Materials and methods for designing, use and manufacture of improved biologic molecules

Abstract

Materials, methods and systems for making and using improved biologic molecules are provided, such as improved biologic multispecific molecules.

Description

ATTORNEY DOCKET NO.253505.000530, JBI6876 MATERIALS AND METHODS FOR DESIGNING, USE AND MANUFACTURE OF IMPROVED BIOLOGIC MOLECULES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 625,395, filed on January 26, 2024, U.S. Provisional Application No.63 / 625,497, filed on January 26, 2024, and U.S. Provisional Application No.63 / 702,377, filed on October 2, 2024, the disclosures of which are herein incorporated by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 16, 2025, is named JBI6876WOPCT1_SL.xml and is 9,996,122 bytes in size. FIELD

[0003] Provided herein are inter alia materials, methods, designs, reagents, and systems for enhanced multispecific molecules and their uses. Such molecules include molecules that have enhanced pairing between a first polypeptide and a second polypeptide. For example, in one aspect, the multispecific molecules of the present invention have a P374 mutation (Eu numbering) in an immunoglobulin heavy chain constant domain 3 (CH3 domain) (e.g., an IgG CH3 domain) of each of the first polypeptide and the second polypeptide, and one or more heterodimerization mutations in the first and / or second polypeptides. In another aspect, the multispecific molecules of the present invention have a P151 mutation (Eu numbering) in an immunoglobulin heavy chain constant domain 1 (CH1 domain) (e.g., an IgG CH1 domain) of at least one of the first and the second polypeptide, optionally a P141 mutation in a paired immunoglobulin light chain constant (CL) domain (e.g., an IgG CL domain), and one or more heterodimerization mutations in the first and / or second polypeptides. In some embodiments, the multispecific molecules can have a P374 mutation in a CH3 domain of each of the first polypeptide and the second polypeptide, a P151 mutation in a CH1 domain of at least one of the first polypeptide and the second polypeptide, optionally a P141 mutation in a paired Page 1 of 370 307304067v1immunoglobulin light chain constant (CL) domain (e.g., an IgG CL domain), and one or more heterodimerization mutations in the first and / or second polypeptides. In some embodiments, the heterodimerization mutations can be in a CH3 domain of the first polypeptide and / or in a CH3 domain of the second polypeptide and can enhance pairing of the first and second polypeptides. In some embodiments, the heterodimerization mutations can be in a first CH1 domain and / or its paired immunoglobulin light chain constant (CL) domain (e.g., an IgG CL domain) and / or in a second CH1 domain and its paired CL domain and can enhance specific pairing of the first CH1 domain and its paired CL domain and / or the second CH1 domain and its paired CL domain. In some embodiments, the one or more heterodimerization mutations can be (i) in a first CH3 domain and in a second CH3 domain and (ii) in a first CH1 domain and / or its paired CL domain and / or in a second CH1 domain and / or its paired CL domain. In some embodiments, one or more heavy chain domains of the multispecific molecules described herein are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of a third polypeptide and a fourth polypeptide. In some embodiments, the multispecific molecules further comprise substitutions at Q38 and Q39 (Chothia numbering) of an immunoglobulin variable region as described herein. The combination of the proline mutations, the heterodimerization mutations in the various domains, and other mutations as described herein provides, for example, a means for enhanced selective assembly of cognate chains in multiparatopic multispecific molecules. BACKGROUND

[0004] Antibodies are a mainstay in protein-based therapeutics. See, Antibodies (Basel).2019 Dec; 8(4): 55.; Published online 2019 Dec 3. doi: 10.3390 / antib8040055; Antibody Structure and Function: The Basis for Engineering Therapeutics; Mark L. Chiu, Dennis R. Goulet, Alexey Teplyakov, and Gary L. Gilliland. While advances have been made in antibody design and manufacturing, there remain present challenges. See, e.g., Madsen et al., Design and engineering of bispecific antibodies: insights and practical considerations, Front. Bioeng. Biotechnol.2024: 12:1352014. doi: 10.3389 / fbioe.2024.1352014. For example, widespread unpredictability in antibody development has been reported. See Cell 2022 Jul 21;185(15):2789-2805 at doi:Page 2 of 370 307304067v110.1016 / j.cell.2022.05.029 and Nature 635, 26-28, 2024 at doi.org / 10.1038 / d41586-024-03590- 0. SUMMARY

[0005] Provided herein are superior materials and methods for designing, use and manufacture of improved biologic molecules. The inventors addressed challenges in the relevant arts which have been, inter alia, design and manufacture of multispecific molecules more effectively in commercially advantaged ways with superior cellular secretion of desired molecular species, modularity, expression, titer, monodispersity, heterodimer formation, pairing efficiencies, biophysical properties, target species, reduction in heterogeneity, etc. For example, in one aspect of the invention, provided herein are multispecific molecules having enhanced pairing; for example multispecific molecules comprising one or more polypeptides each comprising a constant domain comprising an immunoglobulin heavy chain constant domain 3 (CH3 domain), and optionally a variable domain (e.g., variable light chain domain (VL) or variable heavy chain domain (VH)) that binds a target antigen, and, in lieu of a wild-type antibody light chain constant domain (CL) and antibody heavy chain constant domain 1 (CH1), have variant CL and / or CH1 and / or CH3 domains as described herein.

[0006] In one aspect, the multispecific molecules comprise a first polypeptide and a second polypeptide, each comprising an immunoglobulin heavy chain constant domain 3 (CH3 domain), wherein (i) the first polypeptide and the second polypeptide each comprise a P374 mutation (Eu numbering) in its respective CH3 domain, and (ii) either the first polypeptide or the second polypeptide comprises one or more heterodimerization mutations, wherein said one or more heterodimerization mutations favor heterodimerization of the first polypeptide and the second polypeptide over homodimerization of two first polypeptides and two second polypeptides.

[0007] In another aspect, the multispecific molecules comprise a first polypeptide and a second polypeptide, each comprising an immunoglobulin heavy chain constant domain 1 (CH1 domain), wherein (i) at least one of the first polypeptide and the second polypeptide comprises a P151 mutation (Eu numbering) in its respective CH1 domain, and (ii) either the first polypeptide or the second polypeptide comprises one or more heterodimerization mutations, wherein said one or more heterodimerization mutations favor heterodimerization of the first polypeptide and thePage 3 of 370 307304067v1second polypeptide over homodimerization of two first polypeptides and two second polypeptides.

[0008] In some embodiments, the one or more heterodimerization mutations can be in a first CH3 domain and in a second CH3 domain and can enhance pairing of the first and second CH3 domains. In some embodiments, the one or more heterodimerization mutations can be in a first CH1 domain and / or its paired CL domain, and / or in a second CH1 domain and / or its paired CL domain and can enhance specific pairing of the first CH1 domain and its paired CL domain and / or the second CH1 domain and its paired CL domain. In some embodiments, the one or more heterodimