and be bound to or paired with another polypeptide comprising a VH domain, wherein the VL domain and the VH domain form an antigen-binding site.

[0044] In another aspect, provided herein is a molecule comprising at least a first polypeptide and a second polypeptide according to any of the foregoing.

[0045] In some embodiments, (A) a first polypeptide may comprise any of the first variant CH3 domains described above, and (B) a second polypeptide may comprise any of the second variant CH3 domains described above. The first and second variant CH3 domains may differ by at least one amino acid. The first and second polypeptides are optionally linked or paired to each other via a disulfide bond.

[0046] In some embodiments, the molecules may comprise one or more of the following features: (A) the first polypeptide may further comprise a first antigen-binding domain; (B) the second polypeptide may further comprise a second antigen-binding domain; (C) the heteromeric molecule may further comprise a third polypeptide that may optionally comprise a third antigen-binding domain, optionally, the third polypeptide may be bound to or paired with the first polypeptide; and / or (D) the heteromeric molecule may further comprise a fourth polypeptide that may optionally comprise a fourth antigen-binding domain, optionally, the fourth polypeptide may be bound to or paired with the second polypeptide.

[0047] In some embodiments, the heteromeric molecule may include one or more of the following characteristics (I)-(II):

[0048] Feature (I): (I-1-i) the first polypeptide may comprise a first antigen-binding domain capable of forming a first antigen-binding site specific for a first epitope, and / or (I-1-ii) the heteromeric molecule may comprise a third polypeptide comprising a third antigen-binding domain capable of forming a third antigen-binding site specific for a third epitope, or (I-2) the first polypeptide may comprise a first antigen-binding domain and the heteromeric molecule may comprise a third polypeptide comprising a third antigen-binding domain, and the first antigen-binding domain and the third antigen-binding domain may form a first antigen-binding site specific for a first epitope.

[0049] Feature (II): (II-1-i) the second polypeptide may comprise a second antigen-binding domain that can form a second antigen-binding site specific for a second epitope, and / or (I-1-ii) the heteromeric molecule may comprise a fourth polypeptide that comprises a fourth antigen-binding domain that forms a fourth antigen-binding site specific for a fourth epitope, or (II-2) the second polypeptide may comprise a second antigen-binding domain and the heteromeric molecule may comprise a fourth polypeptide that comprises a fourth antigen-binding domain, and the second antigen-binding domain and the fourth antigen-binding domain may form a second antigen-binding site specific for a second epitope.

[0050] Optionally, the first epitope, the second epitope, the third epitope, and / or the fourth epitope are individually the same as or different from each other. In certain embodiments, the first epitope, the second epitope, the third epitope, and / or the fourth epitope may all be different from each other. In certain embodiments, the first epitope, the second epitope, the third epitope, and / or the fourth epitope may all be the same. In certain embodiments, the first epitope is the same as the third epitope, which may be different from the second epitope and / or the fourth epitope. In certain embodiments, a molecule may comprise a first antigen-binding site specific for a first epitope (e.g., formed by a first antigen-binding domain and a third antigen-binding domain) and a second antigen-binding site specific for a second epitope (e.g., formed by a second antigen-binding domain and a fourth antigen-binding domain) (in certain cases, the third antigen-binding domain and the fourth antigen-binding domain may be absent), wherein the first and second epitopes may be different from each other, optionally making the molecule a bispecific antibody or antigen-binding antibody fragment.

[0051] In some embodiments, the (A) first polypeptide may further comprise: (i) a first antigen-binding domain specific for a first epitope, (ii) a first CH1 domain or a first variant CH1 domain, (iii) a first CH2 domain or a first variant CH2 domain, and / or (iv) a first CL domain. Optionally, in (ii), the first CH1 domain may comprise a wild-type CH1 amino acid sequence or may comprise one or more amino acid substitutions compared to the wild-type CH1 amino acid sequence. Optionally, in (iii), the first CH2 domain may comprise a wild-type CH2 amino acid sequence or may comprise one or more amino acid substitutions compared to the wild-type CH2 amino acid sequence. Optionally, in (iv), the first CL domain may comprise a wild-type CL amino acid sequence or may comprise one or more amino acid substitutions compared to the wild-type CL amino acid sequence.

[0052] In some embodiments, the (B) second polypeptide may further comprise (i) a second antigen-binding domain specific for a second epitope, (ii) a second CH1 domain or a second variant CH1 domain, (iii) a second CH2 domain or a second variant CH2 domain, and / or (iv) a second CL domain. Optionally, in (ii), the second CH1 domain may comprise a wild-type CH1 amino acid sequence or may comprise one or more amino acid substitutions compared to the wild-type CH1 amino acid sequence. Optionally, in (iii), the second CH2 domain may comprise a wild-type CH2 amino acid sequence or may comprise one or more amino acid substitutions compared to the wild-type CH2 amino acid sequence. Optionally, in (iv), the second CL domain may comprise a wild-type CL amino acid sequence or may comprise one or more amino acid substitutions compared to the wild-type CL amino acid sequence.

[0053] In some embodiments, (A) the first antigen-binding domain may be a VH domain, a VL domain, an scFv, a nanobody, or a combination thereof, and / or (B) the second antigen-binding domain may be a VH domain, a VL domain, an scFv, a nanobody, or a combination thereof.

[0054] Optionally, when the first antigen-binding domain and the third antigen-binding domain form an antigen-binding site specific for a first epitope (e.g., in embodiment (I-2) above), (i) the first antigen-binding domain may be a VH and the third antigen-binding domain may be a VL, or (ii) the first antigen-binding domain may be a VL and the third antigen-binding domain may be a VH. Further, optionally, when the second antigen-binding domain and the fourth antigen-binding domain form an antigen-binding site specific for a second epitope (e.g., in embodiment (II-2) above), (i) the second antigen-binding domain is a VH and the fourth antigen-binding domain is a VL, or (ii) the second antigen-binding domain is a VL and the fourth antigen-binding domain is a VH.

[0055] In some embodiments, the molecule may be a multispecific antibody or antigen-binding antibody fragment, and optionally the multispecific antibody or antigen-binding antibody fragment may comprise a structure shown in any one of Figures 2-8, and optionally the multispecific antibody or antigen-binding antibody fragment comprises an IgG, and further optionally an IgG1, IgG2, IgG3, or IgG4.

[0056] Thus, in yet another aspect, multispecific antibodies and antigen-binding antibody fragments are also provided herein, where optionally the multispecific antibody or antigen-binding antibody fragment comprises IgG, and further optionally IgG1, IgG2, IgG3, or IgG4. The multispecific antibody or antigen-binding antibody fragment may be obtained by any of the molecules comprising a first polypeptide and a second polypeptide described above, and further optionally may be bispecific.

[0057] In yet another aspect, provided herein are polynucleotides encoding any of the foregoing.

[0058] In some embodiments, such polynucleotide(s) may encode (i) any of the first variant CH3 domain polypeptides described above or heavy chain polypeptides comprising such first variant CH3 domain polypeptides, (ii) any of the polypeptides described above, (iii) any of the molecules described above, and / or (iv) any of the multispecific antibodies or antigen-binding antibody fragments described above.

[0059] In yet another aspect, provided herein is a vector comprising any of the polynucleotide(s) described above.

[0060] In yet another aspect, provided herein is a cell that may comprise (i) any of the first variant CH3 domain polypeptides described above, (ii) any of the polypeptides described above, (iii) any of the molecules described above, (iv) any of the multispecific antibodies or antigen-binding antibody fragments described above, (v) any of the polynucleotide(s) described above, and / or (vi) any of the vectors described above.

[0061] In yet another aspect, provided herein are compositions comprising any of the foregoing.

[0062] In some embodiments, a composition may comprise (I) (i) any of the above-described first variant CH3 domains or heavy chain polypeptides comprising such first variant CH3 domain polypeptides, (ii) any of the above-described polypeptides, (iii) any of the above-described molecules, (iv) any of the above-described multispecific antibodies or antigen-binding antibody fragments, (v) any of the above-described polynucleotide(s), (vi) any of the above-described vectors, and / or (vii) any of the above-described cells; and (II) a pharmaceutically or diagnostically acceptable carrier.

[0063] In another aspect, provided herein are methods for generating CH3 domain libraries, which can be, for example, CH3 domain-encoding polynucleotide libraries or CH3 domain polypeptide libraries, and libraries generated using such methods.

[0064] In some embodiments, methods for generating a CH3 domain-encoding polynucleotide library can include incorporating mutations or randomizing the nucleic acid at one or more predetermined nucleotide positions within a CH3 domain-encoding polynucleotide, either in silico or in vitro, wherein the one or more predetermined nucleotide positions are within a codon encoding an amino acid at one or more predetermined CH3 domain positions.

[0065] In some embodiments, one or more of the predetermined CH3 domain positions may be located at or near the CH3-CH3 interface. In some cases, a CH3 domain position may be considered to be located at or near the CH3-CH3 interface if the amino acid residue at the CH3 domain position meets the following criteria: (i-1) a side-chain solvent-exposed surface area (SASA) of 15% or more in the CH3 monomer (i.e., the CH3 is not paired with another CH3 domain), (i-2) in the CH3-CH3 dimer form, there are about 8.2 Å or less of the paired CH3 domain atoms, and (i-3) in the CH3-CH3 dimer form, the residue is not facing away from the paired CH3 domain or is not solvent-exposed. In some embodiments, one or more of the predetermined CH3 domain positions may be predicted to affect CH3-CH3 interactions. In some cases, such predictions may be made in silico or in vitro. In some cases, a CH3 domain position can be predicted to influence CH3-CH3 interactions if the CH3 domain position meets the following criteria: (ii-1) it is the first, second, or third adjacent position to a CH3 domain position considered to be at the CH3-CH3 interface, and / or (ii-2) in the CH3-CH3 dimer form, it has an interchain beta carbon-beta carbon distance of about 8.2 Å or less (in the case of glycine, the alpha carbon was used instead) from the CH3 domain position of the paired CH3 domain that is at the CH3-CH3 interface.

[0066] In some embodiments, one or more of the predetermined CH3 domain positions may be selected from positions 364, 366, 368, 370, 399, 400, 405, 407, and / or 409 according to EU numbering.

[0067] In some embodiments, one or more predetermined CH3 domain positions may be selected from CH3-CH3 interface positions, which are defined as amino acid positions where (1) the amino acid side chain solvent-exposed surface area (SASA) in the CH3 monomer is 15% or greater, (2) the interchain distance is 8.2 Å or less, and (3) the amino acid side chain is not pointing away from the partner chain or is not solvent exposed (criteria assessed by manual inspection).

[0068] In some embodiments, incorporating mutations at one or more predetermined nucleotide positions or randomizing the nucleic acid at those positions can include introducing a degenerate codon, optionally a degenerate RMW codon representing the six naturally occurring amino acids (D, T, A, E, K, and N), or a degenerate NNK codon representing all 20 naturally occurring amino acid residues.

[0069] In some embodiments, a CH3 domain-encoding polynucleotide library can be used to identify one or more sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides, where the first variant CH3 domain polypeptide preferentially forms heterodimers with a second variant CH3 domain polypeptide that differs from the first variant CH3 domain polypeptide by at least one amino acid.

[0070] In some embodiments, the one or more predetermined CH3 domain positions are: (i) 366, (ii) 366 and 407, (iii) 364, 366, and 409, (iv) 366, 368, and 407, (v) 368, (vi) 407, (vii) 366 and 368, (viii) 366 and 409, (ix) 368 and 370, (x) 368 and 407, (xi) 399 and 405, (xii) 400 and 409, (xiii) 364, 407, and 409, (xiv) 366, 368, and 370, (xv) 366, 399, and 405, (xvi) 366, 400, and 409, (xvii) 366, 407, and 409, (xviii) 368, 400, and 409, (xix) 399, 405, and 407, (xx) 400, 407, and 409, (xxi) 366, 399, 405, and 407, (xxii) 366, 399, 405, and 409, (xxiii) 366, 400, 407, and 409, (xxiv) 366, 368, 399, 405, and 407, or (xxv) 366, 368, 400, 407, and 409, wherein in each of the foregoing, the substitution positions are according to EU numbering.

[0071] In some embodiments, CH3 domain-encoding polynucleotide libraries generated by any of the foregoing methods are also encompassed by the present disclosure.

[0072] In some embodiments, a method for generating a CH3 domain polypeptide library may include obtaining, in silico or in vitro, a plurality of CH3 domain polypeptides corresponding to a plurality of CH3 domain-encoding polynucleotides from any of the above-described CH3 domain-encoding polynucleotide libraries.

[0073] Alternatively, in some embodiments, a method for generating a CH3 domain polypeptide library can include incorporating substitutions in silico or in vitro at one or more predetermined CH3 domain amino acid positions in a plurality of CH3 domain polypeptides.

[0074] In such embodiments, one or more of the one or more predetermined CH3 domain amino acid positions (i) may be at or near a CH3-CH3 interface, and optionally, a CH3 domain position is considered to be at or near a CH3-CH3 interface if the amino acid residue at the CH3 domain position satisfies the following: (i-1) in the CH3 monomer form, the side chain solvent-accessible surface area (SASA) is 15% or greater; (i-2) in the CH3-CH3 dimer form, there are about 8.2 Å or less of the atoms of the paired CH3 domain; and (i-3) in the CH3-CH3 dimer form, the residue does not point away from the paired CH3 domain; and (ii) may be predicted to affect CH3-CH3 interactions. Optionally, the prediction is made in silico or in vitro, and further optionally, a CH3 domain position is predicted to influence CH3-CH3 interactions if it satisfies the following: (ii-1) it is in the first, second, or third adjacent position to a CH3 domain position present at the CH3-CH3 interface, and / or (ii-2) in the CH3-CH3 dimer form, it has an interchain beta carbon-beta carbon distance of about 8.2 Å or less from the CH3 domain position of the paired CH3 domain present at the CH3-CH3 interface; and / or (iii) it may be selected from positions 364, 366, 368, 370, 399, 400, 405, 407, and / or 409 according to EU numbering.

[0075] Optionally, such a CH3 domain polypeptide library can be for identifying one or more sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides, wherein the first variant CH3 domain polypeptide preferentially forms heterodimers with a second variant CH3 domain polypeptide that differs from the first variant CH3 domain polypeptide by at least one amino acid, optionally by at least one amino acid substitution.

[0076] In some embodiments, the CH3 domains of the CH3 domain library can comprise a predetermined number of CH3 domain amino acid substitutions. In certain embodiments, the predetermined number can be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more; 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less; 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4; 1 to 3; 1 to 2; and / or 1, 2, 3, 4, or 5.

[0077] In some embodiments, the one or more predetermined CH3 domain positions are: (i) 366, (ii) 366 and 407, (iii) 364, 366, and 409, (iv) 366, 368, and 407, (v) 368, (vi) 407, (vii) 366 and 368, (viii) 366 and 409, (ix) 368 and 370, (x) 368 and 407, (xi) 399 and 405, (xii) 400 and 409, (xiii) 364, 407, and 409, (xiv) 366, 368, and 370, (xv) 366, 399, and 405, (xvi) 366, 400, and and 409, (xvii) 366, 407, and 409, (xviii) 368, 400, and 409, (xix) 399, 405, and 407, (xx) 400, 407, and 409, (xxi) 366, 399, 405, and 407, (xxii) 366, 399, 405, and 409, (xxiii) 366, 400, 407, and 409, (xxiv) 366, 368, 399, 405, and 407, or (xxv) 366, 368, 400, 407, and 409, wherein in each of the foregoing, the substitution positions are according to EU numbering.

[0078] CH3 domain polypeptide libraries produced by any of the foregoing methods are also encompassed by the present disclosure.

[0079] In another aspect, provided herein is a method of identifying one or more sets of a first variant CH3 domain polypeptide and a second variant CH3 domain polypeptide, wherein the first variant CH3 domain polypeptide preferentially forms heterodimers with the second variant CH3 domain polypeptide.

[0080] In some embodiments, the method comprises the steps of: (a) providing (a-1) a first polypeptide or set of first polypeptides, each comprising a wild-type or variant CH3 domain, and (a-2) a second set of polypeptides or a plurality of sets of second polypeptides, each comprising a wild-type or variant CH3 domain, optionally wherein the plurality of sets of (a-1) and (a-2) are provided in silico or in vitro; and (b) quantifying binding or pairing preferences (e.g., predicted binding strengths) between the CH3 domains of the first and second polypeptides in one or more of the plurality of sets of step (a), optionally wherein quantification is performed in silico and / or in vitro, and further optionally wherein quantification is performed by liquid chromatography-mass spectrometry. and (c) selecting one or more sets of first and second variant CH3 domains that provide a desired binding or pairing preference, optionally a desired binding strength and / or a desired percentage of heterodimers, and optionally a similar or greater binding or pairing preference and / or percentage of CH3 domain heterodimers compared to a reference set of variant CH3 domain polypeptides. The first and second variant CH3 domains may optionally differ by at least one amino acid, optionally one amino acid substitution, at a predetermined CH3 domain position.

[0081] In some embodiments, the CH3 domain polypeptide of the first polypeptide may be obtained from a first CH3 domain polypeptide library according to any of the CH3 domain polypeptide libraries described herein, or may be expressed from a first CH3 domain library according to any of the CH3 domain-encoding polynucleotide libraries disclosed herein.

[0082] In some embodiments, the CH3 domain polypeptide of the second polypeptide may be obtained from a second CH3 domain polypeptide library according to any of the CH3 domain polypeptide libraries described herein, or may be expressed from a second CH3 domain-encoding polynucleotide library according to any of the CH3 domain libraries disclosed herein.

[0083] Optionally, (I) the CH3 domain of the first polypeptide may comprise the substitution S354C and the variant CH3 domain of the second polypeptide may comprise the substitution Y349C. Alternatively, (II) the CH3 domain of the first polypeptide may comprise the substitution Y349C and the variant CH3 domain of the second polypeptide may comprise the substitution S354C.

[0084] In some embodiments, when the CH3 domain polypeptide of the first polypeptide is obtained from the above-described first CH3 domain polypeptide library or expressed from the above-described first CH3 domain-encoding polynucleotide library, the CH3 domain polypeptide of the second polypeptide is obtained from the above-described second CH3 domain polypeptide library or expressed from the above-described second CH3 domain-encoding polynucleotide library, and the combination of one or more predetermined CH3 domain positions in the first library and the second library can be, for example, as follows:

[0085] In certain embodiments of the method, the one or more predetermined CH3 domain positions of the first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of position 366, and the one or more predetermined CH3 domain positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of position 407.

[0086] In certain embodiments, one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or one or more domain-encoding polynucleotide libraries may include or consist of position 366, and one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 407.

[0087] In certain embodiments, one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or one or more domain-encoding polynucleotide libraries may include or consist of positions 368, and one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 409.

[0088] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may comprise or consist of position 407, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may comprise or consist of position 366.

[0089] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 407, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of position 366.

[0090] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 409, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of position 368.

[0091] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 409, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 368 and 370.

[0092] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 368 and 370, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 409.

[0093] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 400 and 409, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 399 and F405.

[0094] In certain embodiments, the one or more predetermined CH3 domain positions of the first CH3 domain library may include or consist of positions 399 and 405, and the one or more predetermined CH3 domain polypeptide or domain-encoding polynucleotide positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 400 and 409.

[0095] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 364, 407, and 409, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366, 368, and 370.

[0096] In certain embodiments, the one or more predetermined CH3 domain positions of the first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366, 368, and 370, and the one or more predetermined CH3 domain positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 364, 407, and 409.

[0097] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 364, 366, and 409, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366, 368, and 407.

[0098] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366, 368, and 407, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 364, 366, and 409.

[0099] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 368, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366, 407, and 409.

[0100] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366, 407, and 409, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366 and 368.

[0101] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 368 and 407, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 364, 366, and 409.

[0102] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain library polypeptide or domain-encoding polynucleotide may include or consist of positions 364, 366, and 409, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 368 and 407.

[0103] In certain embodiments, the one or more predetermined CH3 domain positions of a first CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 366, 399, and 405, and the one or more predetermined CH3 domain positions of a second CH3 domain polypeptide or domain-encoding polynucleotide library may include or consist of positions 400, 407, and 409; positions 366, 400, 407, and 409; or positions 366, 368, 400, 407, and 409.

[0104] In certain embodiments, the one or more predetermined CH3 domain positions of the first CH3 domain polypeptide or domain-encoding polynucleotide library comprise or consist of: positions 400, 407, and 409; positions 366, 400, 407, and 409; or positions 366, 368, 400, 407, and 409, and the one or more predetermined CH3 domain positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library comprise: positions 366, 399, and 405.

[0105] In certain embodiments, the one or more predetermined CH3 domain positions of the first CH3 domain polypeptide or domain-encoding polynucleotide library comprise or consist of positions 366, 400, and 409, and the one or more predetermined CH3 domain positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library comprise positions 399, 405, and 407; positions 366, 399, 405, and 407; or positions 366, 368, 399, 405, and 407.

[0106] In certain embodiments, the one or more predetermined CH3 domain positions of the first CH3 domain polypeptide or domain-encoding polynucleotide library include or consist of positions 399, 405, and 407; positions 366, 399, 405, and 407; or positions 366, 368, 399, 405, and 407, and the one or more predetermined CH3 domain positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library include or consist of positions 366, 400, and 409.

[0107] In certain embodiments, the one or more predetermined CH3 domain positions of the first CH3 domain polypeptide or domain-encoding polynucleotide library include or consist of positions 368, 400, and 409, and the one or more predetermined CH3 domain positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library include or consist of positions 366, 399, 405, and 409.

[0108] In certain embodiments, the one or more predetermined CH3 domain positions of the first CH3 domain polypeptide or domain-encoding polynucleotide library include or consist of positions 366, 399, 405, and 409, and the one or more predetermined CH3 domain positions of the second CH3 domain polypeptide or domain-encoding polynucleotide library include or consist of positions 368, 400, and 409.

[0109] In each of the foregoing, the substitution positions follow EU numbering.

[0110] In certain embodiments of the method, (a-1) the first polypeptide may comprise or be linked to a first label, and (a-2) the second polypeptide may comprise or be linked to a second label. In some cases, the quantifying step (b) may comprise detecting the first label and / or the second label.

[0111] In certain embodiments of the method, the quantification step (b) may comprise at least one of liquid chromatography-mass spectrometry (LC-MS), AlphaLISA®, ion exchange chromatography (IEX), and / or flow cytometry.

[0112] In certain embodiments, the identification method may further comprise selecting one or more sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides based on one or more properties of the antibody comprising the set of first and second polypeptides selected in step (c).Exemplary properties include (i) (i-1) production yield, optionally assessed in one or more cell types, optionally mammalian cells, e.g., Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK) cells, yeast cells, insect cells, and / or plant cells, and / or (i-2) suitability for one or more antibody purification methods, optionally including Protein A affinity purification; (ii) the degree of aggregation, optionally quantified using chromatography, optionally size exclusion chromatography (SEC) or electrophoresis, optionally SDS-PAGE, and optionally the presence of multimers of full-sized antibodies; and (iii) the CH1 domain, optionally assessed using LC-MS. (iv) melting temperature (Tm) and / or aggregation temperature (Tagg), optionally measured using differential scanning fluorometry (DSF) and / or differential scanning calorimetry (DSC) and / or an instrument, optionally using Uncle®, optionally Tagg266; (v) isoelectric point ("pI"); (vi) level of interaction with a multispecific reagent ("PSR"), optionally measured by the method described in WO2014 / 179363; (vii) optionally using hydrophobic interaction chromatography ("HIC"), optionally using Estep; P, et al. MAbs. 2015 May-Jun;7(3):553-561, (viii) optionally, self-interactions measured by (viii-1) affinity capture self-interaction nanoparticle spectroscopy (AC-SINS), optionally as described in Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92, or (viii-2) dynamic light scattering (DLS), (ix) stability to high or low pH stress, (x) solubility, (xi) production cost and / or time, (xii) other stability parameters, (xiii) shelf life, (xiv) in vivo half-life, and / or (xv) immunogenicity.In some embodiments, any such properties may depend on (a) the particular structure of the molecule or multispecific antibody or antigen-binding antibody fragment structure incorporating the variant CH3 domain set, and / or (b) the variable domains that provide the particular binding specificity. Thus, in some cases, when attempting to design a multispecific antibody or antigen-binding antibody fragment with a particular / given antigen specificity, multiple variant CH3 domain sets may be tested in a particular antibody or antibody fragment structure and / or antigen specificity setting.

[0113] Thus, in another aspect, provided herein is a method of screening for a set of first and second variant CH3 domain polypeptides suitable for a multispecific antibody or antigen-binding antibody fragment with a given antigen specificity (e.g., having any of the structures described herein).

[0114] In some embodiments, the method comprises: (a) expressing a plurality of multispecific antibodies and / or antigen-binding antibody fragments, each comprising a different set of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides; and (b) selecting a suitable set of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides for the multispecific antibody or antigen-binding antibody fragment based on one or more antibody properties of the plurality of multispecific antibodies and / or antigen-binding antibody fragments expressed in step (a).

[0115] In some embodiments, one or more antibody properties may be selected from properties (i)-(xv) above.

[0116] In yet another aspect, provided herein are methods for producing heteromeric molecules, such as multispecific antibodies or antigen-binding antibody fragments. Such methods can be driven by cFAE. Optionally, the heteromeric molecules can comprise IgG, and optionally, IgG1, IgG2, IgG3, or IgG4 constant regions.

[0117] In some embodiments, the heteromeric molecule produced or intended to be produced comprises: (A) a first polypeptide comprising a first variant CH3 domain polypeptide (or an immunoglobulin heavy chain polypeptide comprising said first variant CH3 domain polypeptide); and (B) a second polypeptide (an immunoglobulin heavy chain polypeptide comprising said first variant CH3 domain polypeptide) comprising a second variant CH3 domain polypeptide.

[0118] In such heteromeric molecules, (a) the first variant CH3 domain polypeptide and the second variant CH3 domain polypeptide may preferentially pair with each other and may differ by at least one amino acid, and (b) the first polypeptide and the second polypeptide may be linked or paired with each other, optionally via at least one disulfide bond.

[0119] In some embodiments, the method may include (i) incubating under reducing environment or conditions (such as a solution containing a reducing agent) (i-1) a first parent molecule comprising at least two of the first polypeptides linked or paired to each other, optionally via at least one disulfide bond, and (i-2) a second parent molecule comprising at least two of the second polypeptides linked or paired to each other, optionally via at least one disulfide bond.

[0120] In this step (i), when the heteromeric molecule is a multispecific antibody or antigen-binding antibody fragment, the parent molecules may be corresponding monospecific parent antibodies (such as IgG), and the parent antibodies may be incubated under reducing conditions, allowing for dissociation of the inter-heavy chain pairings (e.g., disulfide bonds) in each of the parent antibodies, but not between the heavy and light chains.

[0121] In some embodiments, the method may then include (ii) placing the incubation product of step (i) in a less reducing or non-reducing environment, and in some embodiments, this step (ii) may remove the reducing agent if one is present in the reducing environment.

[0122] In certain embodiments of the production methods, the first variant CH3 domain polypeptide and / or the second variant CH3 domain polypeptide may be any of the sets of first and second variant CH3 domains described herein. In certain embodiments, the set of first and second variant CH3 domain polypeptides may not be the set referred to herein as "KiH" or "RG-FG."

[0123] In certain embodiments, a heteromeric molecule may comprise one or more of the following features: (A) the first polypeptide further comprises a first antigen-binding domain; (B) the second polypeptide further comprises a second antigen-binding domain; (C) the heteromeric molecule further comprises a third polypeptide optionally comprising a third antigen-binding domain, optionally wherein the third polypeptide is bound to or paired with the first polypeptide; and / or (D) the heteromeric molecule further comprises a fourth polypeptide optionally comprising a fourth antigen-binding domain, optionally wherein the fourth polypeptide is bound to or paired with the second polypeptide.

[0124] In certain embodiments of the production methods, the heteromeric molecule may further comprise (C) a third polypeptide optionally comprising a third antigen-binding domain, and / or (D) a fourth polypeptide optionally comprising a fourth antigen-binding domain.

[0125] In certain embodiments of the production methods, the heteromeric molecule may be a multispecific antibody or antigen-binding antibody fragment, which may optionally comprise any of the structures shown in Figures 2-8.

[0126] In certain embodiments, (a) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 366 and the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 407; (b) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 407 and, optionally, the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 366; (c) the amino acid substitution in the first variant CH3 domain polypeptide comprises amino acid substitutions at positions 349 and 366 and, optionally, the amino acid substitution in the second variant CH3 domain polypeptide comprises amino acid substitutions at positions 354 and 407; (d) the first variant CH3 domain polypeptide comprises (e) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 366, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 366, or (f) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 407, wherein the amino acid substitution at position 349 is Y349C and the amino acid substitution at position 354 is S354C. In each of the foregoing, the substitution positions are according to EU numbering.

[0127] In further embodiments, (a) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of T366V, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of Y407V; (b) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of Y407V, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of T366V; or (c) the amino acid substitution in the first variant CH3 domain polypeptide comprises Y349C and T366V, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises S354C and Y407V. (d) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C and Y407V, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C and T366V, (e) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and T366V, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C and Y407V, or (f) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and Y407V, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C and T366V. In each of the foregoing, the substitution positions are according to EU numbering.

[0128] In certain embodiments of the production methods, the heteromeric molecule may be a multispecific antibody or antigen-binding antibody fragment comprising one or more of the following characteristics (I) and (II):

[0129] (I) (I-1-i) the first polypeptide comprises a first antigen-binding domain that forms a first antigen-binding site specific for a first epitope, and / or (I-1-ii) the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain that forms a third antigen-binding site specific for a third epitope, optionally the first epitope being the same as or different from the third epitope, or (I-2) the first polypeptide comprises a first antigen-binding domain and the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain, wherein the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for the first epitope, and / or

[0130] (II) (II-1-i) the second polypeptide comprises a second antigen-binding domain that forms a second antigen-binding site specific for a second epitope, and / or (I-1-ii) the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain that forms a fourth antigen-binding site specific for a fourth epitope, optionally the second epitope being the same as or different from the fourth epitope; or (II-2) the second polypeptide comprises a second antigen-binding domain and the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain, wherein the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for the second epitope.

[0131] In some embodiments, the incubation in step (i) may be carried out at a temperature of about 15°C to about 40°C, about 20°C to about 40°C, about 25°C to about 35°C, about 28°C to about 32°C, or about 29°C to about 31°C, or about 30°C.

[0132] In certain embodiments, the incubation in step (i) may be carried out for about 30 minutes to about 20 hours, about 1 hour to about 15 hours, about 2 hours to about 10 hours, about 3 hours to about 7 hours, or about 4 hours to about 6 hours, or about 5 hours.

[0133] In certain embodiments, the incubation in step (i) may be carried out at about 30° C. for about 5 hours.

[0134] In some embodiments, the reducing environment may include at least one reducing agent, optionally at least one weak reducing agent.

[0135] In certain embodiments, the reducing environment may include at least one reducing agent selected from 2-mercaptoethylamine (2-MEA), β-mercaptoethanol (BME), L-cysteine, dithiothreitol (DTT), or dithionite.

[0136] In certain embodiments, the reducing agent may not be glutathione.

[0137] In certain embodiments, the reducing environment is about 25 to about 125 mM, about 50 mM to about 100 mM, about 70 to about 80 mM, or about 75 mM 2-MEA, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM BME, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM BME. The solution may contain at least one reducing agent selected from about 0 μM or about 100 μM L-cysteine, about 15 to about 400 μM, about 20 to about 200 μM, about 25 to about 100 μM, about 30 to about 70 μM, or about 50 μM DTT, or 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM dithionite.

[0138] In certain embodiments, the reducing environment may optionally include at least about 75 mM 2-MEA.

[0139] In some embodiments, in the first antibody and / or the second antibody incubated in step (i), at least two of the first polypeptides may be bound or paired to each other via at least one disulfide bond, and / or at least two of the second polypeptides may be bound or paired to each other via at least one disulfide bond.

[0140] In some embodiments, the first antibody and / or the second antibody may be produced in mammalian cells, yeast cells, insect cells, plant cells, or bacterial cells.

[0141] In some embodiments, the first antibody and / or the second antibody may be produced in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

[0142] In some embodiments of the production method, the placing in step (ii) may be performed by buffer exchange, optionally exchanging the buffer into phosphate buffered saline (PBS).

[0143] In some embodiments, the placing in step (ii) may be performed by buffer exchange, optionally into PBS, by desalting.

[0144] In some embodiments, the placing in step (ii) may be performed by buffer exchange by diafiltration, optionally into PBS.

[0145] In some embodiments, the disposing in step (ii) may be carried out by the addition of an oxidizing agent.

[0146] In some embodiments, the production method may further comprise (iii) incubating the product of step (ii) in a less reducing or non-reducing environment.

[0147] In certain embodiments, incubation may be performed at a temperature of about 1°C to about 20°C, about 2°C to about 10°C, about 3°C ​​to about 5°C, or about 4°C. In certain embodiments, incubation may be performed for about 12 hours to about 154 hours, about 24 hours to about 96 hours, about 36 hours to about 72 hours, or about 48 hours. In particular embodiments, incubation may be performed at about 4°C for about 48 hours.

[0148] In some embodiments, the production method may further comprise (iv) analyzing the amount of multispecific antibodies or antigen-binding antibody fragments in the product of step (ii) and / or step (iii), and / or purifying the multispecific antibodies or antigen-binding antibody fragments from the product of step (ii) and / or step (iii).

[0149] In certain embodiments, the analysis and / or purification is performed via chromatography, optionally LC-MS, IEX, and / or SEC.

[0150] In yet another aspect, provided herein are heteromeric molecules such as multispecific (e.g., bispecific) antibodies and antigen-binding antibody fragments produced by the production methods described herein. Optionally, such multispecific antibodies or antigen-binding antibody fragments may comprise IgG, and optionally IgG1, IgG2, IgG3, or IgG4.

[0151] In some embodiments, the multispecific antibody or antigen-binding antibody fragment may comprise a structure according to any of the structures described herein or shown in Figures 2-8.

[0152] In yet another aspect, provided herein is a polynucleotide(s) encoding (i) the first and second polypeptides of any of the above-described heteromeric molecules, and / or (ii) any of the above-described heteromeric molecules.

[0153] In another aspect, provided herein are polynucleotide vector(s) comprising such polynucleotide(s).

[0154] In another aspect, provided herein is a cell comprising: (i) a first polypeptide and a second polypeptide of any of the above-described heteromeric molecules; (ii) any of the above-described heteromeric molecules; (iii) any of the polynucleotide(s) encoding (i) and / or (ii); and / or (vi) a vector(s) comprising any one or more of (iii).

[0155] In another aspect, provided herein is a composition comprising: (I) (i) a first polypeptide and a second polypeptide of any of the heteromeric molecules described above; (ii) any of the heteromeric molecules described above; (iii) any of the polynucleotide(s) encoding (i) and / or (ii); and / or (vi) a vector(s) comprising any one or more of (iii); and / or (v) a cell comprising any one or more of (i)-(iv); and (II) a pharmaceutically or diagnostically acceptable carrier.

[0156] In another aspect, further provided herein is a method for screening for a set of first and second variant CH3 domain polypeptides suitable for producing a multispecific antibody or antigen-binding antibody fragment. Such a multispecific antibody or antigen-binding antibody fragment may be any of the heteromeric molecules produced using the methods for producing heteromeric molecules described above. Such a multispecific antibody or antigen-binding antibody fragment may have a given antigen specificity of interest.

[0157] In some embodiments, the method may comprise (a) producing a plurality of multispecific antibodies and / or antigen-binding antibody fragments using the methods for producing heteromeric molecules described above.

[0158] In certain embodiments, the plurality of multispecific antibodies and / or antigen-binding antibody fragments may each comprise a different set of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides.

[0159] In some embodiments, the method may include (b) selecting a set of first and second variant CH3 domain polypeptides suitable for producing multispecific antibodies or antigen-binding antibody fragments based on one or more properties of the plurality of multispecific antibodies and / or antigen-binding antibody fragments produced in step (a).

[0160] In certain embodiments, at least one of the one or more characteristics may be selected from any of characteristics (i)-(xv) above. [Brief explanation of the drawings]

[0161] [Figures 1A-1C]A schematic diagram illustrating the overall advantages of heterodimerized CH3 domains is provided. The desired bispecific antibody comprises (a) a half antibody specific for epitope A comprising heavy chain A (comprising a VH (solid black)) and light chain A (comprising a VL (horizontally striped)), and (b) a half antibody specific for epitope B comprising heavy chain B (comprising a VH (checkered)) and light chain B (comprising a VL (vertically striped)). Figure 1A shows an exemplary production of such a bispecific antibody when heavy chain A, light chain A, heavy chain B, and light chain A all comprise wild-type constant domains. When these four chains are co-expressed, co-delivered, or mixed in an approximately 1:1:1:1 ratio, and when complete promiscuity exists in heavy-chain and heavy-chain pairing, 10 different antibody products can be generated with the respective percentages shown. Approximately 12.5% ​​of the products correspond to the desired bispecific antibody (boxed). FIG. 1B shows an exemplary production of a bispecific antibody similar to that shown in FIG. 1A , except that the CH3 domains of heavy chain A (CH3 domain A (diagonal stripes)) and heavy chain B (CH3 domain B (dotted lines)) are variant CH3 domains that differ from each other and preferentially form heterodimers (i.e., heterodimers between CH3 domain A and CH3 domain B). Existing heavy chain CH3 heterodimerization technologies include those listed in Table 1, such as the "knob-into-hole" technology (see, e.g., U.S. Pat. No. 5,731,168). When such heavy chain A, light chain A, heavy chain B, and light chain B are co-expressed, co-delivered, or mixed in an approximately 1:1:1:1 ratio, where CH3 domain A and CH3 domain B exclusively allow heterodimerization, four different antibody products can be produced at the respective percentages shown. Approximately 25% of the products correspond to the desired bispecific antibody (boxed). Figure 1C provides two schematic diagrams (left and right) generally illustrating the benefits of heterodimerized CH3 domains in producing full-sized antibodies (IgG, IgE, or IgD) from two of the preformed half antibodies, including, but not limited to, methods relying on Fab arm exchange (FAE) or controlled FAE (cFAE).Bispecific antibodies can be produced by combining a half antibody specific for epitope A with a half antibody specific for epitope B. When both heavy chain A and heavy chain B contain wild-type CH3 domains (schematic diagram on the left), only 50% of the product (assuming complete promiscuity in the pairing between the half antibodies) will be the desired bispecific antibody. However, as shown in the scheme on the right, when the variant CH3 domains, CH3 domain A (diagonal stripes) and CH3 domain B (dotted lines), are distinct and preferentially form CH3-CH3 heterodimers (i.e., heterodimers between CH3 domain A and CH3 domain B), the product is biased toward the desired bispecific antibody. In the most ideal set of CH3 domains, which form only heterodimers and no homodimers, 100% of the product will be the desired bispecific antibody. Variant CH3 domains that provide heterodimers in more than 50%, if not 100%, facilitate efficient production of bispecific antibodies.

[0162] In the exemplary multispecific antibodies, the solid black represents the VH (VH Domain A) of heavy chain A (specific for epitope A), the horizontal stripes represent the VL (VL Domain A) of light chain A (specific for epitope A), the checkered pattern represents the VH (VH Domain B) of heavy chain B (specific for epitope B), the vertical stripes represent the VL (VL Domain B) of light chain B (specific for epitope B), the diagonal stripes represent the variant CH3 domain in heavy chain A (CH3 Domain A), and the dotted line represents the variant CH3 domain in heavy chain B (CH3 Domain B), where CH3 Domain A and CH3 Domain B preferentially form CH3 heterodimers (i.e., resulting in greater than 50% CH3 heterodimerization). These definitions apply to all figures unless otherwise noted. [Figure 2-8]Exemplary, non-limiting embodiments of various multispecific antibody structures in which the variant CH3 domains disclosed herein may be used are provided. In Figures 2 to 8, unless otherwise indicated, the following applies: (1) each domain is presented as a rectangle, and the text therein indicates the domain name (e.g., CH3, VH1, etc.); (2) a set of multiple domains linked together represents a polypeptide (e.g., a heavy chain polypeptide, a light chain polypeptide, etc.); (3) the orientation of the domains within the polypeptide follows the direction of the text indicating the domain name, from the N-terminus to the C-terminus; (4) even if a linker, hinge, or disulfide bond is not explicitly shown in the figure, a linker or hinge may be used between domains as needed, and disulfide bonds may exist between polypeptides (and / or within domains), presumably to enable accurate formation of an antigen-binding site; (5) the CH2 domain and / or CH3 domain shown in the figure may be omitted whenever possible and may be replaced with a hinge or linker, if appropriate; (6) the diagonal stripes and dotted lines may be variant CH3 domains disclosed herein, including variant CH3 domains that preferentially form CH3-CH3 heterodimers. (7) Rectangles without a pattern (i.e., open) are domains that may individually contain the corresponding wild-type sequence or may contain one or more amino acid substitutions compared to the wild-type sequence; (8) the CH1 domain, CH2 domain, and CH3 domain may individually be of any (heavy chain) isotype; (9) if more than one CH1 domain is present in the structure, the CH1 domains may or may not be of the same isotype; if more than one CH2 domain is present in the structure, the CH2 domains may or may not be of the same isotype; if more than one CH3 domain is present in the structure, the CH3 domains may or may not be of the same isotype; (10) the light chain constant (CL) domain may be a kappa CL domain or a lambda CL domain; (11) if more than one CL domain is present in the structure, all CL domains may be kappa CL or all CL domains may be lambda CL; orOne CL may be a kappa CL and another CL may be a lambda CL domain; (12) when both a kappa CL domain and a lambda CL domain are present, the CH1 domain paired with the CL domain may in some cases be a variant CH1 domain, one of which may be a variant CH1 that preferentially binds to a kappa CL, and the other CH1 domain may be a variant CH1 that preferentially binds to a lambda CL (having a kappa CL and a lambda CL and a kappa-preferred CH1 and a lambda-preferred CH1 in the molecule allows for efficient production); (13) VH-1 and VL-1 form an antigen-binding site for a first epitope, and VH-2 and VL-3 form an antigen-binding site for a second epitope. VH-2 and VL-3 form the antigen-binding site for the second epitope, VH-3 and VL-3 form the antigen-binding site for the third epitope, VH-4 and VL-4 form the antigen-binding site for the fourth epitope, VH-5 and VL-5 form the antigen-binding site for the fifth epitope, and VH-6 and VL-6 form the antigen-binding site for the sixth epitope; (14) all of epitopes 1 through 6 may be different from each other, or may not be different from each other; and (15) for a given VH-VL pair, if the VH alone confers sufficient specificity for the cognate antigen (i.e., nanobody), the VL is not shown in the figure or may be omitted. Figure 2 provides exemplary, non-limiting examples of various multispecific antibody structures in which the variant CH3 domains disclosed herein may be used. The antibody on the top left (boxed) is an exemplary basic full-size bispecific antibody without hinge or disulfide bonds clearly indicated. The boxed antibody may include hinges, for example, between CH1-1 and CH2-1 and between CH1-2 and CH2-2, and disulfide bonds may exist between the hinges (top center). Alternatively, the boxed antibody may include hinges, for example, between CH1-1 and CH2-1 and between CH1-2 and CH2-2, and disulfide bonds may exist between the hinges, between CL-1 and the hinge, and between CL-2 and the hinge (top right). Hinge and disulfide bonds, for example,The antibody structures shown in the upper center and upper right corner may be present in any of the structures shown in the figures and described herein, even if not explicitly indicated. In some variants of the boxed antibodies, the CH2 domain may be absent (center left), or the CH1 and CH2 domains may be absent (bottom left), and hinge and disulfide bonds may be present, as shown in the center center, center right, bottom center, or bottom right. Although not explicitly indicated, any CH1 and / or CH2 domain may be omitted, as desired, in any of the structures or variants thereof in Figures 3-8. Figure 3 provides variants of the antibody structure shown in Figure 2. In Figure 3A, the VH and VL positions have been altered compared to the structure in Figure 2. In Figure 3B, the CH1 and CL positions have been altered compared to the structure in Figure 2. The equivalent variants shown in Figure 3 (swapping VH-VL positions or CH1-CL positions) may also be applied, as desired, to any of the structures or variants thereof shown in Figures 3-8, even if not explicitly indicated. Figure 4 provides variants of the antibody structure boxed in Figure 2. Specifically, a VH-VL pair specific for a third epitope and a VH-VL pair specific for a fourth epitope are added to the N-terminus of the heavy and light chains in different orientations. While both a VH-VL pair specific for a third epitope and a VH-VL pair specific for a fourth epitope are shown, one pair may be added if desired. The equivalent modifications shown in Figure 4 (addition of one or more VH-VL pairs) may also be applied to any of the structures shown in Figures 3-8 or their modifications, as needed, even if not explicitly indicated. Figure 5 provides an additional modification of the boxed antibody structure in Figure 2. Similar to the structure in Figure 4, the VH-VL pair specific for a third epitope and a VH-VL pair specific for a fourth epitope are added in different orientations, and the order of VH and VL on the light chain differs from that of Figure 4. The equivalent modifications shown in Figure 4 (addition of one or more VH-VL pairs) may also be applied, if desired, to any of the structures shown in Figures 2-8 or modifications thereof, even if not explicitly shown. Figure 6 provides further modifications of the boxed antibody structure in Figure 2. Specifically, in Figures 6A-6D, an scFv specific for a third epitope and an scFv specific for a fourth epitope are added. Although two scFvs are shown,If desired, one scFv may be added. In Figure 6A, an scFv is added to the C-terminus of the heavy chain. The four structures in Figure 6A differ in the VH-VL order within each scFv. In Figure 6B, an scFv is added to the C-terminus of the light chain. The four structures in Figure 6B differ in the VH-VL order within each scFv. In Figure 6C, an scFv is added to the N-terminus of the heavy chain. The four structures in Figure 6C differ in the VH-VL order within each scFv. In Figure 6D, an scFv is added to the N-terminus of the light chain. The four structures in Figure 6D differ in the VH-VL order within each scFv. Although not shown in Figures 6A-6D, two scFvs may be added at different positions (e.g., one to the C-terminus of the heavy chain and one to the N-terminus of the light chain). In Figure 6E, four scFvs are added to the N-terminus of the heavy and light chains. The four structures in Figure 6C differ in the VH-VL order within each scFv. The equivalent modification shown in Figure 6 (the addition of one or more scFvs) may be further applied, if desired, to any of the structures shown in Figures 2-8 or their modifications, even if not explicitly indicated. Figures 7A-7B provide further modifications of the boxed antibody structure in Figure 2. Specifically, a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are added to the C-termini of the heavy and light chains in different orientations. While both a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are shown, only one pair may be added if desired. The equivalent modification shown in Figures 7A-7B (the addition of one or more VH-VL pairs) may be further applied, if desired, to all other structures shown in Figures 3-8 or their modifications, even if not explicitly indicated. 8A-8E provide additional exemplary, non-limiting embodiments of various multispecific antibody fragment structures in which the variant CH3 domains disclosed herein may be used and which do not comprise the VH-VL antigen-binding sites of a conventional antibody, but rather comprise one or more scFvs. In Figure 8A, the antibody on the left (boxed) comprises a first heavy chain comprising an scFv (comprising VH-1 and VL-1) specific for a first epitope and a second heavy chain comprising a second scFv (comprising VH-2 and VL-2) specific for a second epitope.8B-8E provide further variations of the antibody structure of FIG. 8A, including additional scFvs. In FIG. 8B, a third scFv specific for a third epitope (comprising VH-3 and VL-3) and a fourth scFv specific for a fourth epitope (comprising VH-4 and VL-4) are attached to the N-terminus of the heavy chain. In FIG. 8C, a third scFv specific for a third epitope (comprising VH-3 and VL-3) and a fourth scFv specific for a fourth epitope (comprising VH-4 and VL-4) are attached to the C-terminus of the heavy chain. In Figure 8D, a first light chain comprising CL-1, a second light chain comprising CL-2, a third scFv (comprising VH-3 and VL-3) specific for a third epitope, and a fourth scFv (comprising VH-4 and VL-4) specific for a fourth epitope are attached to the N-terminus of the light chain. In Figure 8E, a fifth scFv (comprising VH-5 and VL-5) specific for a fifth epitope and a sixth scFv (comprising VH-6 and VL-6) specific for a sixth epitope are attached to the C-terminus of the heavy chain. Although not explicitly shown, the VH-VL order within the scFvs may be reversed if desired. [Figure 9A-9B]Figure 9A shows a proof-of-concept (POC) study of variant CH3 domain selection in Example 1, in which two heterodimerization technologies (KiH and EW-RVT) were evaluated as controls. Figure 9A provides a schematic of the selection of heterodimer-preferring variant CH3 domains by flow cytometry. High FLAG expressers exhibit a more modified Fc containing CH3 heterodimers. A population from a library that stained exceptionally highly with anti-FLAG antibodies (knob-HIS-hole-FLAG:EWHISRVTFLAG:WTHIS-WTFLAG = 1:1:10,000) (indicating expression of heterodimer-preferring variant CH3 domains) is selected and sorted. Figure 9B provides an exemplary flow plot from multiple selection rounds showing enrichment of control heterodimers (KiH and EW-RVT). A variant CH3 domain library with a knob-HIS-hole FLAG:EWHISRVTFLAG:WTHIS-WTFLAG ratio of 1:1:10,000 was sorted for high Flag expressers over two rounds. Sequencing after round 1 (R1) yielded 1 of 91 with a KiH mutation. Sequencing after round 2 (R2) yielded 2 of 91 with a KiH mutation and 2 with an EW-RVT mutation. [Figures 10A-10D]Representative data from cycle 1 of variant CH3 domain selection in Example 2 is shown. Figure 10A provides three library designs in which KiH amino acid positions (position 366 in the first heavy chain and positions 366, 368, and 407 in the second heavy chain) are mutated. In the first library, both the knob and hole positions are mutated, with the strand with hole mutation encoding a FLAG tag and the strand with knob mutation encoding a HIS tag. In the second library, both the knob and hole positions are mutated, with the strand with hole mutation encoding a HIS tag and the strand with knob mutation encoding a FLAG tag. In the third library, the hole position is mutated, but the knob position is not mutated. The strand with hole mutation encodes a FLAG tag and the strand without mutation encodes a HIS tag. DNA sequence variations for each library are provided using site-saturation mutagenesis (SSM). An "X" in white text on a black background represents mutability. Figure 10B provides exemplary flow plots from six selection rounds performed using the first library, Figure 10C provides exemplary flow plots from six selection rounds using the second library, and Figure 10D provides exemplary flow plots from six selection rounds using the third library. [Figures 11A-11C]An exemplary AlphaLISA® analysis of identified variant CH3 domains is shown. Figure 11A (left) provides a schematic of CH3 heterodimer detection by AlphaLISA®. AlphaLISA® was used to determine the relative degree of heterodimerization of Fc fragments by specifically detecting modified Fc fragments containing heterodimeric CH-CH3 sets due to the proximity between HISx6-tagged and FLA-tagged polypeptides. Figure 11A (right) provides results from several samples in the POC set showing clear differences in photon counts between the existing heterodimerized variant CH3 domain sets (KiH and EW-RVT) and the WT CH3 domain set, regardless of which chain contained the FLAG tag. Figure 11B provides a graph showing AlphaLISA® values ​​(photon counts, fold over background (FOB) ("buffer only," i.e., no Fc, was used as background)) for variant CH3 domain positive controls (KiH and EW-RVT, indicated by arrows), negative (WT / WT, indicated by arrows), and variant CH3 domains identified in Example 2 (bars without arrows). Several of the variant CH3 domains identified herein exhibited comparable or superior heterodimerization (see the left bar for EW-RVT). Figure 11C provides a graph plotting AlphaLISA® values ​​against anti-FLAG antibody staining during the final round of flow cytometry-based selection, demonstrating good correlation. The anti-FLAG FOB and AlphaLISA® FOB values ​​for "T366V-HIS;T366 L368 Y407V-FLAG" were 622 and 86, respectively. "T366V-HIS;T366 L368 Y407V-FLAG" was also found in the reverse orientation (i.e., "T366 L368 Y407V-HIS T366V-FLAG"). The anti-FLAG FOB and AlphaLISA® FOB values ​​for "T366 L368 Y407V-HIS T366V-FLAG" were 588 and 39, respectively. [Figures 12A-12B]Figure 12 shows an exemplary size exclusion chromatography (SEC) analysis of variant CH3 domains. Figure 12A provides results from a control sample. Figure 12B provides results from identified variant CH3 domains. All CH3 sets tested produced uniform distributions, indicating low aggregation. [Figures 13A-13C] Figure 13 shows exemplary ion exchange (IEX) analysis of variant CH3 domains. Figure 13A provides results from a control sample, showing peaks corresponding to different antibody species. Figure 13B provides output results from identified variant CH3 domains. CH3 sets VV, LV, LM, IF, and W-SG show chromatograms similar to that of EW-RVT, with a sharp single peak. Figure 13C provides SEC and IEX data for samples W-SY and SEL-L, which have low AlphaLISA® values, in parallel. Low AlphaLISA® values ​​correlated with poor SEC and IEX chromatograms. [Figures 14A-14C]

[0049] Figure 14 shows the production of bispecific antibodies (BsAbs) comprising variant CH3 domains with 354 / 349 disulfide bond substitutions (S354C and Y349C) in HEK293 cells in Example 6. Figure 14A provides a schematic diagram of the different anti-CD3 / anti-HER2 BsAbs produced. Nivolumab (Nivo) was used as a control. Figure 14B provides exemplary SEC chromatograms of each BsAb. Figure 14C provides exemplary IEX chromatograms of each BsAb. [Figures 15A-15D]Subsequent library generation and screening are shown. Figure 15A provides an exemplary flow plot from a selection round in which heterodimerization variant CH3 domains were enriched from the library. Figure 15B shows the selection criteria applied to the set of 430 variant CH3 domains obtained from cycle 2, step 1 to enrich for variant CH3 domains with improved contact rates across the interface, AlphaLISA® values, and Rosetta scores. Sequences were also verified to ensure diversity at mutation positions. Figures 15C and 15D provide t-SNE plots. These plots were used to ensure substitution diversity in variant CH3 domains selected for further production and characterization. Each point represents a set of substitution positions, and points close to each other on the plot contain similar substitution positions. [Figure 16] Figure 8 shows heterodimerization and stability characterization of 48 variant CH3 domains. This graph plots the % monomeric full-size modified Fc (measured by SEC, showing % non-aggregated modified Fc) against the % heterodimer modified Fc for the 48 variant CH3 domains and the control. Based on these results, a set of five variant CH3 domains ("designated") shown in Table 8 was selected as the cycle 2 output. The designated clones exhibited similar heterodimerization and stability as the control. [Figures 17A-17J]Characterization of exemplary BsAbs containing variant CH3 domains produced in HEK293 cells is shown. Figure 17A shows a schematic diagram of the exemplary antibodies produced. For each variant CH3 domain, several different structures were produced: three anti-CD3 / anti-HER2 BsAbs (one in orientation 1, one in orientation 2, and one in orientation 1 with the 354 / 349 substitution added to the CH3 set), two anti-CD20 / anti-CD3 BsAbs (one in orientation 1 and one in orientation 2), and one anti-HEL / anti-BCMA BsAb (the anti-BCMA binding portion is a nanobody). Sequences are provided in Appendix Tables A–D. Heterodimerization efficiency was assessed by comparing structures within the dotted boxes. Figure 17B shows that heterodimerization is consistent between CH3 orientations and variable regions. The percent heterodimer (%) values ​​for the anti-CD3 / anti-HER2 BsAb (lacking the 354 / 349 substitution) are provided. Figure 17C shows IEX chromatograms of different BsAbs and compares the % heterodimer values ​​measured by IEX and LC-MS. When the BsAb contained a nanobody as one of the two antigen-binding domains, IEX provided lower resolution (BCMA VHH x HEL chromatogram). Figure 17D compares the % heterodimer values ​​measured by IEX and LC-MS of different BsAbs that do not contain the 354 / 349 substitution. The % heterodimer values ​​by IEX and LC-MS show good correlation (data points do not include BsAbs containing a nanobody in one Fab arm). Figure 17E compares the % heterodimer values ​​measured by IEX and LC-MS of different BsAbs that contain the 354 / 349 substitution. Figure 17F compares the % heterodimer values ​​measured by LC-MS between BsAbs with and without the 354 / 349 substitution. The 354 / 349 substitution appears to improve heterodimerization (as measured by LC-MS) for most CH3 sets, including the wild-type set. Figure 17G compares AlphaLISA® values ​​for BsAbs with and without the 354 / 349 substitution (CD3xHER2 BsAb and HELxBCMA Fab-VHH BsAb) with % heterodimer values ​​measured by LC-MS or IEX. The rank order of heterodimerization determined by LC-MS and IEX was similar.Figure 17H ​​compares the % heterodimer values ​​measured by IEX, LC-MS, and AlphaLISA® for the LWG and / or SIG sets in Orientation 1, Orientation 2, and Orientation 1 with the additional 354 / 349 substitution. Figure 17I compares the stability of different bsAbs (excluding bsAbs containing a nanobody in one arm) as defined by % monomeric intact Ab measured by SEC on day 0 (the day of HEK production) and the change in % monomeric intact Ab (Δ%) up to day 14. For all BsAbs tested, % monomeric intact Ab values ​​were very low on day 0, and only a slight increase in % monomeric intact Ab values ​​was observed after day 14, indicating minimal aggregation. Figure 17J compares the production yields of different BsAbs (with or without the 354 / 349 substitution) in HEK293 cells. [Figures 18A-18G]Figure 18 shows a comparison of anti-CD3 / anti-HER2 BsAbs containing different CH3 sets (WT, pre-existing CH3 heterodimerization set, cycle 1 output, cycle 2 output, or a combination thereof, with or without CH3 disulfide bond substitution (i.e., 354 / 349 substitution)). Figure 18A compares the % heterodimer values ​​measured by LC-MS and IEX for different BsAbs without the 354 / 349 substitution, which show good correlation. Figure 18B compares the ranking of heterodimerization potential determined by the % heterodimer values ​​measured by LC-MS and IEX for different BsAbs with the 354 / 349 substitution. The rankings determined by these two different methods (LC-MS and IEX) were the same. Figure 18C shows the % heterodimer values ​​measured by LC-MS and IEX for different BsAbs with and without the 354 / 349 substitution, demonstrating that the LWG-SIG set consistently provided high % heterodimer values ​​regardless of the presence or absence of the 354 / 349 substitution. Figure 18D shows the % monomeric intact Ab values ​​measured by SEC for different BsAbs with and without the 354 / 349 substitution. As shown, several BsAbs, such as DVG-VSY and RG-FG, exhibited higher % monomeric intact Ab values ​​and showed less aggregation compared to existing variant CH3 domains (KiH, EW-RVT, or ZW1). Figure 18E provides a graph of % monomeric intact Ab values ​​measured by SEC plotted against % heterodimer values ​​measured by LC-MS for different BsAbs with and without the 354 / 349 substitution, demonstrating that the 354 / 349 substitution increased the % monomeric intact Ab values ​​and % heterodimer values ​​overall. Figure 18F compares the production yield in HEK293 cells for different BsAbs with and without the 354 / 349 substitution, showing that none of the substitution sets appeared to adversely affect production yield. Figure 18G compares the LWG-SIG set and its variant LWG-IG based on % heterodimer values ​​measured by IEX and LC-MS, % monomeric intact Ab values ​​measured by SEC, and production yield in HEK cells. These profiles were similar between LWG-SIG and LWG-IG and were consistent with the Rosetta heterodimer scores. [Figure 19]A summary of exemplary CH3 domain sets ("CH3 set names") identified herein that preferentially form CH3-CH3 heterodimers over homodimers, thus promoting desired Fc pairing, is provided. The amino acid substitutions (positions and amino acid residues) for each CH3 set listed in Figure 19 can be found, for example, in Appendix Tables E-G. These amino acid substitutions may be incorporated into any CH3 domain sequence. Exemplary variant CH3 domain sequences in which the CH3 substitution sets listed in Figure 19 are incorporated into the reference CH3 domain sequence of SEQ ID NO: 1 are also provided in Appendix Tables E-G. These exemplary variant CH3 domain sequences are the sequences used in the Examples herein. The SEQ ID NOs assigned to these exemplary variant CH3 domain sequences are also provided in Figure 19. [Figure 20]

[0033] Figure 14 provides exemplary Tm2 values ​​measured by DSC for Fc-only constructs containing different CH3 sets (WT, pre-existing, cycle 1 output, or cycle 2 output CH3 heterodimerization sets, with or without CH3 disulfide bond substitutions (354 / 349 substitutions) as shown in Table 15). Open circles represent constructs that do not contain the 354 / 349 substitution, and closed circles represent constructs that do contain the 354 / 349 substitution. [Figure 21] The ADI-64950 CH3-CH3 interface is presented with its electron density. (a) Representative electron density within the region of interest of the crystal structure of the IgG1 Fc-only construct ADI-64950, which contains (i) chain A, containing T366S, L368I, and Y407G, and (ii) chain B, containing S364L, T366W, and K409G (chain B also contains the Fc-III knockout substitutions: M252E, I253A, and Y436A). Chain A carbon atoms are in white, chain B carbon atoms are in light gray, nitrogen atoms are in dark gray, oxygen atoms are in black, and sulfur atoms are in very dark gray. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown as a gray mesh contoured at 1.0σ at 2.0 Å resolution. Data for this crystal structure extend to near-atomic resolution of 2.70 Å. (b) Table comparing interface statistics generated from PISA for ADI-64950 (SIG-LWG) and wild-type IgG1 (WT; PDB ID: 5JII). [Figure 22] ADI-64950 provides polar contacts at the CH3-CH3 interface. (a) Polar contacts at the CH3-CH3 interface between chain A and chain B. Chain A carbon atoms are in white, chain B carbon atoms are in light gray, nitrogen atoms are in dark gray, and oxygen atoms are in black. The protein backbone is shown in cartoon form, with residues of interest shown in stick form. Polar contacts are indicated by black dotted lines. (b) Table comparing polar interactions generated from PyMol for ADI-64950 (SIG-LWG) and wild-type IgG1 (WT; PDB ID: 5JII). [Figure 23] Several residues in potential ADI-64950 homodimer off-products are predicted to sterically clash with each other, reducing the likelihood of mispairing. (a-d) Diagrams of the pairing interface surrounding the region of interest. Alignments of chain A to chain B (a) and chain B to chain A (b-d) reveal steric clashes at the CH3-CH3 interface of several residues at substituted and unsubstituted positions in these potential off-products, including (a) Lys409 and Phe405, (b) Asp356 and Tyr349, (c) T366W and Tyr407, and (d) orthologous sets of T366W and Tyr407. Chain A carbon atoms are in white, chain B carbon atoms are in light gray, nitrogen atoms are in dark gray, and oxygen atoms are in black. The protein backbone is shown in cartoon form, with residues of interest in stick form and side chains involved in the clashes shown as transparent molecular surfaces. [Figures 24A-24B] Figure 24A shows a comparison of exemplary results obtained in Example 14 using antibodies comprising two identical CH3 domains from the WT, RL, VV, QR-F, or RG-FG set and (ii) the variable domains of ADI-29235 (white) or ADI-26908 (black). Figure 24A compares the production yields of antibodies produced in CHO cells. Figure 24B provides a graph of % monomeric full-size Ab values ​​measured by SEC. [Figures 25A-25B]Figure 25 shows exemplary results comparing the FAE output of Example 15 with the respective FAE inputs. Figure 25A compares protein recovery after the FAE reaction step to produce the indicated bsAbs containing the WT, RL, VV, or QR-F sets. Figure 25B provides IEX results for the RL, VV, and QR-F sets, with each panel showing an overlay of (i) the chromatograms of the FAE inputs (blue and red), each containing a monospecific parent antibody with the two identical indicated CH3 domains, and (ii) the chromatogram of the corresponding FAE reaction output (green). [Figures 26A-26D] Figure 26A shows exemplary results comparing the FAE output of Example 16 with the respective FAE inputs. Figure 26A provides exemplary SDS-PAGE results comparing protein quality between the FAE input and the FAE output. FAE reactions were performed to produce the indicated bsAbs containing WT, RL, or VV sets. Figure 26B provides exemplary LC-MS results, with each panel showing an overlay of (i) chromatograms (blue and red) of the FAE input, each containing a monospecific parent antibody with the same two indicated CH3 domains, and (ii) a chromatogram (black) of the corresponding FAE reaction output. Figure 26C provides exemplary binding kinetic curves comparing binding to either HER2 or CD3 by the indicated monospecific antibodies in the FAE input and the indicated bsAbs in the corresponding FAE output. Figure 26D provides exemplary binding kinetic curves comparing simultaneous binding to HER2 and CD3 by the indicated monospecific antibodies in the FAE input and the indicated bsAbs in the corresponding FAE output. "HER2→CD3" indicates that the test antibody was first exposed to HER2, then to CD3. "CD3→HER2" indicates that the test antibody was first exposed to CD3, then to HER2. [Figures 27A-27E]Exemplary results from a GSH exposure experiment comparing the VV set and the RL set, as described in Example 17, are shown. Figure 27A provides a schematic diagram of the GSH exposure experiment. In step 1, an anti-HER2, CH3 hetero-IgG1 containing a test CH3 set and an anti-CD3, CH3 hetero-IgG1 containing a test CH3 set are generated by FAE using 2-MEA. In step 2, the anti-HER2, CH3 hetero-IgG1 from step 1 is placed in a mildly reducing environment containing GSH with an anti-CD3 IgG1 containing (i) two identical CH3 domains that are the same as one of the test CH3 sets, (ii) two identical CH3 domains that are the same as the other of the test CH3 sets, or (iii) the test CH3 set (i.e., the anti-HER2, CH3 hetero-IgG1 from step 1), and chain reassociation is assessed by IEX. Figure 27B provides exemplary IEX results from FAE in step 1 using the RL set and the VV set. Each graph panel shows an overlay of a chromatogram of the FAE output with the chromatograms of the two FAE input antibodies. Figures 27C-27E provide exemplary IEX results of GSH exposure in step 2 using the RL set and the VV set. Each graph panel shows an overlay of a chromatogram of the GSH-exposed product with the chromatogram of the two GSH-exposed input antibodies. That is, the input antibodies are an anti-HER2, CH3 heterodimer antibody and an anti-CD3 IgG1 containing (i) two identical CH3 domains that are the same as one of the tested CH3 sets (Figure 27C), (ii) two identical CH3 domains that are the same as the other of the tested CH3 sets (Figure 27D), or (iii) the tested CH3 set (i.e., the anti-HCD3, CH3 heterodimer IgG1 from step 1 (Figure 27E). DETAILED DESCRIPTION OF THE INVENTION

[0163] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0164] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0165] It is understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0166] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably fragments that exhibit the desired antigen-binding activity, also referred to as "antigen-binding antibody fragments"). "Full antibody," "full Ab," "full-size antibody," "full-size Ab," "full-length antibody," "intact antibody," or "whole antibody" and the like encompass molecules having substantially the same structure as a native antibody, which, in the case of IgG, IgD, or IgE, includes two heavy immunoglobulin chains and two light immunoglobulin chains. An "antigen-binding fragment" or "antigen-binding antibody fragment" refers to a portion of an intact antibody, or a combination of portions derived from an intact antibody(ies), that binds to the antigen bound by the intact antibody(ies).

[0167] "Antigen-binding fragment of an antibody" or "antigen-binding antibody fragment" includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein containing an antibody domain (e.g., a VH domain or a CH3 domain) that specifically binds to an antigen to form a complex. Exemplary antibody fragments include, but are not limited to, Fv, fragment antigen-binding ("Fab") fragment, Fab' fragment, Fab' containing a free sulfhydryl group ("Fab'-SH"), F(ab')2 fragment, diabody, linear antibody, single-chain antibody molecule (e.g., single-chain variable fragment ("scFv"), nanobody, or VHH, or only the VH or VL domain), and monospecific or multispecific compounds formed from one or more of the foregoing antibody fragments. In some embodiments, the antigen-binding fragment of a bispecific antibody described herein is an scFv or nanobody. In preferred embodiments, the antigen-binding fragment comprises a set of CH3 domains that preferentially form CH3-CH3 heterodimers.

[0168] Like intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific, trispecific, tetraspecific, etc.). A multispecific antigen-binding fragment of an antibody may comprise at least two different variable domains, each capable of specifically binding to a distinct antigen or to different epitopes of the same antigen.

[0169] A "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., those containing naturally occurring mutations and / or substitutions or that arise during production of the monoclonal antibody preparation), which are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0170] A "multispecific antibody," which may also be referred to herein as a "multispecific compound," refers to an antibody that comprises at least two different antigen-binding domains that recognize and specifically bind to at least two different antigens and / or at least two different epitopes. In some embodiments, a multispecific antibody comprises (1) a first heavy chain and a first light chain that form a cognate pair and bind to a first antigen, and (2) a second heavy chain and a second light chain that form a cognate pair and bind to a second antigen.

[0171] A "bispecific antibody," which may also be referred to herein as a "bispecific compound," is a type of multispecific antibody and refers to an antibody that contains two different antigen-binding domains that recognize and specifically bind to at least two different antigens or at least two epitopes. The at least two epitopes may or may not be present within the same antigen. A bispecific antibody may, for example, target two different surface receptors, two different cytokines / chemokines, receptors, and ligands on the same or different cells (e.g., an immune cell and a cancer cell).

[0172] In some embodiments, the at least two different antigens may be selected from the following antigens (or the at least two different epitopes may be epitopes on any of the following antigens): CD3; 0772P (CA125, MUC16; Genbank Accession Number AF36148), adipophilin (perilipin-2, adipose differentiation-related protein, ADRP, ADFP, MGC10598; NCBI Reference Sequence: NP-001113.2), AIM-2 (absent in melanoma 2, PYHIN4, interferon-inducible protein AIM2; NCBI Reference Sequence: NP-004824.1), ALDH1 A1 (aldehyde dehydrogenase 1 family, member A1, ALDH1, PUMB1, retinaldehyde dehydrogenase 1, ALDC, ALDH-E1, ALHDII, RALDH 1, EC 1.2.1.36, ALDH11, HEL-9, HEL-S-53e, HEL12, RALDH1, acetaldehyde dehydrogenase 1, aldehyde dehydrogenase 1, soluble aldehyde dehydrogenase, hepatic cytosolic ALDH class 1, epididymal luminal protein 12, epididymal luminal protein 9, epididymal secretory sperm-binding protein Li 53e, retinal dehydrogenase 1, RaIDH1, aldehyde dehydrogenase family 1 member A1, aldehyde dehydrogenase, cytoplasmic, EC 1.2.1; NCBI Reference Number: NP-000680.2); alpha-actinin-4 (ACTN4, actinin, alpha 4, FSGS1, focal segmental glomerulosclerosis 1, non-muscle alpha-actinin 4, F-actin cross-linking protein, FSGS, actinin-4, actinin alpha 4 isoform, alpha-actinin-4; NCBI Reference Sequence: NP-004915.2); alpha-fetoprotein (AFP, HPAFP, FETA, alpha-1-fetoprotein, alpha-fetoglobulin, alpha-1-fetoprotein, alpha-fetoglobulin, HP; GenBank: AAB58754.1); amphiregulin (AREG, SDGF, schwannoma-derived growth factor, colorectal cell-derived growth factor, AR, CRDGF; GenBank: AAA51781.1); ARTC1 (ART1, ADP-ribosyltransferase 1, mono(ADP-ribosyl)transferase 1, ADP-ribosyltransferase C2 and C3 toxin-like 1, ART2, CD296, RT6, ADP-ribosyltransferase 2, GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1, EC 2.4.2.31, CD296 antigen; NP), ASLG659; ASPHD1 (aspartate beta-hydroxylase domain containing 1, aspartate beta-hydroxylase domain-containing protein 1, EC 1.14.11., GenBank: AAI44153.1); B7-H4 (VTCN1, V-Set domain-containing T cell activation inhibitor 1; B7H4, B7 superfamily member 1; immune costimulatory protein B7-H4; B7h.5, T cell costimulatory molecule B7x; B7S1; B7X; VCTN1; H4; B7 family member; PRO1291, B7 family member; H4; T cell costimulatory molecule B7x; V-Set domain-containing T cell activation inhibitor 1; protein B7S1; GenBank: AAZ17406.1); BAFF-R (TNFRSF13C, tumor necrosis factor receptor superfamily member 13C; BAFF R, B-cell-activating factor receptor; BAFF receptor; BLyS receptor 3; CVID4; BROMIX; CD268, B-cell-activating factor receptor; prolixin; tumor necrosis factor receptor superfamily member 13C; BR3, CD268 antigen; NCBI sequence NP-443177.1; BAGE-1; BCLX(L); BCR-ABL fusion protein (b3a2); beta-catenin (CTNNB1, catenin (cadherin-associated protein), beta 1, 88 kDa; CTNNB; MRD19, catenin (cadherin-associated protein), beta 1 (88 kDa); armadillo, catenin beta-1; GenBank: CAA61107.1); BING-4 (WDR46, WD repeat domain 46, C6orf11, BING4, WD repeat-containing protein BING4, chromosome 6 open reading frame 11, FP221, UTP7, WD repeat-containing protein 46; NP); BMPR1 B (bone morphogenetic protein receptor type IB, GenBank accession number NM-00120; NP); B-RAF (brevican (BCAN, BEHAB, GenBank accession number AF22905); brevican (BCAN, chondroitin sulfate proteoglycan 7, brain-enriched hyaluronan-binding protein, BEHAB; CSPG7, brevican proteoglycan, brevican core protein, chondroitin sulfate proteoglycan BEHAB; GenBank: AAH27971.1); CALCA (calcitonin-related polypeptide alpha, CALC1, calcitonin Nin-1, calcitonin, alpha-type CGRP, calcitonin gene-related peptide I, CGRP-I, CGRP, CGRP1, CT, KC, calcitonin / calcitonin-related polypeptide, alpha, katacalcin; NP); CASP-5 (CASP5, caspase-5, apoptosis-related cysteine ​​peptidase, caspase-5, apoptosis-related cysteine ​​protease, protease ICH-3, protease TY, ICE(rel)-111, ICE(rel)III, ICEREL-III, ICH-3, caspase-5, TY protease, EC 3.4.22.58, ICH3, EC 3.4.22; NP); CASP-8; CD19 (CD19 - B-lymphocyte antigen CD19 isoform 2 precursor, B4, CVID3 [Homo sapiens], NCBI Reference Sequence: NP-001761.3); CD20 (CD20 - B-lymphocyte antigen CD20, transmembrane 4-domain, subfamily A, member 1, B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7; NCBI Reference Sequence: NP-690605.1); CD21 (CD21 (CR2 (complement receptor) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792 Genbank accession number M2600); (CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, LybB, SIGLEC-2, FLJ22814, Genbank accession number AK02646); CD22; CD33 (CD33 molecule, CD33 antigen (Gp67), sialic acid-binding Ig-like lectin 3, sialic acid-binding Ig-like lectin 3, SIGLEC3, gp67, SIGLEC-3, myeloid cell surface antigen CD33, p67 , Siglec-3, CD33 antigen; GenBank: AAH28152.1); CD45; CD70 (CD70 - tumor necrosis factor (ligand) superfamily, member 7; surface antigen CD70; Ki-24 antigen; CD27 ligand; CD27-L; tumor necrosis factor ligand superfamily member 7; NCBI reference sequence for Homo sapiens species: NP-001243.1); CD72 (CD72 (B cell differentiation antigen CD72, Lyb-; 359 aa, μl: 8.66, MW: 40225, TM: 1 [P] gene chromosome: 9p13.3, GenBank accession number NP-001773); CD79a (CD79a (CD79A, CD79a, immunoglobulin-related alpha, covalently interacts with Ig beta (CD79B) to form Ig B cell-specific protein that forms a complex with M molecules on the surface and transmits signals involved in B cell differentiation), μl: 4.84, MW: 25028, TM: 2[P] gene chromosome: 19q13.2, Genbank accession number NP-001774.1; CD79b (CD79b (CD79B, CD79b, IGb (immunoglobulin-related beta), B29, Genbank accession number NM-000626 or 1103867); Cdc27 (cell division cycle 27, D0S1430E, D17S978E, Anaphase-promoting complex subunit 3, anaphase-promoting complex subunit 3, ANAPC3, APC3, CDC27Hs, H-NUC, CDC27 homolog, cell division cycle 27 homolog (S. Cerevisiae), HNUC, NUC2, anaphase-promoting complex, protein 3, cell division cycle 27 homolog, cell division cycle protein 27 homolog, Nuc2 homolog; GenBank: AAH11656.1); CDK4 (cyclin-dependent kinase 4, cell division protein kinase 4, PSK-J3, EC 2.7.11.22); CMM3, EC 2.7.11; NCBI Reference Sequence: NP-000066.1; CDKN2A (cyclin-dependent kinase inhibitor 2A, MLM, CDKN2, MTS1, cyclin-dependent kinase inhibitor 2A (inhibits melanoma, P16, CDK4), cyclin-dependent kinase 4 inhibitor A, multiple tumor suppressor 1, CDK4I, MTS-1, CMM2, P16, ARF, INK4, INK 4A, P14, P14ARF, P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16, CDK4 inhibitor P16-INK4, negative cell cycle regulator beta, p14ARF, p16-INK4, p16-INK4a, p16INK4A, p19ARF; NP; CEA; CLL-1 (CLEC12A, MICL, and DCAL) , encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, including roles in cell adhesion, cell-cell signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced ​​transcript variants of this gene have been described, although the full-length nature of some of these variants has not been determined. This gene is closely related to other CTL / CTLD superfamily members within the natural killer gene complex region on chromosome 12p13 (Drickamer, K Curr.Opin.Struct.Biol.9:585-90

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[2004] ;Marshall AS,et al J.Biol.Chem.279:14792-80, 2004. CLL-1 has been shown to be a type II transmembrane receptor containing a single C-type lectin-like domain (not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain, and a short cytoplasmic tail containing ITIM motifs; CLPP (caseinolytic mitochondrial matrix peptidase proteolytic subunit, endopeptidase Clp, EC 3.4.21.92, PRLTS3, ATP-dependent protease ClpAP (E. coli), ClpP (caseinolytic protease ATP-dependent proteolytic subunit, E. coli) homolog, ClpP caseinolytic peptidase ATP-dependent proteolytic subunit homolog (E. coli), ClpP caseinolytic protease ATP-dependent proteolytic subunit homolog (E. coli), human proteolytic subunit, ATP-dependent protease ClpAP, proteolytic subunit, human ClpP caseinolytic peptidase ATP-dependent proteolytic subunit, ClpP caseinolytic peptidase, ATP-dependent proteolytic subunit homolog, ClpP caseinolytic protease, ATP-dependent proteolytic subunit. Homologue, putative ATP-dependent Clp protease proteolytic subunit, mitochondrial; NP); COA-1; CPSF; CRIPTO (CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, GenBank accession number NP-003203 or NM-00321); Cw6; CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, is involved in HIV-2 infection, and may also be involved in the development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, μl: 8.54, MW: 41959, TM: 7 [P] gene chromosome: 11q23.3, GenBank accession number NP-001707); CXORF61 CXORF61 - chromosome X open reading frame 61 [Homo sapiens], NCBI Reference Sequence: NP-001017978.1); cyclin D1 (CCND1, BCL1, PRAD1, D11S287E, B-cell CLL / lymphoma 1, B-cell lymphoma 1 protein, BCL-1 oncogene, PRAD1 oncogene, cyclin D1 (PRAD1: parathyroid adenomatosis 1), G1 / S-specific cyclin D1, parathyroid adenomatosis 1, U21B31, G1 / S-specific cyclin-D1, BCL-1; NCBI Reference Sequence: NP-444284.1); cyclin-A1 (CCNA1, CT146, cyclin A1; GenBank: AAH36346.1); dek-can fusion protein; DKK1 (Dickkopf WNT signaling pathway inhibitor 1, SK, hDkk-1, Dickkopf (Xenopus Laevis) homolog 1, Dickkopf 1 homolog (Xenopus Laevis), DKK-1, Dickkopf 1 homolog, Dickkopf-related protein-1, Dickkopf-1-like, Dickkopf-like protein 1, Dickkopf-related protein 1, Dickkopf-1, Dkk-1; GenBank: AAQ89364.1); DR1 (transcriptional downregulator 1, TBP-binding (negative cofactor 2), negative cofactor 2-beta, TATA-binding protein-associated phosphoprotein, NC2, NC2-beta, protein Dr1, NC2-beta, transcriptional downregulator 1; NCBI Reference Sequence: NP-001929.1);DR13 (major histocompatibility complex, class II, DR beta 1, HLA-DR1B, DRw10, DW2.2 / DR2.2, SS1, DRB1, HLA-DRB, HLA class II histocompatibility antigen, DR-1 beta chain, human leukocyte antigen DRB1, lymphocyte antigen DRB1, MHC class II antigen, MHC class II HLA-DR beta 1 chain, MHC class II HLA-DR beta cell surface glycoprotein, MHC class II HLA-DRw10-beta, DR-1, DR-12, DR-13, DR-14, DR-16, DR-4, DR-5, DR-7, DR-8, DR-9, DR1, DR12, DR13, DR14, DR16, DR4, DR5, DR7, DRB, DR9, DRw11, DRw8, HLA-DRB2, clone P2-beta-3, MHC class II antigen DRB1*1, MHC class II antigen DRB1*10, MHC class II antigen DRB1*11, MHC class II antigen DRB1*12, MHC class II antigen DRB1*13, MHC class II antigen DRB1*14, MHC class II antigen DRB1*15, MHC class II antigen DRB1*16, MHC class II antigen DRB1*3, MH MHC class II antigen DRB1*4, MHC class II antigen DRB1*7, MHC class II antigen DRB1*8, MHC class II antigen DRB1*9; NP; E16 (E16(LAT1, SLC7A5, Genbank accession number NM-00348); EDAR (EDAR - tumor necrosis factor receptor superfamily member EDAR precursor, EDA-A1 receptor; downless homolog; ectodysplasia receptor; ectodermal dysplasia receptor; anhidrotic ectodysplasin receptor 1, DL; ECTD10A; ECTD10B; ED1R; ED3; ED5; EDA-A1R; EDA1R; EDA3; HRM1 [Homo sapiens]; NCBI reference sequence: NP-071731.1); EFTUD2 (elongation factor Tu GTP-binding domain-containing 2, elongation factor Tu GTP-binding domain-containing protein 2, hSNU114, SNU114 homolog, U5 SnRNP-specific protein, 116KDa, MFDGA, KIAA0031, 116KD, U5 SnRNP-specific protein, 116KDa U5 small nuclear ribonucleoprotein component, MFDM, SNRNP116, Snrp116, Snu114, U5-116KD, SNRP116, U5-116KDa; GenBank: AAH02360.1); EGFR (epidermal growth factor receptor, ERBB, proto-oncogene c-ErbB-1, receptor tyrosine-protein kinase ErbB-1, ERBB1, HER1, EC 2.7.10.1, epidermal growth factor receptor (avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog), erythroblastic leukemia virus (V-Erb-B) oncogene homolog (avian), PlG61, avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog, cell growth inhibitory protein 40, cell proliferation-inducing protein 61, mENA, EC 2.7.10; GenBank: AAH94761.1); EGFR-G719A; EGFR-G719C; EGFR-G719S; EGFR-L858R; EGFR-L861 Q; EGFR-57681; EGFR-T790M; elongation factor 2 (EEF2, eukaryotic translation elongation factor 2; EF2, polypeptidyl-tRNA translocase; EF-2; SCA26, EEF-2; NCBI reference sequence: NP-001952.1); ENAH (hMena) (Enabled homolog (Drosophila); MENA, mammalian enabled; ENA, NDPP1, protein enabled homolog; GenBank: AAH95481.1) (results for "ENAH" only, not "ENAH (hMena)"); EpCAM (epithelial cell adhesion molecule, M4S1, MIC18, tumor-associated calcium signal transducer 1, TACSTD1, TROP1, adenocarcinoma-associated antigen, cell surface glycoprotein Trop-1, epithelial glycoprotein 314, major gastrointestinal tumor-associated protein GA733-2, EGP314, KSA, DIAR5, HNPCC8, monoclonal antibody AUA1 specific antigen, EGP-2, EGP40, ESA, KS1 / 4, MK-1, human epithelial glycoprotein-2, membrane component, chromosome 4, surface marker (35 kD glycoprotein), EGP, Ep-CAM, GA733-2, M1S2, CD326 antigen, epithelial cell surface antigen, hEGP314, KS 1 / 4 antigen, ACSTD1; GenBank: AAH14785.1); EphA3 (EPH receptor A3; ETK1, ETK; TYRO4, HEK, Eph-like tyrosine kinase 1, tyrosine-protein kinase receptor ETK1; EK4, EPH-like kinase 4, EC 2.7.10.1; EPHA3, HEK4, ephrin type A receptor 3, human embryonic kinase 1, TYRO4 protein tyrosine kinase; hEK4, human embryonic kinase; tyrosine-protein kinase TYRO4, EC 2.7.10; GenBank: AAH63282.1); EphB2R; epiregulin (EREG, ER, proepiregulin; GenBank: AAI36405.1); ETBR (EDNRB, endothelin receptor type B; HSCR2, HSCR, endothelin receptor nonselective; ET-B, ET-BR; ETRB, ABCDS; WS4A; ETB, endothelin B receptor; NP); ETV6-AML1 fusion protein; EZH2 (zeste homolog enhancer 2 (Drosophila), lysine N-methyltransferase 6; ENX-1, KMT6 EC 2.1.1.43; EZH1, WVS, zeste (Drosophila) homolog enhancer 2; ENX1, EZH2b, KMT6A; WVS2, histone-lysine N-methyltransferase; EZH2, zeste homolog enhancer 2; EC 2.1.1;GenBank:AAH10858.1); FcRH1 (FCRL1, Fc receptor-like 1; FCRH1, Fc receptor homolog 1; FcR-like protein 1; immunoreceptor translocation-associated protein 5; IFGP1, IRTA5; hIFGP1, IFGP family protein 1; CD307a, Fc receptor-like protein 1; immunoglobulin superfamily Fc receptor; Gp42; FcRL1, CD307a antigen; GenBank: AAH33690.1); FcRH2 (FCRL2, Fc receptor-like 2; SPAP1, SH2 domain-containing phosphatase anchor protein 1; Fc receptor homolog 2; FcR-like protein 2; immunoglobulin receptor translocation-associated protein 4; FCRH2; IFGP4; IRTA4; IFGP family protein 4; SPAP1A; SPAP1 B, SPAP1C, CD307b, Fc receptor-like protein 2, immunoreceptor translocation-associated protein 4, immunoglobulin superfamily Fc receptor, Gp42, SH2 domain-containing phosphatase anchor protein 1, FcRL2, CD307b antigen; GenBank: AAQ88497.1); FcRH5 (FCRL5, Fc receptor-like 5; IRTA2, Fc receptor homolog 5, FcR-like protein 5, immunoreceptor translocation-associated protein 2; BXMAS1, FCRH 5, CD307, CD307e, PRO820, Fc receptor-like protein 5, immunoglobulin superfamily receptor translocation-associated 2 (IRTA2), FCRL5, CD307e antigen; GenBank: AAI01070.1; FLT3-ITD; FN1 (fibronectin 1, cold-insoluble globulin; FN, migration-stimulating factor; CIG, FNZ; GFND2, LETS, ED-B; FINC, GFND; MSF, fibronectin; GenBank: AAI43764.1); G250 (MN, CAIX, carbonic anhydrase IX, carbonic dehydratase, RCC-associated protein G250, carbonic dehydratase IX, membrane antigen MN, renal cell carcinoma-associated antigen G250, CA-IX, P54 / 58N, pMW1, RCC-associated antigen G250, carbonic anhydrase 9; NP); (also known as "G250" instead of "G250 / MN / CAIX"); GAGE-1, 2, 8; GAGE-3, 4, 5, 6, 7; GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1 L; GDNFR-alpha 1; GFR-alpha; U95847; BC014962; NM-145793 NM-005264); GEDA (Genbank accession number AY26076); GFRA1 - GDNF family receptor alpha-1; GDNF receptor alpha-1; GDNFR-alpha-1; GFR-alpha-1; RET ligand 1; TGF-beta-related neurotrophic factor receptor 1 [Homo sapiens]; ProtKB / Swiss-Prot:P56159.2; glypican-3 (GPC3, glypican 3, SDY S, glypican proteoglycan 3, intestinal protein OCI-5; GTR2-2, MXR7, SGBS1, DGSX, OCI-5. SGB, SGBS, heparan sulfate proteoglycan, secreted glypican-3, OCI5; GenBank: AAH35972.1); GnTVf; gp100 (PMEL, pre-melanosome protein; SILV, D12S53E, PMEL17; SIL, melanocyte protein Pmel 17, melanocyte lineage-specific antigen GP100, melanoma-associated ME20 antigen, silver locus protein homolog, ME20-M, ME20M, P1, P100, silver (mouse homolog)-like, silver homolog (mouse), ME20, SI, melanocyte protein Mel 17, melanocyte protein PMEL, silver mouse homolog of melanosome matrix protein 17; GenBank: AAC60634.1); GPC; GP. NMB (glycoprotein (transmembrane) Nmb, glycoprotein NMB, glycoprotein Nmb-like protein, osteoactivin, transmembrane glycoprotein HGFIN, HGFIN, NMB, transmembrane glycoprotein, transmembrane glycoprotein NMB; GenBank: AAH32783.1); GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP-078807.1; NM-024531.3); GPR19 (G protein-coupled receptor 19; Mm.478; NP-006134.1; NM-006143.2); GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR1; NP-115940.2; NM-032551.4); HAVCR1 (hepatitis A virus cell cell receptor 1, T-cell immunoglobulin mucin family member 1, kidney injury molecule 1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1, T-cell immunoglobulin mucin receptor 1, T-cell membrane protein 1, HAVCR, HAVCR-1, T-cell immunoglobulin domain and mucin domain protein 1, HAVcr-1, T-cell immunity and mucin domain-containing protein 1; GenBank: AAH13325.1); HER2 (ERBB2, V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2), NGL, NEU, neuro / glioblastoma-derived oncogene homolog, metastatic lymph node gene 19 protein, proto-oncogene C-ErbB-2, proto-oncogene Neu, tyrosine kinase cell surface receptor HER2, MLN 19, p185erbB2, EC 2.7.10.1, V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2 (neuronal / glioblastoma-derived oncogene homolog), CD340, HER-2, HER-2 / neu, TKR1, C-Erb B2 / Neu protein, herstatin, neuroblastoma / glioblastoma-derived oncogene homolog, receptor tyrosine-protein kinase ErbB-2, V-Erb-B2 erythroblastic leukemia viral oncogene homolog 2, neuronal / glioblastoma-derived oncogene homolog, MLN19, CD340 antigen, EC 2.7.10; NP); HER-2 / neu (another name for the above); HERV-K-MEL; HLA-DOB (beta subunit of MHC class II molecules (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, μl: 6.56, MW: 30820, TM: 1 [P] gene chromosome: 6p21.3, GenBank accession number NP-002111); hsp70-2 (HSPA2, heat shock 70 kDa protein 2, heat shock 70 kDa protein 2, HSP70-3, heat shock associated 70 KDa protein 2, heat shock 70 KDa protein 2; GenBank: AAD21815.1); IDO1 (indoleamine 2,3-dioxygenase 1, IDO, INDO, indoleamine-pyrrole 2,3-dioxygenase, IDO-1, indoleamine-pyrrole 2,3 dioxygenase, indoleamine 2,3 dioxygenase, indole 2,3 dioxygenase, EC 1.13.11.52; NCBI Reference Sequence: NP-002155.1); IGF2B3; IL13R alpha 2 (IL13RA2, interleukin-13 receptor, alpha 2, cancer / testis antigen 19, interleukin-13 binding protein, IL-13R-alpha-2, IL-13RA2, IL-13 receptor subunit alpha-2, IL-13R subunit alpha-2, CD213A2, CT19, IL-13R, IL13BP, interleukin-13 binding protein, interleukin-13 receptor alpha 2 chain, interleukin-13 receptor subunit alpha-2, IL 13R, CD213a2 antigen; NP); IL20Rα; intestinal carboxylesterase; IRTA2 (also known as FcRH5); kallikrein 4 (KLK4, kallikrein-related peptidase 4; PRSS17; EMSP1, enamel matrix serine proteinase 1; kallikrein-like protein 1; serine protease 17; KLK-L1; PSTS; AI2A1, kallikrein 4 (prostase, enamel matrix, prostate); ARM1; EMSP, androgen-regulated message 1; enamel matrix serine protease 1; kallikrein; kallikrein-4; prostase; EC 3.4.21; GenBank: AAX30051.1); KIF20A (kinesin family member 20A, RAB6KIFL, RAB6 interacting, kinesin-like (Rab kinesin 6), mitosis a; LAGE-1; LDLR-fucosyltransferase AS fusion protein; lengusin (LGSN, lengusin, glutamine synthetase domain-containing lens protein; GLULD1, glutamate ammonia ligase domain-containing protein 1; LGS, glutamate ammonia ligase (glutamine synthetase) domain-containing 1, glutamate ammonia ligase ( glutamine synthetase) domain-containing 1, lens glutamine synthetase-like; GenBank: AAF61255.1; LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR6; NP-003658.1; NM-003667.2); LY64 (lymphocyte antigen 64 (RP10, a type I membrane protein of the leucine-rich repeat (LRR) family that regulates B cell activation and apoptosis; loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661 aa, μl: 6.20, MW: 74147, TM: 1 [P] Gene chromosome: 5q12, GenBank accession numbers NP-005573; Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-; NP-002337.1; NM-002346.2); Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT; NP-067079.2; NM-021246.2); LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ3522; NP-059997.3; NM-017527.3); LyPD1 -LY6 / PLAUR domain containing 1, PHTS [Homo sapiens], GenBank: AAH17318.1); MAGE-A1 (melanoma antigen family A, 1 (directs expression of antigen MZ2-E, MAGE1, melanoma antigen family A1, MAGEA1, melanoma antigen MAGE-1, melanoma-associated antigen 1, melanoma-associated antigen MZ2-E, antigen MZ2-E, cancer / testis antigen 1.1, CT1.1, MAGE-1 antigen, cancer / testis antigen family 1, member 1, cancer / testis antigen family 1, member 1, MAGE1A; NCBI reference sequence: NP-004979.3); MAGE-A10 (MAGEA10, melanoma antigen family A, 10, MAGE10, MAGE-10 antigen, melanoma-associated antigen 10, cancer / testis antigen 1.10, CT1.10, cancer / testis antigen family 1, member 10, cancer / testis antigen family 1, member 10; NCBI reference sequence: NP-001238757.1); MAGE-A12 (MAGEA12, melanoma antigen family A, 12, MAGE12, cancer / testis antigen 1.12, CT1.12, MAGE12F antigen, cancer / testis antigen family 1, member 12, cancer / testis antigen family 1, member 12, melanoma-associated antigen 12, MAGE-12 antigen; NCBI reference sequence: NP-001159859.1); MAGE-A2 (MAGEA2, melanoma antigen family A, 2, MAGE2, Cancer / testis antigen 1.2, CT1.2, MAGEA2A, MAGE-2 antigen, cancer / testis antigen family 1, member 2, cancer / testis antigen family 1, member 2, melanoma antigen 2, melanoma-associated antigen 2; NCBI reference sequence: NP-001269434.1); MAGE-A3 (MAGEA3, melanoma antigen family A, 3; MAGE3, MAGE-3 antigen; antigen MZ2-D, melanoma-associated antigen 3; cancer / testis antigen 1.3, CT1.3, cancer / testis antigen family 1, member 3; HIPS, HYPD, MAGEA6, cancer / testis antigen family 1, member 3; NCBI reference sequence: NP-005353.1); MAGE-A4 (MAGEA4, melanoma antigen family A, 4; MAGE4, ​​melanoma-associated antigen 4; cancer / testis antigen 1.4, CT1.4, MAGE-4 antigen, MAGE-41 antigen, MAGE-X2 antigen, MAGE4A, MAGE4B, cancer / testis antigen family 1, member 4; MAGE-41, MAGE-X2, cancer / testis antigen family 1, member 4; NCBI reference sequence: NP-001011550.1); MAGE-A6 (MAGEA6, melanoma antigen family A, 6); MAGE6, MAGE-6 antigen, melanoma-associated antigen 6, cancer / testis antigen 1.6, CT1.6); MAGE3B antigen, cancer / testis antigen family 1, melanoma antigen family A 6, member 6; MAGE-3b, MAGE3B, cancer / testis antigen family 1, member 6; NCBI reference sequence: NP-787064.1); MAGE-A9 (MAGEA9, melanoma antigen family A, 9; MAGE9, MAGE-9 antigen, melanoma-associated antigen 9; cancer / testis antigen 1.9; CT1.9, cancer / testis antigen family 1, member 9; cancer / testis antigen family 1, member 9; MAGEA9A; NCBI reference sequence: NP-005356.1); MAGE-C1 (MAGEC1, melanoma antigen family C, 1; cancer / testis antigen 7.1; CT7.1, MAGE-C1 antigen, cancer / testis antigen family 7, member 1; CT7, cancer / testis antigen family 7, member 1; melanoma-associated antigen C1; NCBI reference sequence: NP-005453.2); MAGE-C2 (MAGEC2, melanoma antigen family C, 2; MAGEE1, cancer / testis antigen 10, CT10; HCA587, melanoma antigen family E, 1, cancer / testis specific; hepatocellular carcinoma-associated antigen 587; MAGE-C2 antigen; MAGE-E1 antigen; hepatocellular carcinoma antigen 587; melanoma-associated antigen C2; NCBI reference sequence: NP-057333.1); mammaglobin-A (SCGB2A2, secretoglobin, family 2A, member 2; MGB1, mammaglobin 1; UGB2, mammaglobin A; mammaglobin-A, mammaglobin-1, secretoglobin family 2A member 2; NP); MART2 (H HAT, hedgehog acyltransferase; SKI1, T cell-recognized melanoma antigen 2; skinny hedgehog protein 1; Skn, T cell-recognized melanoma antigen 2; protein-cysteine ​​N-palmitoyltransferase HHAT, EC 2.3.1.-; GenBank: AAH39071.1); M-CSF (CSF1, colony-stimulating factor 1 (macrophage); MCSF, CSF-1; ranimostim, macrophage colony-stimulating factor 1; ranimostim; GenBank: AAH21117.1); MCSP (SMCP, sperm mitochondrial-associated cysteine-rich protein; MCS, mitochondrial sheath selenoprotein; HSMCSGEN1, sperm mitochondrial-associated cysteine-rich protein; NCBI reference sequence: NP-109588.2); XAGE-1b / GAGED2a; WT1 (Wilms tumor 1; WAGR; GUD; WIT-2; WT33; amino-terminal domain of EWS; NPHS4; last three zinc fingers of the DNA-binding domain of WT1; AWT1; Wilms tumor protein; EWS-WT1; GenBank: AAB33443.1); VEGF; tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP; NP-000363.1; NM-000372.4; GenBank: AAB60319.1); TrpM4 (BR22450; FLJ20041; TRPM4; TRPM4B; transient receptor potential cation channel, subfamily M, member 4; GenBank accession number NM-01763). ); TRP2-INT2; TRP-2; TRP-1 / gp75 (tyrosinase-related protein 1, 5,6-dihydroxyindole-2-carboxylic acid oxidase, CAS2, CATB, TYRP, OCAS, catalase B, b-protein, glycoprotein 75, EC 1.14.18., melanoma antigen Gp75, TYRP1, TRP, TYRRP, TRP1, SHEP11, DHICA oxidase, EC 1.14.18, GP75, EC 1.14.18.1); triosephosphate isomerase (triosephosphate isomerase 1, TPID, triosephosphate isomerase, HEL-S-49, TIM, epididymal secretory protein Li 49, TPI, triosephosphate isomerase, EC 5.3.1.1; TRAG-3 (CSAG family member 2, cancer / testis antigen family 24, CSAG3B, member 2, CSAG family member 3B, cancer / testis antigen family 24 member 2, cancer / testis antigen 24.2, chondrosarcoma-associated gene 2 / 3 protein, taxol resistance-associated gene 3 protein, chondrosarcoma-associated gene 2 / 3 protein-like, CT24.2, taxol resistance-associated gene 3, TRAG-3, CSAG3A, TRAG3); TMEM46 (shisa homolog 2 (Xenopus laevis; SHISA; NP-001007539.1; NM-001007538.1; TMEM118 (Ring finger protein, transmembrane 2; RNFT2; FLJ1462; NP-001103373.1; NM-001109903.1; TMEFF1 (EGF-like domain and two follistatin-like domain-containing transmembrane protein 1; Tomoregulin; H7365 ;C9orf2;C9ORF2;U19878;X83961;NM-080655;NM-003692;TGF-betaRII (TGFBR2, transforming growth factor, beta receptor II (70 / 80kDa), TGFbeta-RII, MFS2, tbetaR-II, TGFR-2, TGF-beta receptor type IIB, TGF-beta type II receptor, TGF-beta receptor type 2, EC 2.7.11.30, transforming growth factor beta receptor type IIC, AAT3, TBETAR-II, transforming growth factor, beta receptor II (70-80 kD), TGF-beta receptor type II, FAA3, transforming growth factor-beta receptor type II, LDS1 B, HNPCC6, LDS2B, LDS2, RITC, EC 2.7.11, TAAD2; TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to EGF / heregulin family growth factors and follistatin); 374 aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI reference sequence: NP-057276; NCBI gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession numbers AF179274; AY358907, CAF85723, CQ782436; TAG-2; TAG-1 (contactin 2 (axonal), TAG-1, AXT, axonin-1 cell adhesion molecule, TAX, contactin 2 (transiently expressed), TAXI, contactin-2, axonal glycoprotein TAG-1, transiently expressed axonal glycoprotein, transient axonal glycoprotein, axonin-1, TAX-1, TAG1, FAMES; PRF:444868); SYT-SSX1 or -SSX2 fusion protein; survivin; STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate six-transmembrane epithelial antigen 2, six-transmembrane prostate protein, Genbank accession number AF45513; STEAP1 (prostate six-transmembrane epithelial antigen, Genbank accession number NM-01244); SSX-4; SSX-2 (SSX2, synovial sarcoma, X breakpoint 2, X breakpoint 2, SSX, X breakpoint 2B, cancer / testis antigen 5.2, X-chromosome-associated 2, tumor antigen HOM-MEL-40, CT5.2, HD21, cancer / testis antigen family 5, HOM-MEL-40, isoform B, cancer / testis antigen family 5 member 2a, member 2a, protein SSX2, sarcoma, sarcoma, synovium, X-chromosome-linked 2, synovium, synovial sarcoma, X breakpoint 2B, synovial sarcoma, SSX2A; Sp17; SOX10 (SRY (sex-determining region Y)-box 10), mouse, PCWH, DOM, WS4, WS2E, WS4C, dominant megacolon, mouse, human homolog, dominant megacolon, S RY-related HMG-box gene 10, human homolog, transcription factor SOX-10; GenBank: CAG30470.1); SNRPD1 (small nuclear ribonucleoprotein D1, small nuclear ribonucleoprotein D1, polypeptide 16 kDa, polypeptide (16 kD), SNRPD, HsT2456, Sm-D1, SMD1, Sm-D autoantigen, small nuclear ribonucleoprotein D1 polypeptide 16 kDa pseudogene, SnRNP core protein D1, small nuclear ribonucleoprotein Sm D1; SLC35D3 (solute carrier family 35, member D3, FRCL1, Fringe connection-like protein 1, bA55K22.3, Frc, Fringe-like 1, solute carrier family 35 member D3; NCBI GenBank: NC-000006.11 NC-018917.2 NT-025741.16); SIRT2 (sirtuin 2, NAD-dependent deacetylase sirtuin-2, SIRL2, silent signaling regulator 2, regulatory protein SIR2 homolog 2, Sir2-related protein type 2, SIR2-like protein 2, sirtuin type 2, sirtuin (silent mating signaling regulation 2 homolog) 2 (S. cerevisiae), sirtuin-2, sirtuin (silent mating signaling regulation 2, S. cerevisiae, homolog) 2, EC 3.5.1., SIR2;GenBank:AAK51133.1);Sema 5b(FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, 7 thrombospondin repeats (type 1 and type 1-like), transmembrane domain™ and short cytoplasmic domain, (Semaphorin) 5B, GenBank accession number AB04087; seserin1 (SCRN1, SES1, KIAA0193, seserin-1; GenBank: EAL24458.1); SAGE (SAGE1, sarcoma antigen 1, cancer / testis antigen 14, CT14, putative tumor antigen; NCBI reference sequence: NP-061136.2); RU2AS (KAAG1, kidney-associated antigen 1, RU2AS, RU2 antisense gene protein, kidney-associated antigen 1; GenBank: AAF23613.1); RNF43-E3 ubiquitin-protein ligase RNF43 precursor [homologous sapiens], RNF124; URCC; NCBI Reference Sequence: NP-060233.3; RhoC (RGS5 (regulator of G-protein signaling 5, MSTP032, regulator of G-protein signaling 5, MSTP092, MST092, MSTP106, MST106, MSTP129, MST129; GenBank: AAB84001.1); RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE; NP-066124.1; NM-020975.4); RBAF600 (UBR4, ubiquitin-protein ligase E3 component N-recognin 4, zinc finger, UBR1) Type 1, ZUBR1, E3 ubiquitin-protein ligase UBR4; RBAF600, 600 kDa retinoblastoma protein-associated factor, zinc finger UBR1 type protein 1, EC 6.3.2; N-recognin-4, KIAA0462, p600, EC 6.3.2, KIAA1307; GenBank: AAL83880.1); RAGE-1 (MOK, MOK protein kinase, renal tumor antigen; RAGE, MAPK / MAK / MRK overlapping kinase, renal tumor antigen 1, renal cell carcinoma antigen, RAGE-1, EC 2.7.11.22, RAGE1; UniProtKB / Swiss-Prot: Q9UQ07).1); RAB38 / NY-MEL-1 (RAB38, NY-MEL-1, RAB38, RAS oncogene family member, melanoma antigen NY-MEL-1, Rab-related GTP-binding protein, Ras-related protein Rab-38, rrGTPbp; GenBank: AAH15808.1); PTPRK (DJ480J14.2.1 (protein tyrosine phosphatase, receptor type, K R-PTP-kappa, protein tyrosine phosphatase kappa, protein tyrosine phosphatase kappa), protein tyrosine phosphatase, receptor type, K, protein-tyrosine phosphatase kappa, protein-tyrosine phosphatase, receptor type, kappa, R-PTP-kappa, receptor tyrosine-protein phosphatase kappa, EC 3.1.3.48, PTPK; GenBank: AAI44514.1); PSMA; PSCA hIg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628); PSCA (prostate stem cell antigen precursor, Genbank accession number AJ29743); PRDX5 (peroxiredoxin 5, EC 1.11.1.15, TPx type VI); B166, antioxidant enzyme B166; HEL-S-55, liver tissue 2D-Page spot 71 B; PMP20, peroxisomal antioxidant enzyme; PRDX6, thioredoxin peroxidase PMP20; PRXV; AOEB166, epididymal secretory protein Li 55, Alu corepressor 1, peroxiredoxin-5, mitochondria, peroxiredoxin V, prx-V, thioredoxin reductase, Prx-V, ACR1, Alu corepressor, PLP; GenBank: CAG33484.1); PRAME (melanoma preferentially expressed antigen, melanoma preferentially expressed antigen, MAPE, O1P-4, OIPA, CT130, cancer / testis antigen 130, tumor preferentially expressed melanoma antigen, Opa interacting protein 4, Opa interacting protein O1P4; GenBank: CAG30435.1); pml-RAR alpha fusion protein; PMEL17 (Silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp10 BC001414; BT007202; M32295; M77348; NM-006928; PBF (ZNF395, zinc finger protein 395; PRF-1, Huntington's disease regulatory region binding protein; HD gene regulatory region binding protein 2; protein 2, papillomavirus regulatory factor 1; HD regulatory factor 2; papillomavirus regulatory factor; PRF1; HDBP-2; Si-1-8-14; HDBP2, Huntington's disease gene regulatory region binding protein 2; HDRF-2, papillomavirus regulatory factor PRF-1; PBF; GenBank: AAH012 37.1); PAX5 (paired box 5, paired box homeotic gene 5, BSAP, paired box protein; Pax-5, B cell lineage-specific activator; paired domain gene 5, paired box gene 5 (B cell lineage-specific activator protein), B cell-specific transcription factor; paired box gene 5 (B cell lineage-specific activator); PAP (REG3A, regenerating islet-derived 3 alpha; INGAP, PAP-H, hepato-entero-pancreatic protein; PBBCGF, human islet precursor peptide; REG-Ill, pancreatitis-associated protein 1; Regi, Reg III-alpha, hepato-entero-pancreatic; regenerating islet-derived protein III-alpha; pancreatic beta-cell growth factor; HIP, PAP homologous protein; HIP / PAP, proliferation-inducing protein 34; PAP1, proliferation-inducing protein 42; REG-3-alpha, regenerating islet-derived protein 3-alpha, pancreatitis-associated protein; GenBank: AA. H36776.1); p53 (TP53, tumor protein P53, TPR53, P53, cellular tumor antigen P53, antigen NY-CO-13, mutant tumor protein 53, phosphoprotein P53, P53 tumor suppressor, BCC7, transformation-associated protein 53, LFS1, tumor protein 53, Li-Fraumeni syndrome, tumor suppressor P53); P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), μl: 7.63, MW: 47206, TM: 1[P] gene stain: 17p13.3, Genbank accession number NP-002552; OGT (O-linked N-acetylglucosamine (GlcNAc) transferase, O-GlcNAc transferer P110 subunit, O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase, UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 KDa subunit, UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase, uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase, O-GlcNAc transferase subunit P110, EC 2.4.1.255, O-linked N-acetylglucosamine transferase 110 KDa subunit, EC 2.4.1, HRNT1, EC 2.4.1.186, O-GLCNAC; GenBank: AAH38180.1); 0A1 (osteoarthritis QTL 1, OASD; GenBank: CAA88742.1); NY-ESO-1 / LAGE-2 (cancer / testis antigen 1 B, CTAG1 B, NY-ESO-1, LAGE-2, ESO1, CTAG1, CTAG, LAGE2B, cancer / testis antigen 1, autoimmunogenic cancer / testis antigen NY-ESO-1, anser antigen 3, cancer / testis antigen 6.1, New York esophageal squamous cell carcinoma 1, L antigen family member 2, LAGE2, CT6.1, LAGE2A; GenBank: AAI30365.1); NY-BR-1 (ANKRD30A, ankyrin repeat domain 30A, breast cancer antigen NY-BR-1, serologically defined breast cancer antigen NY-BR-1, ankyrin repeat domain-containing protein 30A; NCBI reference sequence: NP-443723.2); N-ras (NRAS, neuroblastoma RAS viral (V-Ras) oncogene homolog, NRAS1, transforming protein N-Ras, GTPase NRas, ALPS4, N-Ras protein part 4; NS6, oncogene homolog, HRAS1; GenBank: AAH05219.1); NFYC (nuclear transcription factor Y, gamma); HAP5, HSM, nuclear transcription factor Y subunit C; transactivator HSM-1 / 2; CCAAT-binding factor subunit C; NF-YC, CCAAT transcription binding factor subunit gamma; CAAT box DNA-binding protein subunit C; histone H1 transcription factor large subunit 2A; CBFC, nuclear transcription factor Y subunit gamma; CBF-C, transactivator HSM-1; H1TF2A, transcription factor NF-Y, C subunit; neo-PAP (PAPOLG, poly(A) polymerase gamma; neo-poly(A) polymerase; nuclear poly(A) polymerase gamma; polynucleotide adenylyltransferase gamma; SRP RNA 3′ adenylase / Pap2, PAP-gamma, neo-PAP, SRP RNA 3′ adenylase, PAP2, EC 2.7.7.19, PAPG; NCBI reference sequence: NP-075045.2); NCA (CEACAM6, Genbank accession number M1872); Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM-00642); myosin class I; MUM-3; MUM-2 (TRAPPC1, transport protein particle complex 1; BETS, BETS homolog; MUM2, melanoma ubiquitous mutant 2, multiple myeloma protein 2, transport protein particle complex subunit 1; MUM-1f; mucin (MUC1, mucin 1, cell surface associated; PEMT, PUM, CA 15-3, MCKD1, ADMCKD, renal medullary cystic disease 1 (autosomal dominant), ADMCKD1, mucin 1, transmembrane, CD227, breast cancer-associated antigen DF3, MAM6, cancer antigen 15-3, MCD, cancer-associated mucin, MCKD, Krebs-von-den-Lungen-6, MUC-1 / SEC, peanut-reactive urinary mucin, MUC1 / ZD, tumor-associated epithelial membrane antigen, DF3 antigen, tumor-associated mucin, episialin, EMA, H23 antigen, H23AG, mucin-1, KL-6, tumor-associated epithelial mucin, MUC-1, episialin, PEM, CD227 antigen; UniProtKB / Swiss-Prot:P15941.3); MUCSAC (mucin SAC, oligomeric mucus / gel forming, tracheobronchial mucin 'MUC5, TBM, mucin 5, subtypes A and C, tracheobronchial / gastric, leB, gastric mucin, mucin SAC, oligomeric mucus / gel forming pseudogene, Lewis blood group B antigen, LeB, major airway glycoprotein, MUC-SAC, mucin-5 subtype AC, tracheobronchial; MUC1 (mucin 1, cell surface associated, PEMT, PUM, CA 15-3, MCKD1, ADMCKD, renal medullary cystic disease 1 (autosomal dominant), ADMCKD1, mucin 1, transmembrane, CD227, breast cancer-associated antigen DF3, MAM6, cancer antigen 15-3, MCD, cancer-associated mucin, MCKD, Krebs-von-den-Lungen-6, MUC-1 / SEC, peanut-reactive urinary mucin, MUC-1 / X, polymorphic epithelial mucin, MUC1 / ZD, tumor-associated epithelial membrane antigen, DF3 antigen, tumor-associated mucin, episialin, EMA, h23 antigen, H23AG, mucin-1, KL-6, tumor-associated epithelial mucin, MUC-1, episialin, PEM, CD227 antigen; MS G783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM-01776; MRP4 multidrug resistance-associated protein 4 isoform 3, MOAT-B; MOATB [Homo sapiens]; NCBI reference sequence: NP-001288758.1; MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM-00582); MMP-7 (MMP7, matrilysin, MPSL1, matrine, matrix metalloproteinase 7 (matrilysin, uterine), uterine matrilysin, matrix metalloproteinase-7, EC 3.4.24.23, Pump-1 protease, matrine, uterine metalloproteinase, PUMP1, MMP-7, EC 3.4.24, PUMP-1; GenBank: AAC37543.1); MMP-2 (MMP2, matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase), MONA, CLG4A, matrix metalloproteinase 2 (gelatinase A, 72 kD gelatinase, 72 kD type IV collagenase), CLG4, 72 kDa gelatinase, 72 kDa type IV collagenase), matrix metalloproteinase-2, MMP-II, 72 kDa gelatinase, collagenase type IV-A, MMP-2, matrix metalloproteinase-II, TBE-1, neutrophil gelatinase, EC 3.4.24.24, EC 3.4.24; GenBank: AAH02576.1); and Meloe.

[0173] In some embodiments, the at least two different antigens may be selected from the following antigens (or the at least two different epitopes may be epitopes on any of the following antigens): 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, asparagi Phosphate, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin Cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, C D6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD 30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-capped or NIP-capped), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, interferon (INF)-alpha, interferon (INF)-beta, interferon (INF) ... Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, l-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis Y antigen, Lewis Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Mullerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PE CAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRllb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL) Rl Apo-2, DR4), TNFRSFIOB(TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcRl, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TR ANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI). CD120a, p55-60, TNFRSFIB(TNF RII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB). CD137, ILA, TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL). Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LINK, LTg, TNFSF15(TL1A / VEGI), TNFSF18(GITR LINK, AITR TL6, TNFSFIA (TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b). LTa、TNFSF1)、TNFSF3(LTbTNFSF, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, W NT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T-lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T-cell immunoglobulin and mucin protein-3), hormone receptors, and growth factors.

[0174] In certain embodiments, a multispecific (e.g., bispecific) antibody according to the present disclosure may have a first antigen-binding domain with specificity for CD3 and a second binding domain with specificity for a second antigen selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, asparagi Phosphate, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin Cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, C D6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD 30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-capped or NIP-capped), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, interferon (INF)-alpha, interferon (INF)-beta, interferon (INF) ... Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, l-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis Y antigen, Lewis Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Mullerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PE CAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRllb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL) Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFR SF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI). CD120a, p55-60, TNFRSFIB(TNF RII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB). CD137, ILA, TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL). Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LINK, LTg, TNFSF15(TL1A / VEGI), TNFSF18(GITR LINK, AITR TL6, TNFSFIA (TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b). LTa、TNFSF1)、TNFSF3(LTbTNFSF, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, W NT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T-lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T-cell immunoglobulin and mucin protein-3), hormone receptors, and growth factors.

[0175] In certain embodiments, antigen combinations that can be targeted by bispecific (or multispecific) antibodies include CD3 and Her2, CD3 and Her3, CD3 and EGFR, CD3 and CD19, CD3 and CD20, CD3 and EpCAM, CD3 and CD33, CD3 and PSMA, CD3 and CEA, CD3 and gp100, CD3 and gpA33, CD3 and B7-H3, CD64 and EGFR, CEA and HSG, TRAIL-R2 and LTbetaR, EGFR and IGFR, VEGFR2 and VEGFR3, VEGFR2 and PDGFR alpha, PDGFR alpha and PDGFR beta, EGFR and MET, EGFR and EDV-miR16, EGFR and CD64, EGFR and Her2, EGFR and Her3, Her2 domain ECD2 and Her2 domain ECD4, Her2 and Her3, IGF-1R and HER3, CD19 and CD22, CD20 and CD22, CD30 and CD16A, FceRI and CD32B, CD32B and CD79B, BCMA and HEL These may include, but are not limited to, MP65 and SAP-2, IL-17A and IL-23, IL-1 alpha and IL-1 beta, IL-12 and IL-18, VEGF and osteopontin, VEGF and Ang-2, VEGF and PDGFR beta, VEGF and Her2, VEGF and DLL4, FAP and DR5, FcgRII and IgE, CEA and DTPA, CEA and IMP288, and LukS-PV and LukF-PV.

[0176] "Different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules, as well as different and / or distinct epitopes that may be contained within one protein, polypeptide, or molecule. As a result, a bispecific antibody can bind to two epitopes on the same polypeptide.

[0177] The term "epitope" is used herein in the broadest sense to encompass the region or regions of an antigen that interact with a corresponding paratope. A protein or peptide epitope can include amino acid residues that directly interact with the paratope (e.g., via hydrogen bonding or hydrophobic interactions) and non-interacting amino acid residues (e.g., residues that contribute largely to the conformation of the epitope). Epitopes can be defined as structural or functional. A functional epitope is generally an epitope that has residues that directly contribute to a function of the antigen (e.g., affinity for another protein or enzymatic activity). A structural epitope is an epitope that has residues that contribute to the antigen's structure that may not significantly contribute to antigen function. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, an epitope may have specific three-dimensional structural characteristics and / or specific charge characteristics. An antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. The term also refers to the region of an antigen bound by an antibody.

[0178] In some cases, a full-size antibody comprises four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chains each comprise a variable region, such as a heavy chain variable region ("VH"), and a heavy chain constant region ("CH"). In the case of an intact antibody, the CH comprises domains CH1, CH2, and CH3. In the case of an antibody fragment, the CH may comprise a CH1 domain, a CH2 domain, and / or a CH3 domain; in some preferred embodiments, the CH comprises at least a CH1 domain. The variant CH3 domains disclosed herein can be used in combination with one or more wild-type CH2 and / or CH3 domains, or with CH2 and / or CH3 domains comprising one or more amino acid substitutions, e.g., amino acid substitutions that alter or improve antibody stability and / or effector function. The light chains each comprise a variable region, such as a light chain variable region ("VL"), and a light chain constant region ("CL"). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a comparative analysis of two or more CDRs. Thus, the CDRs in the heavy chain are designated "CDRH1," "CDRH2," and "CDRH3," respectively, and the CDRs in the light chain are designated "CDRL1," "CDRL2," and "CDRL3," respectively. In other cases, the antibody may include a multimer thereof (e.g., IgM) or an antigen-binding fragment thereof.

[0179] Numbering of amino acid residues in antibody variable and constant domains may be done according to the EU index or EU numbering system as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Unless otherwise specified, the EU numbering system is used herein.

[0180] According to IMGT (International ImMunoGeneTics Information System for Immunoglobulins or Antibodies, T-cell Receptors, MH, Immunoglobulin Superfamilies IgSF, and MhSF), the CH1 domain is amino acid positions (or simply referred to herein as "positions") 118-215 (EU numbering), and the hinge region is amino acid positions 216-230 (EU numbering). The term "CH1 domain" is used broadly herein to refer to a heavy chain region that includes at least seven consecutive amino acid positions of heavy chain positions 118-215 (EU numbering), and in some cases also includes a portion of the hinge region (a portion of heavy chain positions 216-230 (EU numbering)) (e.g., up to position 218). The CH1 domain reference sequence corresponding to amino acid positions 118 to 220 according to EU numbering is provided herein as SEQ ID NO: 6, which corresponds to the CH1 domain sequence of the human IgG1 allotypes "IGHG1*01 (J00228)," "IGHG1*04 (JN582178)," or "IGHG1*07," and is an exemplary amino acid sequence of a wild-type (WT) CH1 domain.

[0181] CH1 domain reference sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (positions 118-220 according to EU numbering) (SEQ ID NO: 6).

[0182] Alternative CH1 domain reference sequences for human IgG1 include, but are not limited to, SEQ ID NO: 5, which corresponds to the CH1 domain sequence of human IgG1 allotypes "IGHG1*03(Y14737)" or "IGHG1*08".

[0183] Alternative CH1 domain reference sequence (214R compared to SEQ ID NO: 6): (Sequence Listing 1) TIFF2024503034000002.tif5158 TIFF2024503034000003.tif5127 (positions 118 to 220 according to EU numbering) (SEQ ID NO: 5).

[0184] These CH1 domain reference sequences are intended to be exemplary, as it is intended by applicants that the "CH1 domain" sequence encompasses any naturally occurring CH1 domain allotype or allelic variant.

[0185] According to IMGT, the CH2 domain is amino acid positions (or simply referred to herein as "positions") 231-340 (EU numbering). The term "CH2 domain" is used broadly herein to refer to a heavy chain region comprising at least seven consecutive amino acid positions of heavy chain positions 231-340 (EU numbering). A CH2 domain reference sequence corresponding to amino acid positions 231-340 according to EU numbering is provided herein as SEQ ID NO: 7, which is an exemplary amino acid sequence of a wild-type (WT) CH2 domain.

[0186] CH2 domain reference sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 7).

[0187] Again, the listed CH2 domain reference sequences are intended to be exemplary, as it is intended by applicants that the "CH2 domain" sequence include any naturally occurring CH2 domain allotypes or allelic variants.

[0188] According to IMGT, the CH3 domain is amino acid positions (or simply referred to herein as "positions") 341-446 (EU numbering). The term "CH3 domain" is used broadly herein to refer to a heavy chain region comprising at least seven consecutive amino acid positions of heavy chain positions 341-446 (EU numbering). The CH3 domain reference sequence corresponding to amino acid positions 341-446 according to EU numbering is provided herein as SEQ ID NO: 1, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*01 (J00228)" or "IGHG1*08" and is an exemplary amino acid sequence of a wild-type (WT) CH3 domain.

[0189] CH3 domain reference sequence: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1).

[0190] Alternative CH3 domain reference sequences for human IgG1 may include, but are not limited to, SEQ ID NO: 2, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*03 (Y14737)", SEQ ID NO: 3, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*04 (JN582178)", and SEQ ID NO: 4, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*07".

[0191] Alternative CH3 domain reference sequence (356E and 358M compared to SEQ ID NO: 1): (Sequence Listing 2) TIFF2024503034000004.tif5159 TIFF2024503034000005.tif5139 (SEQ ID NO: 2).

[0192] Alternative CH3 domain reference sequence (422I compared to SEQ ID NO: 1): (Sequence Listing 3) TIFF2024503034000006.tif5158 TIFF2024503034000007.tif5137 (SEQ ID NO: 3).

[0193] Alternative CH3 domain reference sequence (431G compared to SEQ ID NO: 1): (Sequence Table 4) TIFF2024503034000008.tif6158 TIFF2024503034000009.tif5140 (SEQ ID NO: 4).

[0194] These CH3 domain reference sequences are intended to be exemplary, as it is intended by applicants that the "CH3 domain" sequence encompasses any naturally occurring CH3 domain allotype or allelic variant.

[0195] Thus, amino acid modifications in a variant CH3 domain polypeptide according to the present disclosure can be compared to and / or incorporated into any parent CH3 domain polypeptide, for example, but not limited to, a wild-type sequence such as SEQ ID NO: 1, or any allelic variant such as SEQ ID NO: 2, 3, or 4.

[0196] There are two major CL isotypes, kappa ("κ") and lambda ("λ"), and such CL domains are referred to herein as kappa CL domains ("CLκ" domains) and lambda CL domains ("CLλ" domains).

[0197] According to IMGT, the CLκ domain is amino acid positions 108-214 (EU numbering). The term "CLκ domain" is used broadly herein to refer to a light chain region comprising at least seven consecutive amino acid positions of kappa light chain positions 108-214 (EU numbering). The CLκ domain reference sequence corresponding to amino acid positions 108-214 (EU numbering) is provided herein as SEQ ID NO: 8, which is an exemplary amino acid sequence of a wild-type (WT) CLκ domain.

[0198] CLκ domain reference sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (positions 108-214 according to EU numbering) (SEQ ID NO: 8).

[0199] According to IMGT, the CLλ domain is amino acid positions 107-215 (EU numbering). The term "CLλ domain" is used broadly herein to refer to a light chain region comprising at least seven consecutive amino acid positions of lambda light chain positions 107-215 (EU numbering). The CLλ domain reference sequence corresponding to amino acid positions 107-215 (EU numbering) is provided herein as SEQ ID NO: 9, which is an exemplary amino acid sequence of a wild-type (WT) CLλ domain.

[0200] CLλ domain reference sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (positions 107 to 215 according to EU numbering) (SEQ ID NO: 9).

[0201] Various standard sequences for the constant domains of human IgG1, IgG2, IgG3, and IgG4 (corresponding to different allotypes) are known in the art and can be found, for example, in Vidarsson et al., Front Immunol. 2014 Oct 20;5:520 and U.S. Patent No. 9,150,663, the disclosures of which are incorporated herein by reference in their entireties. Again, these reference sequences are intended to be exemplary, as the applicant intends the human IgG1, IgG2, IgG3, and IgG4 sequences to encompass all naturally occurring human IgG1, IgG2, IgG3, and IgG4 allotypes.

[0202] As used herein, the term "cognate pair" or "cognate pairing" refers to a pair or association of two antibody chains (e.g., heavy and light chains) that each comprise a variable region (e.g., VH and VL, respectively) and whose combination of variable regions provides the intended binding specificity for a given epitope or antigen. As used herein, the term "non-cognate pair" or "non-cognate pairing" refers to a pair or association of two antibody chains (e.g., heavy and light chains) that each comprise a variable region (e.g., VH and VL, respectively) and whose combination of variable regions does not provide the intended binding specificity for a given epitope or antigen.

[0203] Provided herein are engineered variant CH3 domains that contain at least one amino acid substitution that prevents or reduces the formation of CH3-CH3 homodimers and preferentially forms CH-CH3 homodimers.

[0204] The terms "CH3 domain set" or "CH3 set" are used interchangeably and refer to a combination of two CH3 domains. When a CH3 set contains two non-wild-type CH3 domains (i.e., two variant CH3 domains), such a CH3 set may also be referred to as a "variant CH3 domain set" (or a "CH3 domain variant set") or a "variant CH3 set" (or a "CH3 variant set"). Each "CH3 set" is given a "CH3 set name" based on the amino acid substitutions contained in the CH3 domains of that set. The set of substitutions contained in the CH3 domains of a set may be referred to as a "CH3 substitution set."

[0205] As used herein, "CH3 set names" are designated by the amino acid position (according to EU numbering) substituted in the CH3 domain of each chain, with a dash separating the heavy chains. For example, a "W-SG" set has a W in the CH3 domain (position 366) of the first heavy chain (referred to as chain A in Figure 19 and the appendix table) and, in addition, an S and a G in the CH3 domain (positions 366 and 407) of the second heavy chain (referred to as chain B in Figure 19 and the appendix table). These two chains are referred to as chain A and chain B in Figure 19 and the appendix table, but the chain names are interchangeable. For example, a "W-SG" set may have a W in the CH3 domain (position 366) of the second heavy chain (or "chain B") and, in addition, an S and a G in the CH3 domain (positions 366 and 407) of the first heavy chain (or "chain A"). "(349 / 354)" and "(354 / 349)" refer to substitutions that allow for CH3 interdomain disulfide bonding. For example, "(349 / 354)" indicates the presence of a Y349C substitution in CH3 of chain A and a S354C substitution in CH3 of chain B. Similarly, "(354 / 349)" indicates the presence of a S354C substitution in CH3 of chain A and a Y349C substitution in CH3 of chain B. Thus, "W-SG(354 / 349)" means that the S354C substitution is present in CH3 with the "W" substation (position 366) and the Y349C substitution is present in CH3 with the "SG" substitutions (positions 366 and 407).

[0206] As used herein, the term "variant CH3 domain" (also referred to as "variant CH3 domain polypeptide," "CH3 domain variant," or "CH3 domain variant polypeptide") is used interchangeably and refers to a CH3 domain having an amino acid sequence in which one or more amino acid substitutions are made relative to the CH3 domain sequence. CH3 sequences in which amino acid substitutions are made include, but are not limited to, the reference CH3 domain sequence SEQ ID NO: 1. In the library screened to identify the described variant CH3 domains, the nucleic acid sequence encoding SEQ ID NO: 1 was mutated. As used herein, the term "Fab arm exchange" or "FAE" refers to the process by which one half molecule (i.e., one heavy and one light chain pair, also referred to as half antibody or half IgG if the antibody is IgG) of an Ig molecule (e.g., IgG, IgE, or IgD) recombines with another half molecule of another Ig molecule. It was found that FAEs are naturally present in human IgG4 molecules and that FAEs can be mimicked in vitro by the addition of weak reducing agents (van der Neut Kolfschoten et al. Science. 2007 Sep 14;317(5844):1554-1557). Site-directed mutagenesis studies substituting IgG4 amino acid residues with their IgG1 counterparts identified that FAE in humans may be driven by residues S228 located in the IgG4 core hinge (van der Neut Kolfschoten et al. Science. 2007 Sep 14;317(5844):1554-1557, Labrijn et al. Nat Biotechnol. 2009 Aug;27(8):767-771.) and R409 located in the IgG4 CH3 domain (Labrijn et al. J Immunol. 2011 Sep 15;187(6):3238-46.).It was later discovered that an IgG1 CH3 domain in which position 409 is substituted with R and another IgG1 CH3 domain in which position 405 is substituted with L preferentially pair with each other, and that the use of such CH3s may be useful for producing bispecific IgG1 molecules (Labrijn et al. Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5145-50).

[0207] As used herein, the term "controlled FAE" or "cFAE" refers to an FAE artificially facilitated by a set of engineered CH3 domains that preferentially form heterodimers. cFAEs can be particularly useful for the efficient production of bispecific antibodies. For example, if a target antibody contains (a) a half antibody specific to epitope A comprising heavy chain A (including VH) and light chain A (including VL), and (b) a half antibody specific to epitope B comprising heavy chain B (including VH) and light chain B (including VL), (a) antibody A comprising two half antibodies specific to epitope A and (b) antibody B comprising two half antibodies specific to epitope B can be first produced. Antibody A and antibody B can then be placed together under mildly reducing conditions, which allows for the reduction of disulfide bonds between the heavy chains, resulting in half antibody molecules. If heavy chain A contains an engineered CH3 domain A and heavy chain B contains an engineered CH3 domain B, and CH3 domain A and CH3 domain B preferentially form CH3-CH3 heterodimers, then upon removal of the mildly reducing conditions, cFAE will preferentially form heterodimers between heavy chain A and heavy chain B over heavy chain A homodimers and heavy chain B homodimers, resulting in the desired bispecific antibody being more abundant than antibody A and antibody B.

[0208] The term "half molecule" or "half antibody" when referring to IgG, IgE, or IgD, which may also be referred to as "half IgG," "half IgE," or "half IgD," respectively, refers to one set of heavy chains and one light chain of the referenced antibody.

[0209] "Preferentially" forming heterodimers or "preferentially" forming heterodimers when referring to CH3 domains means that heterodimers with different, non-identical CH3 domains are formed more frequently (i.e., more frequently or with a higher probability) than homodimers with different, identical CH3 domains. When referring to a set of two different CH3 domains (a first CH3 domain and a second CH3 domain), it means that more heterodimers (between the first CH3 domain and the second CH3 domain) are formed than homodimers (between a dimer of the first CH3 domain and a dimer of the second CH3 domain). For example, when a first CH3 domain and a second CH3 domain that is different from the first CH3 domain are mixed, co-expressed, or co-provided in a ratio of approximately 1:1, the percentage of dimers formed between the first CH3 domain and the second CH3 domain is greater than 50%. The % CH-CH3 heterodimer (e.g., also referred to as "% heterodimer" or "% heterodimers") or degree of heterodimerization may be quantified by any available assay, such as, but not limited to, AlphaLISA®, liquid chromatography mass spectrometry (LC-MS), ion exchange chromatography (IEX), or flow cytometry. The % heterodimer may be about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% when the CH3 domain comprises a CH3 substitution set disclosed herein. In some preferred embodiments, the % heterodimer may be about 70% or greater. In some more preferred embodiments, the % heterodimer may be about 75% or greater. In some more preferred embodiments, the % heterodimer may be about 80% or greater. In some more preferred embodiments, the heterodimer % may be about 85% or greater. In some more preferred embodiments, the heterodimer % may be about 90% or greater. In some more preferred embodiments, the heterodimer % may be about 95% or greater.In some more preferred embodiments, the % heterodimer may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some more preferred embodiments, the % heterodimer may be about 100%.

[0210] The CH3 substitution set or CH3 set, and / or antibodies comprising such CH3 sets, can be characterized by the degree of aggregation (e.g., the presence of multimers of whole antibodies) and / or the amount of half antibodies (i.e., one CH3 per molecule or one heavy chain per molecule) (both of which can be quantified, for example, by chromatography, such as size exclusion chromatography (SEC), or electrophoresis, such as SDS-PAGE); the melting temperature (Tm), which can be measured, for example, by differential scanning fluorimetry (DSF); the production yield in an appropriate cell type (e.g., HEK293 cells or yeast cells); the "pI", isoelectric point ("pI"); the level of interaction with multispecific reagents (PSR), which can be measured as in WO 2014 / 179363; the hydrophobic interactions of the antibody (e.g., Estep P, et al. MAbs. 2015 May-Jun;7(3):553-561.); solubility; production cost and / or time; stability; shelf life; in vivo half-life; and / or immunogenicity. Any of these or other properties may be used in addition to the % heterodimer value when evaluating a given variant CH3 domain set or CH3 set. Thus, a variant CH3 domain or CH3 set that results in a relatively low % heterodimer may be as ideal as another CH3 set with a relatively high % heterodimer value, if the variant CH3 domain or CH3 set offers a good profile for one or more properties. For example, a CH3 set that results in 80% heterodimers at 3% aggregation (3% of the expression product is a multimer of the whole antibody) may be as ideal as a CH3 set that results in 90% heterodimers at 10% aggregation.

[0211] There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. A constant domain according to the present disclosure may be of any antibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. As used herein, a CH3 domain may be derived from the CH3 of an antibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, or IgE. CH3 substitution according to the present disclosure may be made to any CH3 domain sequence, such as, but not limited to, the CH3 reference sequence SEQ ID NO: 1. When a CH1 domain and / or CH2 domain is used in conjunction with a variant CH3 domain of the present disclosure, the CH1 domain and / or CH2 domain can be from any antibody isotype, and the CH1 domain isotype and / or CH2 domain isotype does not necessarily have to be the same as the CH3 domain isotype.

[0212] The term "library" is used herein to encompass any collection of biological materials, such as nucleic acids, peptides, proteins, and their sequence information. For example, a "CH3 domain-encoding polynucleotide library" refers to a collection of polynucleotides encoding different CH3 domain polypeptides or a collection of their polynucleotide sequences, and a "CH3 domain polypeptide library" refers to a collection of different CH3 domain polypeptides or a collection of their amino acid sequences.

[0213] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents. In one embodiment, the carrier is suitable for parenteral, intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the carrier may be a liquid in which the active therapeutic agent is formulated. Excipients generally do not provide any pharmacological activity to the formulation, but may provide chemical and / or biological stability and release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, Gennaro, A. editor, 19th edition, Philadelphia, PA: Williams and Wilkins (1995), incorporated by reference.

[0214] "Conservative amino acid substitutions" are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid with a nonpolar side chain for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitutions of an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substitutions of an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., Ile, Thr, and Val), substitutions of an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0215] Variant CH3 domains and CH3 domain sets As described herein, certain positions within the CH3 domain, as well as certain amino acid substitutions and sets of substitutions, have been found to affect CH3 domain pairing or CH3-CH3 dimer (or Fc heterodimer) formation.

[0216] In some embodiments, the variant CH3 domains described herein may comprise an amino acid substitution at one or more of the following amino acid positions according to EU numbering: 364, 366, 368, 370, 399, 400, 405, 407, and 409. In some embodiments, the variant CH3 domains described herein may comprise an amino acid substitution at any of the positions listed in Table 7, or any combination of positions listed in Table 7.

[0217] The parent CH3 domain into which such amino acid substitutions may be incorporated may comprise a wild-type or naturally occurring CH3 domain sequence, or a variant or engineered version thereof. Exemplary sequences of such parent polypeptides include, but are not limited to, the reference CH3 sequence SEQ ID NO: 1, which corresponds to amino acid positions 341-446 according to EU numbering.

[0218] The resulting variant CH3 domain preferentially forms CH3-CH3 heterodimers over CH3-CH3 homodimers. Such variant CH3 domains may be useful for producing heterodimeric (or multimeric) polypeptides and molecules containing such polypeptides. Such variant CH3 domains may be useful in producing multispecific antibodies and antibody fragments by improving the fidelity of heterologous Fc pairing while maintaining the native IgG structure of bispecific antibodies, which are preferred due to their well-established properties as therapeutic molecules, including long in vivo half-lives and the ability to elicit effector functions. These domain CH3 variants can be used to fully or partially resolve chain mispairing in the generation of multispecific antibodies, e.g., bispecific antibodies, by promoting proper heavy chain-heavy chain pairing. More specifically, multispecific antibodies containing these variant CH3 domains form fewer undesired product-related contaminants, i.e., molecules containing mispaired domains or chains, which can be difficult to eliminate during manufacturing.

[0219] In some embodiments, the amino acid substitutions in the variant CH3 domain are at: (i) 366, (ii) 368, (iii) 407, (iv) 366 and 407, (v) 366 and 368, (vi) 366 and 409, (vii) 368 and 370, (viii) 368 and 407, (ix) 399 and 405, (x) 400 and 409, (xi) 364, 366, and 409, (xii) 364, 407, and 409, (xiii) 366, 368, and 370, (xiv) 366, 368, and 407, (xv) 366, 399, and 405, (xvi) 3 (xvii) 366, 407, and 409; (xviii) 368, 400, and 409; (xix) 399, 405, and 407; (xx) 400, 407, and 409; (xxi) 366, 399, 405, and 407; (xxii) 366, 399, 405, and 409; (xxiii) 366, 400, 407, and 409; (xxiv) 366, 368, 399, 405, and 407; or (xxv) 366, 368, 400, 407, and 409.

[0220] In some embodiments, a variant CH3 domain may further comprise a Y349C or S354C substitution, which allows disulfide formation with another variant CH3 domain containing a S354C or Y349C substitution, respectively. In some embodiments, a variant CH3 domain may comprise one or more of the following amino acid substitutions: S364D, S364L, T366Q, T366R, T366S, T366V, T366W, L368A, L368F, L368S, L368I, K370G, K370Y, D399Q, S400T, F405L, Y407V, Y407G, K409R, K409L, and / or K409G. In some embodiments, a variant CH3 domain may optionally further comprise Y349C or S354C.

[0221] In some embodiments, the amino acid substitutions in the variant CH3 domain are selected from the following substitution combinations: T366W; T366S and Y407G; T366V; Y407V; T366Q and K409R; L368F; T366R and K409G; L368F and K370G; S400T and K409L; D399Q and F405L; S364D, Y407V, and K409G; T366V, L368S, and K370Y; S364L, T366W, and K409G; T366S, L368I, and Y407G; T366W, S400T, and K409L; T366S, L368A, Y407V, D399Q, and F405L; T366W, S400T, and K409L; T366S, Y407G, D399Q, and F40 5L;T366W, D399Q, and F405L;T366S, L368A, Y407V, S400T, and K409L;T366W, D399Q, and F405L;T366S, Y407G, S400T, and K409L;Y407V, S400T, and K409L;T366V, D399Q, and F405L;Y407V, D399Q, and F405L; It may comprise or consist of any one of: T366V, S400T, and K409L; T366Q, K409R, D399Q, and F405L; L368F, S400T, and K409L; Y407V, T366Q, and K409R; T366V and L368F; S364L, T366W, and K409G; or L368I and Y407G. In some embodiments, amino acid substitutions within the variant CH3 domain can include or consist of any one of the following substitution combinations: T366W; T366S and Y407G; T366V; Y407V; T366Q and K409R; L368F; T366R and K409G; L368F and K370G; S400T and K409L; D399Q and F405L; S364D, Y407V, and K409G; T366V, L368S, and K370Y; S364L, T366W, and K409G; T366S, L368I, and Y407G; or L368I and Y407G. In some embodiments, an S354C or Y349C substitution can be further added to any of the substitution combinations.

[0222] In some embodiments, these substitutions are made relative to the reference CH3 domain sequence of SEQ ID NO: 1. In such cases, the amino acid sequence of a variant CH3 domain according to the present disclosure may comprise or consist of the sequence in any one of SEQ ID NOs: 11-16, 21-26, 31-36, 41-46, 51-56, 61-66, 71-76, 81-86, 91-96, 101-106, 111-116, 121-126, 131-136, 141-146, 151-156, and 161-166. In some embodiments, a variant CH3 domain according to the present disclosure may comprise or consist of the sequence in any one of SEQ ID NOs: 11-16, 21-26, 31-36, 41-46, 51-56, 61-66, 71-76, and 161-166. In some preferred embodiments, the variant CH3 domain may comprise an amino acid sequence according to any one of SEQ ID NOs: 11-16 and 71-76.

[0223] In some embodiments, a variant CH3 domain according to the present disclosure may pair with or form a heterodimer with another variant CH3 domain according to the CH3 domains disclosed herein.

[0224] The variant CH3 domain sets according to the present disclosure that preferentially form CH3-CH3 heterodimers are not identical to those previously identified as heterodimerization-preferring CH3 domain sets, such as existing CH3 technologies listed in Table 1. However, any of the inventive CH3 substitution sets described herein may be combined with existing CH3 technologies, such as the CH3 technologies in Table 1. [Table 1]

[0225] In some embodiments, such CH3 heterodimers may comprise any of the following CH3 sets: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, WTL-SAVQL, WTL-SGQL, WQL-SAVTL, WQL-SGTL, VTL-VQL, VQL-VTL, QRQL-FTL, VQR-VF, or LWG-IG. In some embodiments, such CH3 heterodimers may comprise any of the following CH3 sets: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, or LWG-IG. In some embodiments, CH3 sets may be further supplemented with CH3 disulfide bondable substitutions ("354 / 349" substitutions or "349 / 354" substitutions). In such embodiments, such CH3 heterodimers comprise the following CH3 sets: W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY(349 / 354), LWG-SIG(349 / 354), WTL-SA VQL(349 / 354), WTL-SGQL(349 / 354), WQL-SAVTL(349 / 354), WQL-SGTL(349 / 354), VTL- VQL(349 / 354), VQL-VTL(349 / 354), QRQL-FTL(349 / 354), VQR-VF(349 / 354), or LWG-IG (349 / 354), or W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL (354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), WTL-SAVQL(354 / 349), WTL-SGQL( 354 / 349), WQL-SAVTL(354 / 349), WQL-SGTL(354 / 349), VTL-VQL(354 / 349), VQL-VTL(354 / 349), QRQL-FTL(354 / 349), VQR-VF(354 / 349), or LWG-IG(354 / 349).In some embodiments, such CH3 heterodimers can comprise any of the following CH3 sets: W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY(349 / 354), LWG-SIG(349 / 354), LWG-IG(349 / 354), W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL(354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), or LWG-IG(354 / 349). Details of the amino acid positions and residues of the substitutions in these sets are shown in Appendix Tables E-G.

[0226] In some preferred embodiments, a CH3 set according to the present disclosure can be W-SG, LWG-SIG, W-SG(349 / 354), LWG-SIG(349 / 354), W-SG(354 / 349), or LWG-IG(354 / 349).

[0227] In further embodiments, any of the substitution sets described herein can be combined with another CH3 heterodimerization-preferring CH3 substitution or substitution set, for example, any one of the inventive CH3 substitution or substitution sets described herein, or any one of the existing CH3 heterodimerization-preferring substitutions or substitution sets, such as those listed in Table 1, to further enhance or promote CH3 heterodimerization.

[0228] Furthermore, for each specific amino acid substitution in a CH3 domain provided herein for CH3 heterodimerization preference, the resulting amino acid can be further substituted with a conservative amino acid substitution to obtain another variant CH3 domain that provides the same preference for CH3 heterodimerization. Alternatively, for each variant CH3 domain, one or more amino acid positions that were not affected in the variant CH3 domain compared to the wild-type sequence can be modified with a conservative substitution to obtain another variant CH3 domain that provides the same CH3 preference.

[0229] Provided below is a summary of some of the CH3 sets identified as shown in the Examples and that offer at least one superior property, such as higher heterodimerization, than existing CH3 heterodimerization variant CH3 domain sets. For example, as shown in the Examples, when expressed as "modified Fc" on yeast cells, sets (1)-(7) all exhibit superior heterodimerization as measured by flow cytometry. Some additional superior properties (non-exhaustive) of each of (1)-(7) are also provided below.

[0230] (1) “W-SG” set with or without CH3 disulfide substitution The "W-SG" set contains T366W in one CH3 domain and T366S and Y407G in another CH3 domain.

[0231] For example, the "W-SG" set exhibits higher % heterodimer values ​​than the tested controls (EW-RVT and KiH) as measured by AlphaLISA® (see Figure 11B). The "W-SG" set with CH3 disulfide substitutions further exhibits much higher % heterodimer values ​​(100%) than the tested controls (EW-RVT and KiH with CH3 disulfide substitutions) when expressed as BsAbs in HEK cells as measured by LC-MS (see Table 13). Furthermore, the "W-SG" set with and without CH3 disulfide substitutions exhibits less aggregation compared to the respective tested control existing technologies (EW-RVT and KiH with and without CH3 disulfide substitutions) as measured by SEC (see Table 6). In addition, the "W-SG" set with CH3 disulfide substitutions showed higher yields than the existing technologies tested (EW-RVT and KiH with CH3 disulfide substitutions) (see Table 6).

[0232] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 11 and 12, respectively, the amino acid sequences of SEQ ID NOs: 13 and 14, respectively, or the amino acid sequences of SEQ ID NOs: 15 and 16, respectively.

[0233] (2) "VV" set with or without CH3 disulfide substitution The "VV" set contains T366V in one CH3 domain and Y407V in another CH3 domain.

[0234] For example, the "VV" set exhibits higher % heterodimer values ​​than the tested existing technologies (EW-RVT and KiH) as measured by AlphaLISA® (see Figure 11B). The "VV" set also exhibits higher yields than the tested controls (EW-RVT and KiH) when produced in HEK293 cells (see Table 6). Furthermore, the "VV" set, with and without CH3 disulfide substitutions, exhibits less aggregation compared to the respective tested control existing technologies (EW-RVT and KiH, with and without CH3 disulfide substitutions, respectively) as measured by SEC (see Table 6). In addition, the "VV" set, when expressed as a BsAb in HEK293 cells, provides less aggregation and a much higher yield (207 mg / L) than the tested existing technologies (EW-RVT and KiH) as measured by SEC (see Table 13 and Figure 18F).

[0235] Furthermore, when bsAbs containing the "VV" set were produced using the cFAE-mediated production method, 100% of the products were the intended bsAbs, and no mismatches were observed (see Table 18), indicating that the "VV" set is superior to the existing "RL" set in terms of bsAb production efficiency. In addition, it was further found that bsAbs containing the "VV" set were resistant to glutathione exposure (see Figures 28A-28E).

[0236] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 21 and 22, respectively, the amino acid sequences of SEQ ID NOs: 23 and 24, respectively, or the amino acid sequences of SEQ ID NOs: 25 and 26, respectively.

[0237] (3) "QR-F" set with and without CH3 disulfide substitution The "QR-F" set contains T366Q and K409R, and L368F in one CH3 domain.

[0238] For example, the "QR-F" set, when expressed as a modified Fc, exhibits (i) a higher % heterodimer value (100%) as measured by LC-MS, and (ii) a higher Tm (64°C) than the existing technology (KiH) tested (see Table 8). The "QR-F" with CH3 disulfide substitution, when expressed as a BsAb in HEK293 cells, exhibits a much higher % heterodimer (100%) as measured by LC-MS than the KiH control (see Table 10). In addition, the "QR-F" set, with or without CH3 disulfide substitution, provides less aggregation as measured by SEC than the related existing technology controls tested (EW-RVT and KiH with CH3 disulfide substitution) (see Table 13 and Figure 18D).

[0239] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 31 and 32, respectively, the amino acid sequences of SEQ ID NOs: 33 and 34, respectively, or the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.

[0240] (4) “RG-FG” set with or without CH3 disulfide substitution The "RG-FG" set contains T366R and K409G, as well as L368F and K370G in one CH3 domain.

[0241] For example, the "RG-FG" set, when expressed as a modified Fc, exhibits (i) a higher % heterodimer value (90%) as measured by LC-MS, and (ii) a higher Tm (64°C) than the existing technology tested (KiH) (see Table 8). The "RG-FG" set with a CH3 disulfide substitution, when expressed as a BsAb in HEK cells, also exhibits a much higher % heterodimer value (100%) as measured by LC-MS than the controls tested (EW-RVT and KiH with a CH3 disulfide substitution) (see Table 13).

[0242] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 41 and 42, respectively, the amino acid sequences of SEQ ID NOs: 43 and 44, respectively, or the amino acid sequences of SEQ ID NOs: 45 and 46, respectively.

[0243] (5) “TL-QL” set with and without CH3 disulfide substitution The "TL-QL" set contains S400T and K409L, as well as D399Q and F405L in one CH3 domain.

[0244] For example, the "TL-QL" set, when expressed as a modified Fc, exhibits a higher Tm (65 degrees Celsius) than the existing technology tested (KiH) (see Table 8).

[0245] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 51 and 52, respectively, the amino acid sequences of SEQ ID NOs: 53 and 54, respectively, or the amino acid sequences of SEQ ID NOs: 55 and 56, respectively.

[0246] (6) “DVG-VSY” set with or without CH3 disulfide substitution The "DVG-VSY" set contains S364D, Y407V, and K409G, as well as T366V, L368S, and K370Y in one CH3 domain.

[0247] For example, the "DVG-VSY" set with CH3 disulfide substitutions exhibits less aggregation when expressed as a BsAb in HEK293 cells than the existing technology tested (KiH) as measured by SEC (see Table 11). In addition, the "DVG-VSY" set with CH3 disulfide substitutions provides less aggregation than the related existing technology controls tested (EW-RVT and KiH with CH3 disulfide substitutions) as measured by SEC (see Table 13 and Figure 18D).

[0248] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 61 and 62, respectively, the amino acid sequences of SEQ ID NOs: 63 and 64, respectively, or the amino acid sequences of SEQ ID NOs: 65 and 66, respectively.

[0249] (7) “LWG-SIG” set with or without CH3 disulfide substitution The "LWG-SIG" set contains S364L, T366W, and K409G in one CH3 domain of the set, and T366S, L368I, and Y407G in the other CH3 domain of the set.

[0250] For example, the "LWG-SIG" set, when expressed as a modified Fc, exhibits a higher Tm (62.5 degrees Celsius) than the existing technology tested (KiH) (see Table 8). Furthermore, the "LWG-SIG" set, with or without CH3 disulfide bond substitution, when expressed as an anti-CD3 / anti-HER2 BsAb, exhibits a higher % heterodimer as measured by LC-MS or IEX than the existing technology tested (KiH) (see Figure 18C and Table 13).

[0251] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 71 and 72, respectively, the amino acid sequences of SEQ ID NOs: 73 and 74, respectively, or the amino acid sequences of SEQ ID NOs: 75 and 76, respectively.

[0252] Any of the variant CH3 domains described above or herein may be part of a polypeptide, such as a heavy chain polypeptide. Such polypeptides, such as heavy chain polypeptides, are also encompassed by the present invention.

[0253] Polypeptides, molecules, and multispecific antibodies A variant CH3 domain according to the present disclosure may be present within a polypeptide such as an immunoglobulin polypeptide, molecule, and / or a multispecific antibody.

[0254] As used herein, an "immunoglobulin polypeptide" refers to a polypeptide comprising at least one immunoglobulin domain (e.g., a CH3 domain). In certain cases, a first CH3 domain can be present in a first polypeptide. In certain cases, a second CH3 domain can be present in a second polypeptide. If the first CH3 domain and the second CH3 domain are CH3 domains that preferentially form CH3-CH3 heterodimers, a heteromeric (e.g., dimeric) molecule can be formed between the first and second polypeptides. Such heteromeric molecules can be multispecific antibodies having structures such as, but not limited to, those disclosed in Figures 2-8.

[0255] In some embodiments, such heterodimer-preferred CH3 domain sets can be any one of the following: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, WTL-SAVQL, WTL-SGQL, WQL-SAVTL, WQL-SGTL, VTL-VQL, VQL-VTL, QRQL-FTL, VQR-VF, or LWG-IG. In some embodiments, such heterodimer-preferred CH3 domain sets can be any one of the following: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, or LWG-IG. In some embodiments, such heterodimer-preferred CH3 domain sets include CH3 disulfide bond substitutions ("345 / 354" or "354 / 345"), i.e., W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY ... 9 / 354), LWG-SIG(349 / 354), WTL-SAVQL(349 / 354), WTL-SGQL(349 / 354), WQL-SAVTL(349 / 354 ), WQL-SGTL(349 / 354), VTL-VQL(349 / 354), VQL-VTL(349 / 354), QRQL-FTL(349 / 354), VQR-VF( 349 / 354), or LWG-IG(349 / 354), or W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL(354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), WTL-SAVQL(354 / 349), WTL- It may be any of the foregoing, further including SGQL(354 / 349), WQL-SAVTL(354 / 349), WQL-SGTL(354 / 349), VTL-VQL(354 / 349), VQL-VTL(354 / 349), QRQL-FTL(354 / 349), VQR-VF(354 / 349), or LWG-IG(354 / 349).In some embodiments, such CH3 heterodimers can comprise any of the following CH3 sets: W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY(349 / 354), LWG-SIG(349 / 354), LWG-IG(349 / 354), W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL(354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), or LWG-IG(354 / 349). The amino acid substitution positions in these CH3 sets are specified in Appendix Tables E–G).

[0256] Such immunoglobulin polypeptides may further comprise one or more antigen-binding domains (such as a VH, VL, scFv, or nanobody), a CH1 domain, and / or a CH2 domain. An additional CH3 domain (with or without amino acid substitutions) may further be included. Such polypeptides may be part of a multispecific antibody molecule. In some embodiments, a peptide may comprise an antigen-binding domain (such as a VH, VL, scFv, or nanobody) and a variant CH3 domain. In some embodiments, a peptide may comprise an antigen-binding domain (such as a VH, VL, scFv, or nanobody), a CH1 domain, and a variant CH3 domain. In some embodiments, a peptide may comprise an antigen-binding domain (such as a VH, VL, scFv, or nanobody), a CH2 domain, and a variant CH3 domain. In some embodiments, a peptide may comprise an antigen-binding domain (such as a VH, VL, scFv, or nanobody), a CH1 domain, a CH2 domain, and a variant CH3 domain.

[0257] Alternatively, an immunoglobulin polypeptide may not comprise a VH, VL, CH1, or CH2 domain. For example, a first polypeptide may further comprise a first domain in addition to a first CH3. If a second polypeptide further comprises a second domain in addition to a second CH3 that preferentially forms a heterodimer with the first CH3, and heterodimerization between the first and second domains is desired, heterodimerization between the first and second domains is promoted by preferential heterodimerization between the first and second CH3 domains.

[0258] In one embodiment, such polypeptides can optionally utilize other variants outside of the CH3 domain in combination with a variant CH3 domain to further promote preferential pairing between two polypeptides that differ from each other.

[0259] In one embodiment, such polypeptides may optionally utilize a variant CH1 domain in combination with a variant CH3 domain that promotes preferential lambda pairing or preferential kappa pairing. In another embodiment, such polypeptides may optionally utilize a variant CH1 domain in combination with a variant CH3 domain that preferentially pairs with a variant kappa CL domain over another CL domain, such as a wild-type kappa CL domain. In another embodiment, such polypeptides may optionally utilize a variant CH1 domain in combination with a variant CH3 domain that preferentially pairs with a variant lambda CL domain over another CL domain, such as a wild-type lambda CL domain. In yet another embodiment, such polypeptides may optionally utilize a variant kappa or lambda CL domain in combination with a variant CH3 domain that preferentially pairs with a variant CH1 domain over another CH1 domain, such as a wild-type CH1 domain. Such polypeptides may be used to facilitate the generation of antibodies specific for three or more antibodies (e.g., tetrabodies).

[0260] Any such polypeptide can be present in a molecule having a first polypeptide comprising a first variant CH3 domain and a second polypeptide comprising a second variant CH3 domain that preferentially forms a CH3-CH3 heterodimer with the first CH3. In some cases, the first and second polypeptides can be further linked, for example, via one or more disulfide bonds, linkers, etc.

[0261] Such molecules can be multispecific antibodies or antigen-binding fragments having structures such as, but not limited to, those disclosed in Figures 2-8. Multispecific antibodies according to the present disclosure can be bispecific, trispecific, tetraspecific, or specific for five, six, or more epitopes. Multispecific antibodies according to the present disclosure can be bivalent, trivalent, or tetravalent, or can have five, six, or more valencies.

[0262] Polynucleotides, Vectors, Cells, and Compositions Polypeptides, molecules, and / or multispecific antibodies comprising a variant CH3 domain described herein may be encoded by a polynucleotide(s). Such polynucleotide(s) may be DNA or RNA, or a combination thereof.

[0263] Any of the polypeptides described herein can be present in a vector.

[0264] Any CH3 domain, polypeptide, molecule, multispecific antibody, polynucleotide, and / or vector can be present in a cell, e.g., a eukaryotic cell. In some embodiments, such polypeptides can be expressed in mammalian cells, such as HEK293 cells or Chinese hamster ovary (CHO) cells. In some embodiments, the variant CH3 domain is expressed in yeast (e.g., Saccharomyces cerevisiae). In some embodiments, the yeast strain co-expresses one or more polypeptides, such as one or more light chains.

[0265] Any CH3 domain, polypeptide, molecule, multispecific antibody, polynucleotide, vector, and / or cell may be present in a composition. If the composition is a therapeutic composition, the composition may further comprise a pharmaceutically acceptable carrier.

[0266] CH3 domain library and variant CH3 domain screening / selection Methods for generating CH3 domain libraries are also contemplated by the present disclosure, which can be used to screen for CH3 sequences and sets that preferentially form CH3 heterodimers, among others.

[0267] In some embodiments, at least one nucleic acid position within a codon encoding any of the amino acid positions of a CH3 in which an amino acid substitution is present in any of the invention CH3 sets can be varied. For example, such predetermined amino acid positions can be 364, 366, 368, 370, 399, 400, 405, 407, and / or 409 according to EU numbering, or any combination thereof.

[0268] In some embodiments, any amino acid position listed in Table 7 can be varied.

[0269] In some embodiments, any of the amino acid positions considered to be CH3-CH3 "interface positions" can also be varied.

[0270] In certain embodiments, some of the CH3 domains expressed by the library may contain CH3 disulfide bond substitutions (i.e., S354C / Y349C) in addition to the substitutions caused by mutating.

[0271] In some embodiments, variability can be introduced at a given position using degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N) or degenerate NNK codons representing all 20 naturally occurring amino acid residues.

[0272] Also provided herein are methods for identifying one or more variant CH3 domains and CH3 sets that preferentially form CH3 heterodimers.

[0273] In some embodiments, the method may include at least three steps. The first step may be co-expressing in a cell (e.g., yeast cell, mammalian cell) or ex vivo (1) a first polypeptide comprising a first variant CH3 domain expressed from a first library according to any of the libraries described herein, and (2) a second polypeptide comprising a second variant CH3 domain expressed from a second library according to any of the libraries described herein. The second step may be quantifying the amount of CH3 heterodimers and CH3 homodimers. The third step may be selecting one or more CH3 sets that provide the desired % heterodimer.

[0274] In certain embodiments, the first library and the second library may differ at at least one predetermined amino acid position.

[0275] In some embodiments, the predetermined positions in the first library and the predetermined positions in the second library can comprise or consist of any of the positions or sets of positions substituted in the CH3 sets identified herein as preferential heterodimerization.

[0276] The variability can be performed on any available CH3 sequence, i.e., wild-type or modified CH3 sequence. In some embodiments, the variability can be performed on the reference CH3 domain sequence of SEQ ID NO: 1.

[0277] In certain embodiments, the desired % heterodimer can be greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or about 100%.

[0278] In certain embodiments, the desired % heterodimer can be compared to a reference CH3 set, for example, an existing CH3 heterodimerization technology (e.g., Table 1).

[0279] In some embodiments, the first polypeptide may comprise or be expressed with a first tag, and the second polypeptide may comprise or be expressed with a second tag that is different from the first tag, which may allow for specific identification of CH3 heterodimers by techniques such as AlphaLISA®.

[0280] In some embodiments, the second step of quantifying heterodimers and homodimers may use, for example, liquid chromatography-mass spectrometry (LC-MS), AlphaLISA®, ion exchange chromatography (IEX), and / or flow cytometry.

[0281] In certain embodiments, the identification method may further comprise selecting one or more sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides based on one or more antibody properties. Exemplary properties include (i) (i-1) production yield, optionally assessed in one or more cell types, optionally mammalian cells, e.g., CHO and HEK cells, yeast cells, insect cells, and / or plant cells, and / or (i-2) suitability for one or more antibody purification methods, optionally including Protein A affinity purification; (ii) degree of aggregation, optionally quantified using chromatography, optionally SEC or electrophoresis, optionally SDS-PAGE, and optionally the presence of multimers of full-size antibodies; (iii) rate of correct pairing between CH1 domains and / or between CH1 domains and CL domains, optionally correct pairing, optionally assessed using LC-MS; (iv) DSF and These may include, but are not limited to, Tm and / or Tag, optionally Tagg266, measured using DSC and / or instruments, optionally using Uncle®, (v) pI, (vi) level of interaction with PSR, (vii) hydrophobic interactions of the antibody, optionally measured using HIC, (viii) self-interactions, optionally measured by (viii-1) AC-SINS or (viii-2) DLS, (ix) stability against high or low pH stress, (x) solubility, (xi) production cost and / or time, (xii) other stability parameters, (xiii) shelf life, (xiv) in vivo half-life, and / or (xv) immunogenicity.

[0282] Such properties may depend, at least in part, on (a) the particular structure of the molecule or multispecific antibody or antigen-binding antibody fragment structure incorporating the variant CH3 domain set, and / or (b) the variable domains that provide the particular binding specificity. Suitability can be tested in the particular context of the antibody structure and antigen specificity of interest.

[0283] Thus, also provided herein are methods for screening for a set of first and second variant CH3 domain polypeptides suitable for a multispecific antibody or antigen-binding antibody fragment with a given antigen specificity (e.g., having any of the structures described herein).

[0284] In some embodiments, the method comprises: (a) expressing a plurality of multispecific antibodies and / or antigen-binding antibody fragments comprising different sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides; and (b) selecting one or more sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides based on one or more antibody properties of the plurality of multispecific antibodies and / or antigen-binding antibody fragments expressed in step (a).

[0285] In some embodiments, one or more antibody properties may be selected from properties (i)-(xv) above.

[0286] Methods for producing cFAE-mediated multispecific antibodies or antigen-binding antibody fragments, and multispecific antibodies and antigen-binding antibody fragments produced by such methods Also contemplated by the present disclosure are methods for producing heteromeric molecules comprising a CH3 set (which in some embodiments may be any of the CH3 sets described herein) that preferentially form CH3 heterodimers. The heteromeric molecules may be any of the heteromeric molecules or multispecific antibodies and antigen-binding antibody fragments described herein, optionally having a structure shown in any one of Figures 2-8.

[0287] In some embodiments, a heteromeric molecule of interest (which the present methods are intended to produce) may comprise: (A) a first polypeptide (e.g., a first heavy chain) comprising a first variant CH3 domain polypeptide; and (B) a second polypeptide (e.g., a second heavy chain) comprising a second variant CH3 domain polypeptide, wherein the first and second polypeptides may optionally be linked or paired to each other via at least one disulfide bond.

[0288] The examples herein demonstrated that the "RL" set provides superior bsAb production efficiency without causing any mismatching, as shown in Table 18. In addition, it was further found that bsAbs containing the "VV" set are resistant to glutathione exposure (see Figures 28A-28E).

[0289] Thus, in certain embodiments, the CH3 set incorporated into the heteromeric molecule or multispecific antibody or antigen-binding antibody fragment produced by the present methods can be a "VV" set. In certain embodiments, the CH3 set can include additional substitutions in addition to the "VV" set substitutions, such as, but not limited to, disulfide modifications at positions 349 and 354 described herein (i.e., a VV(349 / 354) set or a "VV(354 / 349)" set).

[0290] In some embodiments, the method may comprise (i) incubating under a reducing environment: (i-1) a first antibody (which may also be referred to as a first parent antibody or a first monospecific parent antibody) comprising at least two of the first polypeptides linked or paired to each other, optionally via at least one disulfide bond; and (i-2) a second antibody (which may also be referred to as a second parent antibody or a second monospecific parent antibody) comprising at least two of the second polypeptides linked or paired to each other, optionally via at least one disulfide bond.

[0291] In some embodiments, the first polypeptide may further comprise a first antigen-binding domain. In some embodiments, the second polypeptide may further comprise a second antigen-binding domain. In some embodiments, the heteromeric molecule may further comprise a third polypeptide comprising a third antigen-binding domain, optionally, the third polypeptide may be bound to or paired with the first polypeptide. In some embodiments, the heteromeric molecule may further comprise a fourth polypeptide optionally comprising a fourth antigen-binding domain, optionally, the fourth polypeptide may be bound to or paired with the second polypeptide.

[0292] In certain embodiments, the first polypeptide may comprise a first antigen-binding domain that forms a first antigen-binding site specific for a first epitope, and / or the heteromeric molecule may comprise a third polypeptide that comprises a third antigen-binding domain that forms a third antigen-binding site specific for a third epitope. Optionally, the first epitope may be the same as or different from the third epitope. In alternative embodiments, the first polypeptide may comprise a first antigen-binding domain and the heteromeric molecule may comprise a third polypeptide that comprises a third antigen-binding domain, wherein the first and third antigen-binding domains form a first antigen-binding site specific for the first epitope.

[0293] In certain embodiments, the second polypeptide may comprise a second antigen-binding domain that forms a second antigen-binding site specific for a second epitope, and / or the heteromeric molecule may comprise a fourth polypeptide that comprises a fourth antigen-binding domain that forms a fourth antigen-binding site specific for a fourth epitope. Optionally, the second epitope may be the same as or different from the fourth epitope. In alternative embodiments, the second polypeptide may comprise a second antigen-binding domain and the heteromeric molecule may comprise a fourth polypeptide that comprises a fourth antigen-binding domain, wherein the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for the second epitope.

[0294] The first and second antibodies may be produced in any suitable cell type, including, but not limited to, mammalian, yeast, insect, plant, or bacterial cells, more specifically, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

[0295] In certain embodiments, the first antibody and the second antibody may be incubated at a temperature of about 15°C to about 40°C, about 20°C to about 40°C, about 25°C to about 35°C, about 28°C to about 32°C, or about 29°C to about 31°C, or about 30°C. In certain embodiments, the first antibody and the second antibody may be incubated for about 30 minutes to about 20 hours, about 1 hour to about 15 hours, about 2 hours to about 10 hours, about 3 hours to about 7 hours, about 4 hours to about 6 hours, or about 5 hours. In certain embodiments, the first antibody and the second antibody may be incubated at about 30°C for about 5 hours.

[0296] In certain embodiments, the first antibody and the second antibody can be incubated in the presence of at least one reducing agent, optionally at least one weak reducing agent. Ideally, the at least one reducing agent or reducing environment is capable of reducing the disulfide bond between two heavy chains (or between a first polypeptide and a second polypeptide), but not between a heavy chain and a light chain. Various reducing agents have been shown to provide this reducing function in the context of FAEs (see, e.g., van der Neut Kolfschoten et al. Science. 2007 Sep 14;317(5844):1554-1557). Exemplary reducing agents include, but are not limited to, 2-mercaptoethylamine (2-MEA), β-mercaptoethanol (BME), L-cysteine, dithiothreitol (DTT), or dithionite.

[0297] In certain embodiments, the at least one reducing agent is about 25 to about 125 mM, about 50 mM to about 100 mM, about 70 to about 80 mM, or about 75 mM 2-MEA, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM BME, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM L-cysteine, about 15 to about 400 μM, about 20 to about 200 μM, about 25 to about 100 μM, about 30 to about 70 μM, or about 50 μM DTT, or The dithionite may be selected from about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM. In certain embodiments, the reducing environment may include at least about 75 mM 2-MEA.

[0298] In some embodiments, the method may then include (ii) placing the incubation product of step (i) in a less reducing or non-reducing environment.

[0299] In certain embodiments, this step (ii) may allow pairing between the first and second variant CH3 domains, and thus pairing between the first and second polypeptides.

[0300] In certain embodiments, the disposition may be via buffer exchange, allowing for the removal of reducing conditions such as reducing agents. For example, the buffer may be exchanged with PBS.

[0301] In certain embodiments, buffer exchange may be performed by desalting or diafiltration.

[0302] In another embodiment, the disposing may be performed by adding an oxidizing agent.

[0303] In some embodiments, the product of step (ii) may be incubated in a low-reducing or non-reducing environment. In certain embodiments, incubation may occur at a temperature of about 1°C to about 20°C, about 2°C to about 10°C, about 3°C ​​to about 5°C, or about 4°C. In certain embodiments, incubation may occur for about 12 hours to about 154 hours, about 24 hours to about 96 hours, about 36 hours to about 72 hours, or about 48 hours. In certain embodiments, incubation may occur at about 4°C for about 48 hours.

[0304] In some embodiments, the product of step (ii) and / or step (iii) may be analyzed for the amount of the desired multispecific antibody or antigen-binding antibody fragment in the product of step (ii) and / or step (iii). In some embodiments, the product of step (ii) and / or step (iii) may be purified to obtain a purified multispecific antibody or antigen-binding antibody fragment. In certain embodiments, such analysis and / or purification may be performed by chromatography, such as, but not limited to, LC-MS, IEX, and / or SEC. In certain embodiments, mispairings may not be observed by LC-MS.

[0305] Any multispecific antibodies and antigen-binding antibody fragments produced by the production methods are further encompassed by the present disclosure.

[0306] Examples are provided below to illustrate the present invention. These examples are not intended to limit the invention to any particular application or theory of operation. [Example]

[0307] In the Examples described herein, a CH3 domain reference sequence (SEQ ID NO: 1) was used as the wild-type CH3 domain sequence of IgG1, and various amino acid substitutions were incorporated into the reference sequence to test for heterodimerization potential. Some of the sequences used in the Examples are provided in Appendix Tables A-G and in the Sequence Listing. While SEQ ID NO: 1 was used as the CH3 domain reference sequence in the Examples, the present invention regarding CH3 domain sequence modifications may also be applied to other CH3 domain reference sequences, such as, but not limited to, SEQ ID NO: 2, 3, or 4 (for IgG1), or another standard CH3 sequence for IgG1, IgG2, IgG3, or IgG4.

[0308] Unless otherwise stated, CH1 and CH2 reference sequences (SEQ ID NO: 6 and SEQ ID NO: 7, respectively) were used in the examples where applicable.

[0309] Example 1: Evaluation of flow cytometry-based selection of modified Fc-displaying yeast libraries as a readout of CH3 domain dimerization using existing CH3 heterodimerization technology (proof-of-concept study). To screen for heterodimer-preferring variant CH3 domains, we designed a yeast library system in which each cell displayed a "modified Fc" (a portion of the Fc encompassing positions D221 to K447 (EU numbering), a hinge (SPPS instead of CPPC), a modified CH2 domain (N297A), and a portion of the CH3 domain with either the wild-type or variant sequence) and initially analyzed it by flow cytometry to enrich for cell populations containing CH3 heterodimers.

[0310] To initially evaluate this system as a method for identifying variant CH3 domains that preferentially form CH3 heterodimers, the system was tested using existing variant CH3 domain sets, W-SAV (i.e., KiH) and EW-RVT (see Table 1), as controls. These two CH3 sets were confirmed to exhibit a high preference for CH3 heterodimer formation when used in bispecific antibodies, as measured by AlphaLISA® and liquid chromatography-mass spectrometry (LC-MS) (using methods described herein).

[0311] A library of variant CH3 domains was constructed and expressed in the engineered yeast strain. Specifically, two heavy chain expression plasmids (pAD6234 and pAD6233) were constructed, each containing a "modified Fc." pAD6234 contained a FLAG tag, while URA and pAD6233 contained a HIS tag and TRP.

[0312] Three sets of pAD6234 and pAD6233 were generated. The first set was the KiH substitution set (knob HIS hole FLAG ), and the second set encodes the EW-RT set (EW HIS RVT FLAG ), and the third set encodes a WT-WT set (also referred to as the "WT set") (WT HIS -WT FLAG ) encoded a CH3 domain containing

[0313] Yeast proof-of-concept (POC) libraries were generated by transfecting yeast cells with the first, second, and third plasmid sets at a 1:1:10,000 ratio, growing them as previously described (see, e.g., WO2009036379, WO2010105256, WO2012009568, Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663-70), and subjected to flow cytometry-based selection of high FLAG expressers (representing CH3 heterodimer expressers) (schematic diagram in Figure 9A).

[0314] Briefly, after expression of the modified Fc, the engineered yeast cells (approximately 10 7 ~10 8 ) were stained with anti-HIS FITC (Invitrogen, Carlsbad, California, Catalog No. MA1-81891) diluted 1:100 and anti-FLAG APC (BioLegend, San Diego, California, Catalog No. 637308) diluted 1:500 in PBSF for 15 minutes at 4°C. After two washes with ice-cold wash buffer, the cell pellet was resuspended in 0.4 mL of PBSF and transferred to a strainer-capped sort tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sort gates were determined to enrich for heterodimers. The library was selected over two rounds. Clones bearing both the HIS and FLAG tags were sequenced from the fourth and fifth rounds and analyzed for sequence uniqueness. Cells were plated on medium lacking uracil and tryptophan to generate single isolates and sequence them.

[0315] As expected, cells expressing the CH3 heterodimer (KiH or EW-RVT) were significantly enriched after two selection rounds (Figure 9B), demonstrating that the modified Fc library coupled with flow cytometry-based selection can indeed be used to identify CH3 heterodimer-preferring variant CH3 domains.

[0316] Example 2: Cycle 1 selection using a variant CH3 domain library saturating one or more of the four KiH positions. In this example, the variant CH3 domain selection system described in Example 1 was used to identify novel variant CH3 domains with amino acid substitutions at one or more of the KiH substitution positions (KiH has a W at position 366 (the "knob" position) in one CH3 and an S, A, and V at positions 366, 368, and 407 (the "hole" positions) in another CH3; see Table 1).

[0317] Specifically, two pools of variant CH3 domain DNA fragments were generated and inserted into expression plasmids (Figure 10A). The first pool was generated by allowing substitution of all 20 amino acids at positions T366 in the first CH3 domain and T366, L368, and Y407 in the second CH3 domain. Two variations were made within the first pool, one of which was a hole-position variation in the strand bearing the Flag tag (knob 1). SSM -HIS Hall SSM -FLAG) (Library 1 in Figure 10A), and the other one has a knob position variable in the strand with a Flag tag (Hole SSM -HIS knob SSM -FLAG) (Library 2 in Figure 10A). A second pool was generated by fixing the first CH3 domain constant position T336W for all 20 amino acids and varying positions T366, L368, and Y407 in the second CH3 domain (Knob-HIS-Hole). SSM -FLAG) (Library 3 in Figure 10A) A library was constructed using DNA shuffling as previously described (Stemmer, Proc. Natl. Acad. Sci., 91 (1994), pp. 10747-10751). The library was expanded and subjected to six selection rounds, and the selection products were sequenced as described in Example 1 (Figures 10B-10D).

[0318] As shown in Table 2, selection did not result in a disruption of order. [Table 2]

[0319] At the end of round 6, 86 unique CH3 heterodimers were found. The top substitution combinations at the knob-into-hole (KiH) positions, along with their frequency of occurrence (repeats), are provided in Table 3. [Table 3]

[0320] In the following examples, 86 unique CH3 heterodimer sequences were generated in yeast and characterized by AlphaLISA®, ion exchange chromatography (IEX), and size exclusion chromatography (SEC). Melting temperatures were also determined.

[0321] Example 3: AlphaLISA® analysis of the CH3 set identified in Example 2. The 86 unique CH3 heterodimers identified in Example 2 were analyzed by AlphaLISA® (Figure 11A, left). AlphaLISA® was used to determine the relative degree of heterodimerization of Fc fragments. Briefly, 0.5 nM of the fragments were added to Perkin Elmer AlphaLISA immunoassay buffer (10x) along with 10x biotin-a-Flag (5 μl, 20 nM final test concentration) as a 0.5 nM final test concentration solution and placed in a 384-well AlphaPlate (Perkin Elmer). Then, 10x acceptor bead solution (5 μl) with a-His was added, and the plate was covered with a black cover and incubated at room temperature for 1 hour. Next, 10x (5 μl) donor bead (SA-coated) solution was added to the assay in the dark and incubated at room temperature for 30 minutes. The plate was read using the EnSpire Alpha program (Perkin Elmer).

[0322] Data on existing CH3 heterodimerization substitution sets demonstrated that CH3 heterodimerization can be measured using AlphaLISA® (Figure 11A, right). These results are provided in Figure 11B. Five CH3 sets exhibited AlphaLISA® values ​​higher than the positive controls (KiH and EW-RT) (Table 4). AlphaLISA® values ​​(fold over background) were further plotted against α-Flag APC values ​​(fold over background) on the x-axis in Figure 11C. The "VV" set was found in both orientations (366V for FLAG and 407V for FLAG). [Table 4]

[0323] Example 4: Size exclusion chromatography (SEC) analysis of the CH3 set selected in Example 3. The expression and quality of purified antibodies containing one of the five CH3 sets ("VV") identified in Example 3 were tested in both orientations (a total of six variants) and evaluated by size-exclusion chromatography (SEC). A higher main peak percentage indicates higher quality. Briefly, column chromatography (TSKgel Super SW3000 column) was monitored using an Agilent 1100 HPLC. Prior to use, the column was preconditioned with highly glycosylated, aggregated IgG to minimize potential antibody-column interactions and equilibrated with wash buffer (200 mM sodium phosphate, 250 mM sodium chloride, pH 6.8). Approximately 2-5 μg of protein sample was injected onto the column, and the flow rate was adjusted to 0.400 mL / min. Protein migration was monitored at a wavelength of 280 nm. The total assay time was approximately 11 min. Data were analyzed using ChemStation software.

[0324] The SEC chromatographs of the WT and control CH3 sets (W-SAV (i.e., KiH) and EW-RVT) are shown in Figure 12A, and the SEC chromatographs of the CH3 sets selected in Example 3 are shown in Figure 12B.

[0325] Example 5: Ion Exchange (IEX) Chromatography Analysis of Cycle 1 Output Ion exchange chromatography (IEX) was performed on a subset of clones. All chromatographic separations were performed on a computer-controlled AKTA Avant 150 preparative chromatography system equipped with an integrated pH electrode allowing in-line pH monitoring and a Mono S 5 / 50 GL column. The cation exchange buffer consisted of 15.6 mM CAPS, 9.4 mM CHES, 4.6 mM TAPS, 9.9 mM HEPPSO, 8.7 mM MOPSO, 11.0 mM MES, 13.0 mM acetate, 9.9 mM formate, and 10 mM NaCl. The pH was adjusted to 4.0 (Buffer A) or 11.0 (Buffer B) using NaOH. 500 μg of protein was buffer exchanged into 25% Buffer B and filtered through a 0.2 mm filter. Before each separation, the column was equilibrated with 10 column volumes of 25% Buffer B. The protein was then loaded onto the column via a capillary loop, followed by a 10 column volume wash with 25% buffer B, a linear pH gradient from 25% to 100% buffer B over 20 column volumes, and a 10 column volume hold at 100% B.

[0326] IEX chromatographs of the WT and control CH3 sets (W-SAV (i.e., KiH) and EW-RVT) are shown in Figure 13A, and SEC chromatographs of selected CH3 sets in Example 3 are shown in Figure 14B. Several CH3 sets with low AlphaLISA® values ​​exhibited poor quality as measured by SEC and IEX.

[0327] Example 6: Production of cycle 1 output in HEK293 The effects of the identified variant CH3 domains on a control bispecific co-antibody in an IgG-like format (two Fab regions linked at the N-terminus of a dimeric Fc molecule) were also evaluated. The W-SG substitution set (containing T366W in one CH3 and T366S and Y407G in the other) and the VV substitution set (containing T366V in one CH3 and Y407V in the other) were selected as exemplary test sets and produced in HEK293 cells as anti-Her2 / anti-CD3 bispecific antibodies. VH-CH1 sequences derived from two antibodies, ADI-29235 (anti-HER2) and ADI-26908 (anti-CD3), were used. The wild-type CH3 set, the W-SAV set (i.e., KiH), and the EW-RVT set were included as controls. Additionally, a CH3 domain substitution (S354C / Y349C) was introduced to promote the desired heterodimeric pairing of the heavy chains. The CH3 sets tested are summarized in Table 5.

[0328] The DNA plasmids were verified by Sanger sequencing and then transfected into HEK293 cells using standard protocols. Transfected HEK cells were cultured in CD optiCHO medium (Invitrogen), and supernatants were harvested 6 days after transfection and subjected to Protein A-based affinity purification. [Table 5]

[0329] Anti-Her2 / anti-CD3 bispecific antibodies produced in HEK293 cells, including control antibodies (WT or containing CH3 sets containing W-SAV(KiH) or EW-RVT substitutions), are summarized in Figure 14A.

[0330] The percentage of heterodimers (HC1 / HC2 heterodimers) and homodimers (HC1 homodimers and HC2 homodimers) among the full-sized antibodies, as well as the presence of half antibodies ("1 / 2 Ab," i.e., containing only one heavy chain (HC)), were analyzed by LC-MS. To assess CH3 heterodimerization using LC-MS, antibody samples were digested with PNGaseF glycosidase to remove N-linked glycans and then injected onto an Acquity ultra-performance liquid chromatography (UPLC) system (Waters) equipped with a Thermo Scientific MabPac RP® 4 μm column (2.1 × 100 mm) maintained at 80°C. After injection, the sample was eluted from the column using a 13-minute gradient of 20–55% acetonitrile (mobile phase A: 0.1% formic acid / HO solution; mobile phase B: 0.1% formic acid / acetonitrile solution) at a flow rate of 0.3 mL / min. Species eluted from the column were detected with a Q Exactive mass spectrometer (Thermo) in positive electrospray ionization mode. Instrument parameters were set as follows: spray voltage 3.5 kV, capillary temperature 350 °C, sheath gas flow rate 35, auxiliary gas flow rate 10, and S-lens RF level 90. MS spectra were acquired with a scan range of 750–4000 m / z. The acquired MS data were analyzed using Biopharma Finder software (Thermo Scientific), followed by manual inspection to ensure accurate assignment and relative quantification accuracy. The relative quantification of each heterodimeric and homodimeric species was calculated based on the intensity of the peak relative to the sum of all heterodimeric and homodimeric peak intensities.

[0331] The HEK-produced product was also analyzed by Protein A-based size-exclusion chromatography (SEC) and ion-exchange (IEX) chromatography, and the chromatographic profiles from SEC and IEX are shown in Figures 14B and 14C. The LC-MS results, SEC results, and resulting titers are summarized in Table 6. "(354 / 349)" means that HC1 contained Y349C and HC2 contained S354C. [Table 6]

[0332] Example 7: Cycle 2 library generation based on CH3-CH3 interface position and cycle 1 output. To further explore variant CH3 domains that preferentially form CH3-CH3 heterodimers, we designed a new CH3 domain library based on amino acid positions at the CH3-CH3 interface (interface positions) and KiH positions (including cycle 1 output substitutions). First, to identify interface positions, 32 high-resolution aligned wild-type CH3 crystal structures were assembled from the Protein Data Bank (PDB) and used in a structure-guided approach to identify CH3 interface residues for diversification. Interface residues for variability were defined as those with 1) a side-chain SASA (solvent-exposed surface area) of 15% or more in the monomer, 2) adjacent atom contact distances of 8.2 Å or less (a distance set to capture the distance between known knob-in-hole mutations), and 3) residues that are not facing away from the partner strand or solvent-exposed (as determined by manual inspection). Applying these rules, 24 variable positions at the CH3 interface were identified.

[0333] Next, we designed a library to test one or two "anchor" mutations on one side of the interface (on strand A) against one, two, or three "neighbor" mutations on the other side of the interface (on strand B). For each anchor position A on strand A, a set of neighbor positions B was identified as the subset of interface positions on strand B that contact position A (with an interstrand Cb-Cb distance of less than 8.2 Å (Cb = beta carbon). In the case of glycine, the C-alpha atom was used because glycine does not have a Cb atom). We then generated all possible singlet, doublet, and triplet mutation combinations within the neighbor (B) set. Dimers and trimers containing pairs of residues not within an intrastrand Cb-Cb distance cutoff of 8.0 Å or less were filtered out. The resulting set identified the following neighbor mutations for testing: 24 singlets, 39 doublets, and 16 triplets.

[0334] The set of neighbor / anchor pairing positions was divided into 14 library pools for screening by the following steps: 1) neighbor / anchor pairing positions were sorted by increasing diversity (singlets, doublets, triplets) and by common position in the protein, and 2) neighbor / anchor pairs were combined into pools (selecting the closest pool as measured by interstrand contact distance) until a limit of diversity was reached. Each individual library pool contained approximately 10 6 In addition, two pools were constructed based on the output obtained in Example 1 (T366V / Y407V ("VV") and T366W / T366S Y407G ("W-SG")). The anchor and neighbor positions and position combinations that were variegated, as well as the DNA and amino acid sequence diversity possible through variegation in some of the pools, are summarized in Table 7. DNA sequence diversity was calculated as follows: Diversity = ((N アンカー単量体 ×32 1 +N アンカー二量体 ×32 2 )×(N ネイバー単量体× 32 1 +N ネイバー二量体 ×32 2 +N ネイバー三量体× 32 3 )). [Table 7]

[0335] Example 8: Cycle 2 Selection Step 1: Selection using modified Fc displayed on yeast. Library DNA was synthesized using the BioXp system and transformed into yeast as described above. Clones were selected using anti-His and anti-Flag reagents for high His and Flag signals. The library was selected over five rounds (Figure 15A). Clones from rounds 4 and 5 were sequenced and analyzed for sequence uniqueness. A set of 430 unique variant CH3 domains was obtained.

[0336] The 430 CH3 sets were characterized by IEX (subset) and AlphaLISA as previously described. Rosetta scoring was also used to characterize the 430 CH3 sets. ΔΔG, defined as the change in interfacial binding energy (predicted by Rosetta), was determined as described in Barlow et al., J Phys Chem B (2018), pp. 5389-5399. Briefly, ΔΔG was calculated for the input PDB crystal structure and averaged (1l6x, 2iwg, 4wi2, 5gsq). Next, the ΔΔG score for the heterodimer state was calculated along with the ΔΔG scores for both possible homodimer states. Finally, the Rosetta heterodimerization score (RHS) was calculated: RHS = ΔΔG ヘテロ二量体 -Min(ΔΔG ホモ二量体A , ΔΔG ホモ二量体B ).

[0337] Based on the uniqueness, IEX, AlphaLISA®, and Rosetta characterization data, 48 CH3 sets were selected for further production in HEK293 cells in Example 9. A comparison of three variables, IEX (percent interchain contacts), AlphaLISA® values, and Rosetta scores, between the selected and unselected CH3 sets is provided in Figure 15B.

[0338] The set was further characterized by sequence uniqueness using t-SNE (t-distributed stochastic neighbor embedding) visualization. t-SNE plots were constructed to visualize the position space in a 2D plot, with each point representing a set of mutation positions, and points close to each other on the plot contained similar mutation positions. The clones selected for carryover spanned a wide range of the design space, as visualized in the t-SNE plot (Figures 15C and 15D). The clones selected for carryover spanned a wide range of the design space, as visualized in the t-SNE plot.

[0339] Example 9: Cycle 2 Selection Step 2: Selection using modified Fc production in HEK293 cells. The set of 48 variant CH3 domains selected in Example 8 were cloned as CH2-CH3 constructs, produced in HEK293 cells, and further characterized using LCMS (as described above), melting temperature, and 14-day stability.

[0340] Melting temperatures (Tm) were measured by differential scanning fluorometry (DSF). Twenty microliters of sample was mixed with 10 μl of 20x Sypro orange (Sigma-Aldrich) at 0.1–1 mg / ml and then subjected to a controlled temperature ramp from 40 to 95°C in 0.5°C intervals in a C1000 thermocycler (BioRad) to collect Fret signals. Melting temperatures were obtained by taking the negative of the first derivative of the raw signal.

[0341] For accelerated stability testing, samples were incubated in HBS at 40°C for 14 days, with samples taken on days 0, 1, 2, 7, and 14. Samples were then analyzed for aggregation by SEC. For SEC analysis, the running buffer composition was 200 mM sodium phosphate, 250 mM sodium chloride, pH 7.0. The accelerated stability slope was calculated from the percent aggregation measured by SEC.

[0342] Based on additional characterization data, five CH3 sets were selected as cycle 2 outputs and further produced as IgG-like bispecific antibodies (BsAbs) in Example 10. Figure 16 provides plots showing the % heterodimer (measured by LC-MS) and stability (measured by SEC) of the tested CH3 sets, with the filled data points representing the variant CH3 domain sets designated for bispecific antibody production in HEK293 cells. Table 8 summarizes the five cycle 2 outputs, along with controls (wild-type and W-SAV (i.e., KiH)), with their respective % heterodimer values ​​measured by LC-MS and melting temperatures, Tm, measured by differential scanning fluorimetry (DSF). [Table 8]

[0343] Example 10: Characterization of cycle 2 output produced as bispecific antibodies (BsAbs) in HEK293 cells. The five cycle 2 output variant CH3 domain sets selected in Example 9 were produced as BsAbs with three different Fv sets, and heterodimerization efficiency was evaluated in an IgG-like format. As shown in Figure 17A, a total of five different structures per output variant CH3 domain set were used, with three Fv sets in different orientations (Orientation 1 or Orientation 2): anti-CD3 / anti-HER2 (Adimab), anti-CD20 / anti-CD3 (Regeneron), or anti-HEL / anti-BCMA (Nanjing Legend Bio / Janssen). The anti-BCMA antigen-binding domain is a nanobody (VHH).

[0344] The wild-type CH3 domain set (i.e., dimer of reference sequence SEQ ID NO: 1) of either the bispecific or monospecific antibody was used as a negative control, and the W-SAV (i.e., KiH) substitution set used in the anti-CD3 / anti-HER2 bispecific antibody was used as a positive control. Additionally, anti-CD3 / anti-HER2 bispecific antibodies were produced containing either the wild-type CH3 set in orientation 1 or the cycle 2 output variant CH3 domain set, which additionally incorporated the S354C / Y349C substitutions.

[0345] In total, 41 different antibodies were produced, as summarized in Table 9. The heavy and light chain sequences of the antibodies in Table 9 are provided in Appendix Tables A-D. [Table 9-1] [Table 9-2]

[0346] The antibodies in Table 9 produced in HEK293 cells were analyzed by LC-MS (% heterodimer and % homodimer of full-size antibody), IEX (% heterodimer), and AlphaLISA® as described above. The results are summarized in Table 10. [Table 10-1] [Table 10-2]

[0347] The antibodies in Table 9 produced in HEK293 cells were further analyzed by SEC for accelerated stability, i.e., % intact antibody on Protein A purified samples at day 0 (the day of production) ("% monomeric intact Ab") and change in % intact antibody ("% monomeric intact Ab") up to day 14. The SEC results and process yields from production in HEK293 cells are summarized in Table 11. [Table 11-1] [Table 11-2] The % heterodimer data, % monomeric full-size antibody data, and yields were further compared between specific sets of antibodies from the 41 antibodies (Figures 17B-17J). In these comparisons, the % heterodimers measured by LC-MS were comparable between BsAbs with the same CH3 substitution when comparing "anti-CD3 / anti-HER2 orientation 1," "anti-CD3 / anti-HER2 orientation 2," "anti-CD20 / anti-CD3 orientation 1," "anti-CD20 / anti-CD3 orientation 2," and "anti-HEL / anti-BCMA orientation 1" (Figure 17B). When comparing the heterodimer % measured by LC-MS with the heterodimer % measured by IEX for BsAbs, LC-MS and IEX provided different heterodimer % values ​​(e.g., 54% and 41%, respectively) for anti-HEL / anti-BCMA antibodies (BsAbs containing a nanobody in one Fab arm), where the anti-BCMA binding moiety is a nanobody ("VHH") instead of a VH / VL pair (Figure 17C). In contrast, the heterodimer % values ​​by LC-MS and IEX correlated well for other antibodies with two VH / VL pairs (Figure 17D). The correlation between the heterodimer % values ​​by LC-MS and IEX was not evident for some of the anti-CD3 anti-HER2 BsAbs with a 354 / 349 disulfide bond (Figure 17E).

[0348] When the % heterodimer values ​​measured by LC-MS in the CH3 sets with and without the 354 / 349 substitution were compared, most CH3 sets with the 354 / 349 substitution (except "TL-QL") showed higher % heterodimer values ​​than the corresponding CH3 sets without the 354 / 349 substitution (Figure 17F), indicating that the disulfide bond between S354C and Y349C enhances heterodimerization in most sets (except "TL-QL").

[0349] The results show that when the % heterodimer values ​​measured by AlphaLISA® were compared with those measured by LC-MS or IEX, the order (i.e., rank) of the CH3 sets with respect to heterodimerization potential was maintained even if there were some discrepancies in the % values ​​(Figure 17G).

[0350] Overall, regardless of the method used to determine the heterodimer %, "LWG-SIG" in Orientation 1 or Orientation 2, with or without the 354 / 349 substitution, appears to provide the highest heterodimer % among the various CH3 sets tested (Figure 17H).

[0351] The stability of the BsAbs was also compared based on the % monomeric intact Ab at day 0 and the change in % monomeric intact Ab (Δ% monomeric intact Ab) by day 14, as measured by SEC. As shown in Figure 17I, regardless of whether the antibody was "anti-CD3 / anti-HER2 orientation 1," "anti-CD3 / anti-HER2 orientation 2," "anti-CD20 / anti-CD3 orientation 1," "anti-CD20 / anti-CD3 orientation 2," or "anti-HEL / anti-BCMA orientation 1," the % monomeric intact antibody values ​​were low (i.e., less aggregation) at day 0, and only slight aggregation occurred by day 14.

[0352] Finally, we compared the production yields of BsAbs in HEK293 cells. As shown in Figure 17J, production yields were similar regardless of whether the antibody was "anti-CD3 / anti-HER2 orientation 1," "anti-CD3 / anti-HER2 orientation 2," "anti-CD3 / anti-HER2 orientation 1 with 354 / 349 substitutions," "anti-CD20 / anti-CD3 orientation 1," "anti-CD20 / anti-CD3 orientation 2," or "anti-HEL / BCMA orientation 1."

[0353] Example 11: Simultaneous characterization of BsAbs containing cycle 1 output substitutions, cycle 2 output substitutions, or a combination of cycle 1 and cycle 2 output substitutions. In Example 11, anti-CD3 anti-HER2 BsAbs containing cycle 1 output substitutions (W-SG or VV) and cycle 2 output substitutions (QR-F, RG-FG, TL-QL, DVG-VSY, or LWG-SIG) were compared side-by-side with and without the 354 / 349 substitution. In addition, BsAbs containing several combinations of cycle 2 output substitutions with cycle 1 output substitutions or KiH substitutions (WTL-SAVQL, WTL-SGQL, WQL-SAVTL, WQL-SGTL, VTL-VQL, VQL-VTL, QRQL-FTL, or VQR-VF) were tested side-by-side, along with a modified version of the cycle 2 output substitution (LWG-IG).

[0354] The BsAbs used in Example 11 are summarized in Table 12. All BsAbs were produced in HEK293 cells. [Table 12-1] [Table 12-2]

[0355] The BsAbs in Table 12 produced in HEK293 cells were analyzed for % heterodimer by LC-MS and IEX. The results are summarized in Table 13. [Table 13]

[0356] The bsAbs in Table 12 produced in HEK293 cells and Protein A purified were further analyzed for % monomeric intact Ab by SEC. The SEC results and process yields from production in HEK293 cells are summarized in Table 14. [Table 14]

[0357] The heterodimer % data, monomeric full-size antibody % data, and yields were further compared among specific antibody sets among the 31 antibodies. This comparison revealed that when the heterodimer % values ​​measured by LC-MS and IEX were compared for BsAbs containing wild-type or cycle 1 or 2 output CH3 domains without the 354 / 349 substitution, a good correlation was observed between the LC-MS and IEX values ​​(Figure 18A). Furthermore, when the same comparison was performed for BsAbs containing wild-type or cycle 1 or 2 output CH3 domains further containing the 354 / 349 substitution, the order (i.e., rank) of the CH3 sets with respect to heterodimerization potential was maintained even if there were some discrepancies in the % values ​​(Figure 18B).

[0358] As observed in Example 10, among the CH3 sets identified in cycle 1 and cycle 2 selections (i.e., cycle 1 outputs (W-SG and VV) and cycle 2 outputs (QR-F, RG-FG, TL-QL, DVG-VSY, and LWG-SIG) with and without the 354 / 349 substitution, and the existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1), LWG-SIG consistently provided the highest heterodimerization rates, both with and without the 354 / 349 substitution (Figure 18C). W-SG, RG-FG, and QR-F also exceeded the heterodimerization rates of the existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1) when combined with the 354 / 349 substitution.

[0359] Interestingly, some of the cycle 1 and cycle 2 output CH3 sets, such as VV and QR-F (with or without the 354 / 349 substitution), showed less aggregation compared to the existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1) as measured by SEC in Protein A purified production products (Figure 18D).

[0360] When comparing CH3 sets with and without the 354 / 349 substitution, the CH3 set with the 354 / 349 substitution showed overall higher heterodimer values ​​as measured by LC-MS and higher monomeric intact Ab values ​​as measured by SEC (Figure 18E), indicating that the addition of the 354 / 349 substitution promoted CH3 heterodimerization and improved stability.

[0361] The production yields were compared between different CH3 sets with and without the 354 / 349 substitution. As shown in Figure 18F, none of the CH3 substitution sets appeared to reduce the production yield. Furthermore, it was observed that certain CH3 sets, such as VV, appeared to have higher yields than the existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1). In addition, as can be seen from Table 13, many of the combined substitution sets (combinations of cycle 1 substitutions with cycle 2 substitutions or cycle 2 substitutions with KiH substitutions), such as WTL-SAVQL, WTL-SGQL, WQL-SGTL, and VQL-VTL, especially the LWG-IG substitution, provided superior production yields than the existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1).

[0362] A new CH3 set, "LWG-IG," which is a variant of the cycle 2 output LWG-SIG, showed similar overall properties to LWG-SIG, except that LWG-IG provided a higher production yield than LWG-SIG, as shown in Figure 18G. This similarity between LWG-SIG and LWG-IG is consistent with the Rosetta heterodimer scores of LWG-SIG and LWG-IG.

[0363] The invention CH3 domain substitution sets described in the Examples, e.g., cycle 1 output and cycle 2 output, with or without CH3 disulfide bond substitutions (which may be 354 / 349 substitutions or 349 / 354 substitutions), as well as additional variant CH3 domains tested in Example 11 (i.e., combinations of cycle 1 output substitutions with cycle 2 output substitutions, combinations of cycle 2 output substitutions with cycle 2 output substitutions, combinations of cycle 2 output substitutions with KiH substitutions, and LWG-IG), and many of their related variations, are provided in Appendix Tables E-G. Exemplary variant CH3 domain sequences incorporating such CH3 substitution sets into the reference CH3 domain sequence of SEQ ID NO: 1 are also provided in Appendix Tables E-G. The exemplary sequences are those used in the Examples herein. However, it should be noted that these sequences are exemplary and the same CH3 substitution sets may be incorporated into any CH3 domain sequence, i.e., may not be limited to SEQ ID NO: 1. A summary of the SEQ ID NOs assigned to these exemplary variant CH3 domain sequences is provided in Figure 19.

[0364] Example 12: Simultaneous evaluation of the impact on Tm of cycle 1 and cycle 2 output sets. In Example 12, two variant CH3 domain sets from cycle 1 (W-SG and VV), and four variant CH3 domain sets from cycle 2 (QR-F, RG-FG, DVG-VSY, and LWG-SIG), with or without the 354 / 349 substitution, as well as existing CH3 sets (W-SAV (also referred to as KiH), VYAV-VLLW (also referred to as ZW1), and EW-RVT), were produced in HEK293 cells as CH2-CH3 constructs (i.e., Fc-only constructs), and the effect of the CH3 substitution on melting temperature (Tm) measured by differential scanning calorimetry (DSC) was analyzed.

[0365] method Protein Expression: The heterodimer fc-only construct of the CH3 mutant set contained both a HIS tag and a FLAG tag, and the heterodimer was expressed as a HIS tag and a FLAG purification tag. The protein was transiently transfected into HEK cells as described above.

[0366] Primary acquisition: Transiently transfected HEK cultures were harvested by centrifugation at 2400g for 5 minutes. The supernatant was decanted from the cell pellet, and after a second spin at 2400g for 5 minutes, the resin was loaded onto Ni Sepharose 6 Fast Flow resin (Cytiva 1753180) equilibrated with 10 column volumes of 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 buffer. Bound proteins were then washed with 5 column volumes of equilibration buffer containing 2 mM imidazole and eluted with 5 column volumes of equilibration buffer containing 250 mM imidazole. The eluate was immediately desalted into 25 mM HEPES, 150 mM sodium chloride, pH 7.2 using Sephadex G...

Claims

1. a first variant immunoglobulin heavy chain constant region 3 ("CH3") domain polypeptide ("first variant CH3 domain polypeptide") or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide, wherein said first variant CH3 domain polypeptide comprises one or more amino acid substitutions comprising or consisting of an amino acid substitution at one or more of the following amino acid positions according to EU numbering: 366, 407, 364, 368, 370, 399, 400, 405, and 409, according to EU numbering, and optionally (I) the first variant CH3 domain polypeptide preferentially forms a heterodimer with a second variant CH3 domain polypeptide, the second variant CH3 domain polypeptide being (a) differs from the first variant CH3 domain polypeptide by at least one amino acid; and (b) comprising an amino acid substitution at one or more of the following positions according to EU numbering: 407, 366, 364, 368, 370, 399, 400, 405, and 409; and / or (II) (i) the first variant CH3 domain polypeptide further comprises the amino acid substitution S354C and the second variant CH3 domain polypeptide further comprises the amino acid substitution Y349C; or (ii) the first variant CH3 domain polypeptide further comprises the amino acid substitution Y349C and the second variant CH3 domain polypeptide further comprises the amino acid substitution S354C; A first variant CH3 domain polypeptide or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide.

2. (1) the amino acid substitution in the first variant CH3 domain polypeptide is (i) 366th place, (ii) 407th place, (iii) positions 366 and 407; (iv) 364th, 366th, and 409th; (v) positions 366, 368, and 407; (vi) 368th place, (vii) positions 366 and 368, (viii) positions 366 and 409, (ix) positions 368 and 370, (x) positions 368 and 407; (xi) positions 399 and 405, (xii) positions 400 and 409; (xiii) positions 364, 407, and 409; (xiv) 366th, 368th, and 370th, (xv) positions 366, 399, and 405, (xvi) positions 366, 400, and 409; (xvii) positions 366, 407, and 409, (xviii) positions 368, 400, and 409; (xix) positions 399, 405, and 407, (xx) 400th, 407th, and 409th places, (xxi) 366th, 399th, 405th, and 407th, (xxii) 366th, 399th, 405th, and 409th; (xxiii) 366th, 400th, 407th, and 409th, (xxiv) 366th, 368th, 399th, 405th, and 407th, or (xxv) comprising or consisting of amino acid substitutions at positions 366, 368, 400, 407, and 409; In each of the foregoing, the substitution positions are according to EU numbering: （２） (i) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 366, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of amino acid substitutions at positions 366 and 407; (ii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366 and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of an amino acid substitution at position 366; (iii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 407; (iv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409; (v) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 366, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 407; (vi) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 368, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of amino acid substitutions at positions 366 and 409; (vii) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 407, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of an amino acid substitution at position 366; (viii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366 and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 368 or 368 and 370; (ix) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 368 and 370, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366 and 409; (x) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 399 and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 400 and 409; (xi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 400 and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 399 and 405; (xii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 364, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 370; (xiii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 370, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 364, 407, and 409; (xiv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366 and 368, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 407, and 409. (xv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366 and 368; (xvi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 368 and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409; (xvii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 368 and 407. (xviii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409; (xix) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 399, and 405; (xx) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407; (xxi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 400, and 409. (xxii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 368, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409; (xxiii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of amino acid substitutions at positions 368, 400, and 409; (xxiv) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366 and 368, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 407, and 409; (xxv) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366 and 368; (xxvi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368 and 407, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 366, and 409; (xxvii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 368 and 407; (xxviii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409; (xxix) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, and 405; (xxx) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407; (xxxi) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 400, and 409; (xxxii) the amino acid substitutions in the first variant CH3 domain comprise or consist of amino acid substitutions at positions 368, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409; or (xxxiii) the amino acid substitutions in the first variant CH3 domain consist of amino acid substitutions at positions 366, 399, 405, and 409, and optionally the amino acid substitutions in the second variant CH3 domain comprise or consist of amino acid substitutions at positions 368, 400, and 409; In each of the foregoing, the substitution positions are according to EU numbering: （３） (i) if the amino acid substitution in the first variant CH3 domain polypeptide consists of T366Y, then the amino acid substitution in the second variant CH3 domain polypeptide does not consist of Y407T; (ii) if the amino acid substitution in the first variant CH3 domain polypeptide consists of Y407T, then the amino acid substitution in the second variant CH3 domain polypeptide does not consist of T366Y; (iii) if the amino acid substitution in the first variant CH3 domain polypeptide consists of T366W, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of T366S, L368A, and Y407V; (iv) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of T366S, L368A, and Y407V, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of T366W; (v) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and T366W, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of Y349C, T366S, L368A, and Y407V; (vi) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, L368A, and Y407V, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of S354C and T366W; (vii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of S364H and F405A, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of Y349T and T394F; (viii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349T and T394F, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of S364H and F405A; (ix) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of T350V, L351Y, F405A, and Y407V, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of T350V, T366L, K392L, and T394W; (x) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of T350V, T366L, K392L, and T394W, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of T350V, L351Y, F405A, and Y407V; (xi) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of K392D and K409D, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of E356K and D399K; (xii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of E356K and D399K, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of K392D and K409D; (xiii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of D221E, P228E, and L368E, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of D221R, P228R, and K409R, and the first CH3 domain and the second CH3 domain are derived from a human IgG1 CH3 domain; (xiv) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of D221R, P228R, and K409R, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of D221E, P228E, and L368E, and the first CH3 domain and the second CH3 domain are derived from a human IgG1 CH3 domain; (xv) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of C223E, P228E, and L368E, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of C223R, E225R, P228R, and K409R, and the first CH3 domain and the second CH3 domain are derived from a human IgG2 CH3 domain; (xvi) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of C223R, E225R, P228R, and K409R, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of C223E, P228E, and L368E, and the first CH3 domain and the second CH3 domain are derived from a human IgG2 CH3 domain; (xvii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of K360E and K409W, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of Q347R, D399V, and F405T; (xviii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of Q347R, D399V, and F405T, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of K360E and K409W; (xix) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of K360E, K409W, and Y349C, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of Q347R, D399V, F405T, and S354C; (xx) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of Q347R, D399V, F405T, and S354C, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of K360E, K409W, and Y349C; (xxi) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of 366K, or consist of 366K and 351K, then the amino acid substitutions in the second variant CH3 domain polypeptide consist of not consisting of 351D, not consisting of 349E, not consisting of 349D, not consisting of 368E, not consisting of 368D, not consisting of 349E and 355E, not consisting of 349E and 355D, not consisting of 349D and 355E, or not consisting of 349D and 355D; (xxii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of 351D, consist of 349E, consist of 349D, consist of 368E, consist of 368D, consist of 349E and 355E, consist of 349E and 355D, consist of 349D and 355E, or consist of 349D and 355D, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of 366K, or do not consist of 366K and 351K; (xxiii) if the amino acid substitution in the first variant CH3 domain polypeptide consists of F405L, then the amino acid substitution in the second variant CH3 domain polypeptide does not consist of K409R; (xxiv) if the amino acid substitution in the first variant CH3 domain polypeptide consists of K409R, then the amino acid substitution in the second variant CH3 domain polypeptide does not consist of F405L; (xxv) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of K360D, D399M, and Y407A, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of E345R, Q347R, T366V, and K409V; (xxvi) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of E345R, Q347R, T366V, and K409V, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of K360D, D399M, and Y407A; (xxvii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349S, K370Y, T366M, and K409V, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of E356G, E357D, S364Q, and Y407A; and (xxviii) if the amino acid substitutions in the first variant CH3 domain polypeptide consist of E356G, E357D, S364Q, and Y407A, then the amino acid substitutions in the second variant CH3 domain polypeptide do not consist of Y349S, K370Y, T366M, and K409V; In each of the foregoing, the substitution positions are according to EU numbering; and / or （４） (I) preferentially forms a heterodimer with a second variant CH3 domain polypeptide that comprises an amino acid substitution at one or more of the following amino acid positions: 366, 407, 364, 400, and 409, according to EU numbering, and optionally an amino acid substitution at one or more of the following amino acid positions: 407, 366, 368, 370, 399, and 405; or (II) comprises an amino acid substitution at one or more of the following amino acid positions: 407, 366, 368, 370, 399, and 405 according to EU numbering, and optionally forms a heterodimer with a second variant CH3 domain polypeptide comprising an amino acid substitution at one or more of the following amino acid positions: 366, 407, 364, 400, and 409; 2. The first variant CH3 domain polypeptide of claim 1 or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide.

3. The first CH3 domain comprises: (I) one or more of the following amino acid positions according to EU numbering: 366, 407, 364, 400, and 409; or (II) The first variant CH3 domain polypeptide of claim 1 or 2, or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide, comprising amino acid substitutions only at one or more of the following amino acid positions according to EU numbering: 407, 366, 368, 370, 399, and 405.

4. (I) (i) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 366, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 407; (ii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 366; (iii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 366, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 407; (iv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 366. (v) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 368, and 407; (vi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409; (vii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 368, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 409. (viii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 368, or at positions 354, 368, and 370; (ix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, and 370, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 409; (x) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 400, and 409; (xi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 399, and 405. (xii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 368, and 370. (xiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 368, and 370, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 407, and 409. (xiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 368, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 407, and 409. (xv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 368. (xvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409. (xvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, and 407. (xviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 400, 407, and 409, 354, 366, 400, 407, and 409, or 354, 366, 368, 400, 407, and 409; (xix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 400, 407, and 409, 349, 366, 400, 407, and 409, or 349, 366, 368, 400, 407, and 409; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, and 405. (xx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 399, 405, and 407, 354, 366, 399, 405, and 407, or 354, 366, 368, 399, 405, and 407; (xxi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 399, 405, and 407, 349, 366, 399, 405, and 407, or 349, 366, 368, 399, 405, and 407; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 400, and 409. (xxii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, 405, and 409; or (xxiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, 405, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, 400, and 409. (xxiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 349, 366, and 368, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 407, and 409. (xxv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 368. (xxvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409. (xxvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, and 407. (xxviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 400, 407, and 409, 354, 366, 400, 407, and 409, or 354, 366, 368, 400, 407, and 409. (xxix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 400, 407, and 409, 349, 366, 400, 407, and 409, or 349, 366, 368, 400, 407, and 409; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, and 405. (xxx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 399, 405, and 407, 354, 366, 399, 405, and 407, or 354, 366, 368, 399, 405, and 407. (xxxi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 399, 405, and 407, 349, 366, 399, 405, and 407, or 349, 366, 368, 399, 405, and 407; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 400, and 409. (xxxii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, 405, and 409; or (xxxiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, 405, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, 400, and 409; the amino acid substitution at position 349 in said first variant CH3 domain polypeptide is Y349C and the amino acid substitution at position 354 in said second variant CH3 domain polypeptide is S354C; In each of the foregoing, the substitution positions are according to EU numbering; or (II) (i) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 366, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 407; (ii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 366; (iii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 366, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 407. (iv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 366. (v) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 368, and 407; (vi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409; (vii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354 and 368, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 409. (viii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349 and 368, or at positions 349, 368, and 370; (ix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, and 370, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 409; (x) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 400, and 409; (xi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 399, and 405. (xii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 368, and 370. (xiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 368, and 370, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 407, and 409. (xiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 368, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 407, and 409. (xv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 368. (xvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409. (xvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, and 407. (xviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 400, 407, and 409, 349, 366, 400, 407, and 409, or 349, 366, 368, 400, 407, and 409. (xix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 400, 407, and 409, 354, 366, 400, 407, and 409, or 354, 366, 368, 400, 407, and 409; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, and 405. (xx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 399, 405, and 407, 349, 366, 399, 405, and 407, or 349, 366, 368, 399, 405, and 407. (xxi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 399, 405, and 407, or at positions 354, 366, 399, 405, and 407, or at positions 354, 366, 368, 399, 405, and 407; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 400, and 409. (xxii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, 405, and 409; or (xxiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, 405, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, 400, and 409. (xxiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, and 368, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 407, and 409. (xxv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 407, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, and 368. (xxvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, and 407, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409. (xxvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, and 407. (xxviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, and 405, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 400, 407, and 409, 349, 366, 400, 407, and 409, or 349, 366, 368, 400, 407, and 409. (xxix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 400, 407, and 409, or at positions 354, 366, 400, 407, and 409, or at positions 354, 366, 368, 400, 407, and 409; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, and 405. (xxx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 399, 405, and 407, 349, 366, 399, 405, and 407, or 349, 366, 368, 399, 405, and 407. (xxxi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 399, 405, and 407, 354, 366, 399, 405, and 407, or 354, 366, 368, 399, 405, and 407; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 400, and 409. (xxxii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 368, 400, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 366, 399, 405, and 409; or (xxxiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of amino acid substitutions at positions 354, 366, 399, 405, and 409, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of amino acid substitutions at positions 349, 368, 400, and 409; the amino acid substitution at position 354 in said first variant CH3 domain polypeptide is S354C and the amino acid substitution at position 349 in said second variant CH3 domain polypeptide is Y349C; 3. The first variant CH3 domain polypeptide or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide according to claim 1 or 2, in each of the foregoing, wherein the substitution positions are according to EU numbering.

5. comprising one or more of the following amino acid substitutions: T366V, Y407V, S364D, S364L, T366Q, T366R, T366S, T366W, L368A, L368F, L368S, L368I, K370G, K370Y, D399Q, S400T, F405L, Y407G, K409R, K409L, and / or K409G; Optionally, further comprising Y349C or S354C; In each of the foregoing, the substitution positions are according to EU numbering: Optionally, the following set of amino acid substitutions: (i) T366V, (ii) Y407V, (iii) T366W, (iv) T366S and Y407G, (v) S364L, T366W, and K409G, (vi) T366S, L368I, and Y407G, (vii) L368F, (viii) T366V and L368F, (ix) T366Q and K409R, (x) T366R and K409G, (xi) L368F and K370G, (xii) L368I and Y407G, (xiii) S400T and K409L, (xiv) D399Q and F405L, (xv) S364D, Y407V, and K409G, (xvi) T366V, L368S, and K370Y, (xvii) T366V, D399Q, and F405L, (xviii) T366W, D399Q, and F405L, (xix) T366V, S400T, and K409L, (xx) T366W, S400T, and K409L, (xxi) T366Q, Y407V, and K409R, (xxii) L368F, S400T, and K409L, (xxiii) D399Q, Y407V, and F405L, (xxiv) S400T, Y407V, and K409L, (xxv) T366S, D399Q, F405L, and Y407G, (xxvi) T366Q, D399Q, F405L, and K409R, (xxvii) T366S, S400T, Y407G, and K409L, (xxviii) T366S, L368A, D399Q, Y407V, and F405L, or (xxiv) T366S, L368A, S400T, Y407V, and K409L one of Optionally, further comprising Y349C or S354C; In each of the foregoing, the substitution positions are according to EU numbering: Optionally, (i) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of T366V, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of Y407V; (ii) the amino acid substitution in the first variant CH3 domain polypeptide comprises or consists of Y407V, and optionally the amino acid substitution in the second variant CH3 domain polypeptide comprises or consists of T366V; (iii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366W, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366S and Y407G; (iv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366S and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366W; (v) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366S, L368I, and Y407G; (vi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of S364L, T366W, and K409G; (vii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of L368F, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366Q and K409R; (viii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366Q and K409R, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of L368F; (ix) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366R and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of L368F and K370G; (x) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of L368F and K370G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366R and K409G; (xi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of S400T and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of D399Q and F405L; (xii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of D399Q and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of S400T and K409L; (xiii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of S364D, Y407V, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366V, L368S, and K370Y; (xiv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366V, L368S, and K370Y, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of S364D, Y407V, and K409G. (xv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366S, L368A, Y407V, D399Q, and F405L; (xvi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366S, L368A, Y407V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366W, S400T, and K409L; (xvii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366S, Y407G, D399Q, and F405L; (xviii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366S, Y407G, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366W, S400T, and K409L; (xix) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366W, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366S, L368A, Y407V, S400T, and K409L; (xx) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366S, L368A, Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366W, D399Q, and F405L; (xxi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366W, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366S, Y407G, S400T, and K409L; (xxii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366S, Y407G, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366W, D399Q, and F405L; (xxiii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366V, D399Q, and F405L; (xxiv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of Y407V, S400T, and K409L. (xxv) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of Y407V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366V, S400T, and K409L; (xxvi) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of Y407V, D399Q, and F405L; (xxvii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366Q, K409R, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of L368F, S400T, and K409L; (xxviii) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of L368F, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366Q, K409R, D399Q, and F405L; (xxix) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of Y407V, T366Q, and K409R, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of T366V and L368F. (xxx) the amino acid substitutions in the first variant CH3 domain polypeptide comprise or consist of T366V and L368F, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide comprise or consist of Y407V, T366Q, and K409R; (xxxi) the amino acid substitutions in said first variant CH3 domain polypeptide comprise or consist of S364L, T366W, and K409G, and optionally the amino acid substitutions in said second variant CH3 domain polypeptide comprise or consist of L368I and Y407G; or (xxxii) the amino acid substitutions in said first variant CH3 domain polypeptide comprise or consist of L368I and Y407G, and optionally the amino acid substitutions in said second variant CH3 domain polypeptide comprise or consist of S364L, T366W, and K409G; 5. The first variant CH3 domain polypeptide or a heavy chain polypeptide comprising the first variant CH3 domain polypeptide according to any one of claims 1, 2 and 4, in each of the foregoing, wherein the substitution positions are according to EU numbering.

6. (I) (i) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C and T366V, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C and Y407V; (ii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C and Y407V, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C and T366V; (iii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C and T366W, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, and Y407G; (iv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, S354C, and T366W; (v) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, L368I, and Y407G; (vi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, S364L, T366W, and K409G; (vii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C and L368F, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366Q, and K409R. (viii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366Q, and K409R, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C and L368F. (ix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366R, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, L368F, and K370G; (x) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, L368F, and K370G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366R, and K409G; (xi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, D399Q, and F405L; (xii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, S400T, and K409L; (xiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, S364D, Y407V, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366V, L368S, and K370Y. (xiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366V, L368S, and K370Y, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, S364D, Y407V, and K409G. (xv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, L368A, Y407V, D399Q, and F405L; (xvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, L368A, Y407V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366W, S400T, and K409L; (xvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, Y407G, D399Q, and F405L; (xviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, Y407G, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366W, S400T, and K409L. (xix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366W, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, L368A, Y407V, S400T, and K409L. (xx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, L368A, Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366W, D399Q, and F405L; (xxi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366W, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, Y407G, S400T, and K409L. (xxii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, Y407G, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366W, D399Q, and F405L. (xxiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366V, D399Q, and F405L. (xxiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, Y407V, S400T, and K409L. (xxv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, Y407V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366V, S400T, and K409L. (xxvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, Y407V, D399Q, and F405L. (xxvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366Q, K409R, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, L368F, S400T, and K409L. (xxviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, L368F, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366Q, K409R, D399Q, and F405L. (xxix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, Y407V, T366Q, and K409R, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366V, and L368F. (xxx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366V, and L368F, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, Y407V, T366Q, and K409R. (xxxi) the amino acid substitutions in said first variant CH3 domain polypeptide consist of Y349C, S364L, T366W, and K409G, and optionally the amino acid substitutions in said second variant CH3 domain polypeptide consist of S354C, L368I, and Y407G; or (xxxii) the amino acid substitutions in said first variant CH3 domain polypeptide consist of Y349C, L368I, and Y407G, and optionally the amino acid substitutions in said second variant CH3 domain polypeptide consist of S354C, S364L, T366W, and K409G; In each of the foregoing, the substitution positions are according to EU numbering: (II) (i) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and T366V, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C and Y407V; (ii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and Y407V, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C and T366V; (iii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and T366W, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366S, and Y407G; (iv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C and T366W. (v) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366S, L368I, and Y407G; (vi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, S364L, T366W, and K409G; (vii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and L368F, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366Q, and K409R. (viii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366Q, and K409R, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C and L368F. (ix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366R, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, L368F, and K370G; (x) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, L368F, and K370G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366R, and K409G; (xi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, D399Q, and F405L; (xii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, S400T, and K409L; (xiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, S364D, Y407V, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366V, L368S, and K370Y. (xiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366V, L368S, and K370Y, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, S364D, Y407V, and K409G. (xv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366S, L368A, Y407V, D399Q, and F405L; (xvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, L368A, Y407V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366W, S400T, and K409L; (xvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366W, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366S, Y407G, D399Q, and F405L; (xviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, Y407G, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366W, S400T, and K409L; (xix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366W, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366S, L368A, Y407V, S400T, and K409L. (xx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, L368A, Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366W, D399Q, and F405L; (xxi) the amino acid substitutions in said first variant CH3 domain polypeptide consist of S354C, T366W, D399Q, and F405L, and optionally the amino acid substitutions in said second variant CH3 domain polypeptide consist of Y349C, T366S, Y407G, S400T, and K409L. (xxii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, Y407G, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366W, D399Q, and F405L. (xxiii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, Y407V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366V, D399Q, and F405L. (xxiv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, Y407V, S400T, and K409L. (xxv) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, Y407V, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366V, S400T, and K409L. (xxvi) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366V, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, Y407V, D399Q, and F405L. (xxvii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366Q, K409R, D399Q, and F405L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, L368F, S400T, and K409L. (xxviii) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, L368F, S400T, and K409L, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366Q, K409R, D399Q, and F405L. (xxix) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, Y407V, T366Q, and K409R, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366V, and L368F. (xxx) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366V, and L368F, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, Y407V, T366Q, and K409R. (xxxi) the amino acid substitutions in said first variant CH3 domain polypeptide consist of S354C, S364L, T366W, and K409G, and optionally the amino acid substitutions in said second variant CH3 domain polypeptide consist of Y349C, L368I, and Y407G; or (xxxii) the amino acid substitutions in said first variant CH3 domain polypeptide consist of S354C, L368I, and Y407G, and optionally the amino acid substitutions in said second variant CH3 domain polypeptide consist of Y349C, S364L, T366W, and K409G; In each of the foregoing, the substitution positions are according to EU numbering; or (II) (1) the amino acid substitutions in the first variant CH3 domain polypeptide consist of T366W, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of T366S and Y407G; (2) the amino acid substitutions in the first variant CH3 domain polypeptide consist of T366S and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of T366W. (3) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of T366S, L368I, and Y407G. (4) the amino acid substitutions in the first variant CH3 domain polypeptide consist of T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S364L, T366W, and K409G; (5) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C and T366W, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366S, and Y407G. (6) The amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C and T366W. (7) The amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, T366S, L368I, and Y407G. (8) the amino acid substitutions in the first variant CH3 domain polypeptide consist of S354C, T366S, L368I, and Y407G; and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of Y349C, S364L, T366W, and K409G; (9) The amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C and T366W, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, and Y407G. (10) The amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, S354C, and T366W. (11) The amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, S364L, T366W, and K409G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, T366S, L368I, and Y407G; or (12) The amino acid substitutions in the first variant CH3 domain polypeptide consist of Y349C, T366S, L368I, and Y407G, and optionally the amino acid substitutions in the second variant CH3 domain polypeptide consist of S354C, S364L, T366W, and K409G; 6. The first variant CH3 domain polypeptide or a heavy chain polypeptide comprising the first variant CH3 domain polypeptide according to any one of claims 1, 2, 4 and 5, in each of the foregoing, wherein the substitution positions are according to EU numbering.

7. (I) SEQ ID NO: 21, 11, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, or 161 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22, 12, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, or 162, respectively); (II) SEQ ID NO: 22, 12, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, or 162 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 21, 11, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, or 161, respectively); (III) SEQ ID NO: 23, 13, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, or 163 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24, 14, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, or 164, respectively); (IV) SEQ ID NO: 24, 14, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, or 164 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23, 13, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, or 163, respectively); (V) SEQ ID NO: 25, 15, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, or 165 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 26, 16, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, or 166, respectively); or (VI) SEQ ID NO: 26, 16, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, or 166 (optionally the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 25, 15, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, or 165, respectively); and optionally (I) SEQ ID NO:21 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO:22); (II) SEQ ID NO:22 (optionally each of the second variant CH3 domain polypeptides comprises an amino acid sequence according to SEQ ID NO:21); (III) SEQ ID NO: 11 or 71 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 12 or 72, respectively); (IV) SEQ ID NO: 12 or 72 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 11 or 71, respectively); (V) SEQ ID NO: 13 or 73 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 14 or 74, respectively); (VI) SEQ ID NO: 14 or 74 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 13 or 73, respectively); (VII) SEQ ID NO: 15 or 75 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 16 or 76, respectively); or (VIII) The first variant CH3 domain polypeptide of any one of claims 1, 2, 4, and 6, or a heavy chain polypeptide comprising the first variant CH3 domain polypeptide, comprising an amino acid sequence selected from SEQ ID NO: 16 or 76 (optionally, the second variant CH3 domain polypeptide comprises an amino acid sequence according to SEQ ID NO: 15 or 75, respectively).

8. 8. An immunoglobulin polypeptide comprising at least one first variant CH3 domain polypeptide according to any one of claims 1 to 7 or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide.

9. A molecule comprising at least a first polypeptide and a second polypeptide, (A) the first polypeptide comprises a first variant CH3 domain polypeptide of any one of claims 1 to 7; (B) the second polypeptide comprises a second variant CH3 domain polypeptide of any one of claims 1 to 7; further, the first polypeptide and the second polypeptide are optionally linked or paired with each other via a disulfide bond; Optionally, the molecule has the following characteristics: (A) the first polypeptide further comprises a first antigen-binding domain; (B) the second polypeptide further comprises a second antigen-binding domain. (C) the heteromeric molecule further comprises a third polypeptide optionally comprising a third antigen-binding domain, optionally wherein said third polypeptide is bound to or paired with said first polypeptide; and / or (D) the heteromeric molecule further comprises a fourth polypeptide optionally comprising a fourth antigen-binding domain, optionally wherein the fourth polypeptide is bound to or paired with the second polypeptide; Optionally, the molecule is a multispecific antibody or antigen-binding antibody fragment, optionally comprising a structure as shown in any one of Figures 2-8, and further optionally is an IgG, and even further optionally is IgG1, IgG2, IgG3, or IgG4; Optionally, the molecule has the following characteristics: (I) (I-1-i) the first polypeptide comprises a first antigen-binding domain that forms a first antigen-binding site specific for a first epitope, and / or (I-1-ii) the heteromeric molecule comprises a third polypeptide that comprises a third antigen-binding domain that forms a third antigen-binding site specific for a third epitope, or (I-2) the first polypeptide comprises a first antigen-binding domain, the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain, and the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for a first epitope; and / or (II) (II-1-i) the second polypeptide comprises a second antigen-binding domain forming a second antigen-binding site specific for a second epitope, and / or (I-1-ii) the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain forming a fourth antigen-binding site specific for a fourth epitope, or (II-2) The second polypeptide comprises a second antigen-binding domain, and the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain, and the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for a second epitope; Optionally, the first epitope, the second epitope, the third epitope, and / or the fourth epitope are individually identical to or different from each other.

10. 1. A multispecific antibody or antigen-binding antibody fragment, comprising: (A) a first polypeptide comprising a first antigen-binding domain and a first variant CH3 domain polypeptide of any one of claims 1 to 7; (B) a second polypeptide comprising a second antigen-binding domain and a second variant CH3 domain polypeptide according to any one of claims 1 to 7, the first polypeptide and the second polypeptide are bound or paired to each other, and optionally the multispecific antibody or antigen-binding antibody fragment comprises: (1) The structure shown in any one of Figures 2 to 8 (2) IgG, and optionally IgG1, IgG2, IgG3, or IgG4, and / or （３） (I) (I-1-i) the first polypeptide comprises a first antigen-binding domain that forms a first antigen-binding site specific for a first epitope, and / or (I-1-ii) the heteromeric molecule comprises a third polypeptide that comprises a third antigen-binding domain that forms a third antigen-binding site specific for a third epitope, or (I-2) The first polypeptide comprises a first antigen-binding domain, the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain, and the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for a first epitope, and / or (II) (II-1-i) the second polypeptide comprises a second antigen-binding domain forming a second antigen-binding site specific for a second epitope, and / or (I-1-ii) the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain forming a fourth antigen-binding site specific for a fourth epitope, or (II-2) The second polypeptide comprises a second antigen-binding domain, the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain, and the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for a second epitope; Optionally, the first epitope, the second epitope, the third epitope, and / or the fourth epitope are individually identical or different from each other; Optionally, In (I-2), (i) the first antigen-binding domain is an immunoglobulin heavy chain variable region (VH) domain and the third antigen-binding domain is an immunoglobulin light chain variable (VL) domain, or (ii) the first antigen-binding domain is a VL domain and the third antigen-binding domain is a VH domain; and / or In (II-2), (i) the second antigen-binding domain is a VH domain and the fourth antigen-binding domain is a VL domain, or (ii) the second antigen-binding domain is a VL domain and the fourth antigen-binding domain is a VH domain; Further optionally, said multispecific antibody or antigen-binding antibody fragment is bispecific.

11. A polynucleotide or polynucleotides comprising: (i) a first variant CH3 domain polypeptide according to any one of claims 1 to 7, or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide; (ii) an immunoglobulin polypeptide according to claim 8; (iii) a molecule according to claim 9, and / or (iv) a polynucleotide or polynucleotides encoding the multispecific antibody or antigen-binding antibody fragment of claim 10; or A vector comprising said polynucleotide(s).

12. A cell, (i) comprising a first variant CH3 domain polypeptide or a heavy chain polypeptide comprising said first variant CH3 domain polypeptide according to any one of claims 1 to 7; (ii) comprising an immunoglobulin polypeptide according to claim 8; (iii) comprising a molecule according to claim 9; (iv) comprising a multispecific antibody or antigen-binding antibody fragment according to claim 10, (v) comprising the polynucleotide of claim 11; and / or (vi) A cell comprising the vector according to claim 11.

13. 1. A composition comprising: (I) (i 8. A heavy chain polypeptide comprising the first variant CH3 domain polypeptide or the first variant CH3 domain polypeptide according to any one of claims 1 to 7, (ii) an immunoglobulin polypeptide according to claim 8; (iii) a molecule according to claim 9, (iv) the multispecific antibody or antigen-binding antibody fragment of claim 10, (v) the polynucleotide of claim 11, (vi) a vector according to claim 11, and / or (vii) a cell according to claim 12; (II) a pharma- ceutically or diagnostically acceptable carrier.

14. 1. A method of generating a CH3 domain encoding polynucleotide library, or a CH3 domain encoding polynucleotide library generated using said method, said method comprising incorporating mutations or randomizing nucleic acid at one or more predefined nucleotide positions within a CH3 domain encoding polynucleotide, either in silico or in vitro, said one or more predefined nucleotide positions being within a codon encoding an amino acid at one or more predefined CH3 domain positions, and said one or more predefined CH3 domain positions being (i) a CH3 domain position is considered to be at or near a CH3-CH3 interface if the amino acid residue at the CH3 domain position satisfies the following: (i-1) in the CH3 monomer form, the side chain solvent exposed surface area (SASA) is 15% or more; (i-2) in the CH3-CH3 dimer form, there are no more than about 8.2 Å of atoms of the paired CH3 domain; and (i-3) in the CH3-CH3 dimer form, the residue does not point away from the paired CH3 domain. (ii) predicted to affect CH3-CH3 interactions, optionally wherein said prediction is made in silico or in vitro, and further optionally wherein a CH3 domain position is predicted to affect CH3-CH3 interactions if said CH3 domain position satisfies the following: (ii-1) is in the first, second, or third adjacent position to a CH3 domain position present at a CH3-CH3 interface, and / or (ii-2) in a CH3-CH3 dimer form has an interchain beta carbon-beta carbon distance of about 8.2 Å or less from a CH3 domain position of the paired CH3 domain present at the CH3-CH3 interface; and / or (iii) selected from positions 366, 407, 364, 368, 370, 399, 400, 405, and / or 409 according to EU numbering; Optionally, (1) said incorporating said mutations at said one or more predetermined nucleotide positions or randomizing the nucleic acid at those positions comprises introducing a degenerate codon, optionally a degenerate RMW codon representing the six naturally occurring amino acids (D, T, A, E, K, and N), or a degenerate NNK codon representing all 20 naturally occurring amino acid residues; (2) the CH3 domain-encoding polynucleotide library is for identifying one or more sets of a first variant CH3 domain polypeptide and a second variant CH3 domain polypeptide, wherein the first variant CH3 domain polypeptide preferentially forms a heterodimer with the second variant CH3 domain polypeptide that differs from the first variant CH3 domain polypeptide by at least one amino acid; and / or (3) the one or more predetermined CH3 domain positions are (i) 366th place, (ii) 407th place, (iii) positions 366 and 407; (iv) 364th, 366th, and 409th; (v) positions 366, 368, and 407; (vi) 368th place, (vii) positions 366 and 368, (viii) positions 366 and 409, (ix) positions 368 and 370, (x) positions 368 and 407; (xi) positions 399 and 405, (xii) positions 400 and 409; (xiii) positions 364, 407, and 409; (xiv) 366th, 368th, and 370th, (xv) positions 366, 399, and 405, (xvi) positions 366, 400, and 409; (xvii) positions 366, 407, and 409, (xviii) positions 368, 400, and 409; (xix) positions 399, 405, and 407, (xx) 400th, 407th, and 409th places, (xxi) 366th, 399th, 405th, and 407th, (xxii) 366th, 399th, 405th, and 409th; (xxiii) 366th, 400th, 407th, and 409th, (xxiv) 366th, 368th, 399th, 405th, and 407th, or (xxv) comprising or consisting of amino acid substitutions at positions 366, 368, 400, 407, and 409; In each of the foregoing, the substitution positions are according to EU numbering.

15. 1. A method for generating a CH3 domain polypeptide library, or a CH3 domain polypeptide library generated using said method, said method comprising: (I) obtaining, in silico or in vitro, a plurality of CH3 domain polypeptides corresponding to a plurality of CH3 domain-encoding polynucleotides of the CH3 domain-encoding polynucleotide library of claim 14; or (II) incorporating substitutions in silico or in vitro at one or more predetermined CH3 domain amino acid positions within a plurality of CH3 domain polypeptides, wherein the one or more of the one or more predetermined CH3 domain amino acid positions are: (i) a CH3 domain position is considered to be at or near a CH3-CH3 interface if the amino acid residue at the CH3 domain position satisfies the following: (i-1) in the CH3 monomer form, the side chain solvent exposed surface area (SASA) is 15% or more; (i-2) in the CH3-CH3 dimer form, there are no more than about 8.2 Å of atoms of the paired CH3 domain; and (i-3) in the CH3-CH3 dimer form, the residue does not point away from the paired CH3 domain. (ii) predicted to affect CH3-CH3 interactions, optionally wherein said prediction is made in silico or in vitro, and further optionally wherein a CH3 domain position is predicted to affect CH3-CH3 interactions if said CH3 domain position satisfies the following: (ii-1) is in the first, second, or third adjacent position to a CH3 domain position present at a CH3-CH3 interface, and / or (ii-2) in a CH3-CH3 dimer form has an interchain beta carbon-beta carbon distance of about 8.2 Å or less from a CH3 domain position of the paired CH3 domain present at the CH3-CH3 interface; and / or (iii) selected from positions 364, 366, 368, 370, 399, 400, 405, 407, and / or 409 according to EU numbering; Optionally, (1) the CH3 domain polypeptide library is for identifying one or more sets of a first variant CH3 domain polypeptide and a second variant CH3 domain polypeptide, wherein the first variant CH3 domain polypeptide preferentially forms a heterodimer with the second variant CH3 domain polypeptide that differs from the first variant CH3 domain polypeptide by at least one amino acid, optionally by at least one amino acid substitution; (2) the CH3 domain polypeptides of the CH3 domain polypeptide library comprise a predetermined number of CH3 domain amino acid substitutions, and optionally the predetermined number is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more; 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less; 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4; 1-3; 1-2; and / or 1, 2, 3, 4, or 5; and / or (II) wherein the one or more predetermined CH3 domain positions are (i) 366th place, (ii) 407th place, (iii) positions 366 and 407; (iv) 364th, 366th, and 409th; (v) positions 366, 368, and 407; (vi) 368th place, (vii) positions 366 and 368, (viii) positions 366 and 409, (ix) positions 368 and 370, (x) positions 368 and 407; (xi) positions 399 and 405, (xii) positions 400 and 409; (xiii) positions 364, 407, and 409; (xiv) 366th, 368th, and 370th, (xv) positions 366, 399, and 405, (xvi) positions 366, 400, and 409; (xvii) positions 366, 407, and 409, (xviii) positions 368, 400, and 409; (xix) positions 399, 405, and 407, (xx) 400th, 407th, and 409th places, (xxi) 366th, 399th, 405th, and 407th, (xxii) 366th, 399th, 405th, and 409th; (xxiii) 366th, 400th, 407th, and 409th, (xxiv) 366th, 368th, 399th, 405th, and 407th, or (xxv) comprising or consisting of amino acid substitutions at positions 366, 368, 400, 407, and 409; In each of the foregoing, the substitution positions are according to EU numbering.

16. 1. A method for identifying one or more sets of a first variant CH3 domain polypeptide and a second variant CH3 domain polypeptide, wherein the first variant CH3 domain polypeptide preferentially forms a heterodimer with the second variant CH3 domain polypeptide, the method comprising: (a) providing a plurality of sets of (a-1) a first polypeptide comprising a wild-type or variant CH3 domain polypeptide and (a-2) a second polypeptide comprising a wild-type or variant CH3 domain polypeptide, optionally wherein said plurality of sets of (a-1) and (a-2) are provided in silico or in vitro; (b) quantifying binding or pairing preferences between the CH3 domains of the first and second polypeptides in one or more of the plurality of sets of step (a), optionally wherein the quantification is performed in silico and / or in vitro, and further optionally wherein the quantification comprises quantifying in vitro the amount of CH3 domain homodimers and CH3 domain heterodimers formed by the CH3 domains in one or more of the plurality of sets of step (a), optionally by liquid chromatography mass spectrometry (LC-MS), ion exchange chromatography (IEX), AlphaLISA®, and / or flow cytometry; (c) selecting one or more sets of first and second variant CH3 domain polypeptides that provide a desired binding or pairing preference, optionally a desired percentage, of heterodimers, and optionally a similar or greater binding or pairing preference and / or percentage of CH3 domain heterodimers compared to a reference set of CH3 domain polypeptides; Optionally, at a given CH3 domain position, said first variant CH3 domain polypeptide and said second variant CH3 domain polypeptide differ by at least one amino acid, optionally by at least one amino acid substitution; Optionally, (1) (A) the CH3 domain polypeptide of the first polypeptide is obtained from a first CH3 domain polypeptide library of claim 15 or expressed from a first CH3 domain encoding polynucleotide library of claim 14; and / or (B) the CH3 domain polypeptide of the second polypeptide is obtained from a second CH3 domain polypeptide library of claim 15 or expressed from a second CH3 domain encoding polynucleotide library of claim 14; （２） (I) the CH3 domain polypeptide of the first polypeptide comprises a substitution S354C and the CH3 domain polypeptide of the second polypeptide comprises a substitution Y349C; or (II) the CH3 domain polypeptide of the first polypeptide comprises a substitution Y349C, and the CH3 domain polypeptide of the second polypeptide comprises a substitution S354C; and (3). (a-1) the first polypeptide comprises or is linked to a first label; and (a-2) the second polypeptide comprises or is linked to a second label, and optionally the quantification step (b) comprises detecting the first label and / or the second label; （４） the quantifying step (b) comprises at least one of liquid chromatography-mass spectrometry (LC-MS), AlphaLISA®, ion exchange chromatography (IEX), and / or flow cytometry; （５） The method further comprises the step of selecting one or more sets of first and second variant CH3 domain polypeptides based on one or more properties of the antibody comprising the set of first and second polypeptides selected in step (c), wherein the one or more properties are: (i) (i-1) optionally, production yield assessed in one or more cell types, optionally mammalian cells, e.g., CHO and HEK cells, isoforms, insect cells, and / or plant cells, and / or (i-2) optionally suitability for one or more antibody purification methods, including Protein A affinity purification; (ii) the extent of aggregation, optionally quantified using chromatography, optionally size exclusion chromatography (SEC) or electrophoresis, optionally SDS-PAGE, and optionally the presence of multimers of full size antibodies; (iii) the rate of correct pairing between the CH1 domains and / or between the CH1 domain and the CL domain, optionally correct pairing, optionally assessed using LC-MS; (iv) melting temperature (Tm) and / or aggregation temperature (Tag), optionally measured using differential scanning fluorimetry (DSF) and / or differential scanning calorimetry (DSC) and / or using an instrument, optionally an Uncle®, optionally Tagg266; (v) isoelectric point (“pI”); (vi) optionally, the level of interaction with a multispecific reagent ("PSR"), as measured by the methods described in WO 2014 / 179363; (vii) hydrophobic interactions of the antibody, optionally measured using hydrophobic interaction chromatography ("HIC"), optionally as described in Estep P, et al. MAbs. 2015 May-Jun;7(3):553-561; (viii) optionally, the self-interaction as measured by (viii-1) affinity capture self-interaction nanoparticle spectroscopy (AC-SINS), optionally as described in Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92, or (viii-2) dynamic light scattering (DLS); (ix) stability against high or low pH stress; (x) solubility; (xi) production costs and / or times; (xii) other stability parameters; (xiii) shelf life; (xiv) in vivo half-life, and / or (xv) immunogenicity.

17. (A) the CH3 domain polypeptide of the first polypeptide is obtained from a first CH3 domain polypeptide library of claim 15 or expressed from a first CH3 domain encoding polynucleotide library of claim 14; and (B) the CH3 domain polypeptide of the second polypeptide is obtained from a second CH3 domain polypeptide library of claim 15 or expressed from a second CH3 domain encoding polynucleotide library of claim 14; (i) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of position 366, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of position 407; (ii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of position 407, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of position 366; (iii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of position 366, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 407; (iv) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 407, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of position 366; (v) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 364, 366, and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366, 368, and 407; (vi) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366, 368, and 407, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 364, 366, and 409; (vii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of position 368, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 409. (viii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of position 368. (ix) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 368 and 370; (x) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 368 and 370, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 409; (xi) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 400 and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 399 and F405; (xii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 399 and 405, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 400 and 409. (xiii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 364, 407, and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366, 368, and 370. (xiv) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366, 368, and 370, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 364, 407, and 409. (xv) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 368, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366, 407, and 409. (xiv) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366, 407, and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366 and 368. (xv) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 368 and 407, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 364, 366, and 409. (xvi) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 364, 366, and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 368 and 407. (xvii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366, 399, and 405, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 400, 407, and 409; positions 366, 400, 407, and 409; or positions 366, 368, 400, 407, and 409. (xviii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of: 400, 407, and 409; 366, 400, 407, and 409; or 366, 368, 400, 407, and 409; and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of: 366, 399, and 405. (xix) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366, 400, and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 399, 405, and 407; 366, 399, 405, and 407; or 366, 368, 399, 405, and 407. (xx) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of: 399, 405, and 407; 366, 399, 405, and 407; or 366, 368, 399, 405, and 407; and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of: 366, 400, and 409. (xxi) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 368, 400, and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 366, 399, 405, and 409; or (xxii) the one or more predefined CH3 domain positions of the first CH3 domain polypeptide or the first CH3 domain encoding polynucleotide library comprise or consist of positions 366, 399, 405, and 409, and the one or more predefined CH3 domain positions of the second CH3 domain polypeptide or the second CH3 domain encoding polynucleotide library comprise or consist of positions 368, 400, and 409; 17. The method of claim 16, in each of the foregoing, wherein the substitution positions are according to EU numbering.

18. 11. A method for screening for a set of first and second variant CH3 domain polypeptides suitable for a multispecific antibody or antigen-binding antibody fragment according to claim 10 having a given antigen specificity, comprising: (a) expressing a plurality of multispecific antibodies and / or antigen-binding antibody fragments, each comprising a different set of a first variant CH3 domain polypeptide and a second variant CH3 domain polypeptide; (b) selecting a set of first and second variant CH3 domain polypeptides suitable for the multispecific antibody or antigen-binding antibody fragment based on one or more antibody properties of the plurality of multispecific antibodies and / or antigen-binding antibody fragments expressed in step (a), wherein the one or more antibody properties are determined as follows: (i) (i-1) optionally, production yield assessed in one or more cell types, optionally mammalian cells, e.g., CHO and HEK cells, yeast cells, insect cells, and / or plant cells, and / or (i-2) optionally suitability for one or more antibody purification methods, including Protein A affinity purification; (ii) the extent of aggregation, optionally quantified using chromatography, optionally size exclusion chromatography (SEC) or electrophoresis, optionally SDS-PAGE, and optionally the presence of multimers of full size antibodies; (iii) the rate of correct pairing between the CH1 domains and / or between the CH1 domain and the CL domain, optionally correct pairing, optionally assessed using LC-MS; (iv) melting temperature (Tm) and / or aggregation temperature (Tag), optionally measured using differential scanning fluorimetry (DSF) and / or differential scanning calorimetry (DSC) and / or using an instrument, optionally an Uncle®, optionally Tagg266; (v) isoelectric point (“pI”); (vi) optionally, the level of interaction with a multispecific reagent ("PSR"), as measured by the methods described in WO 2014 / 179363; (vii) hydrophobic interactions of the antibody, optionally measured using hydrophobic interaction chromatography ("HIC"), optionally as described in Estep P, et al. MAbs. 2015 May-Jun;7(3):553-561; (viii) optionally, the self-interaction as measured by (viii-1) affinity capture self-interaction nanoparticle spectroscopy (AC-SINS), optionally as described in Liu Y et al., MAbs. Mar-Apr 2014;6(2):483-92, or (viii-2) dynamic light scattering (DLS); (ix) stability against high or low pH stress; (x) solubility; (xi) production costs and / or times; (xii) other stability parameters; (xiii) shelf life; (xiv) in vivo half-life, and / or (xv) immunogenicity.

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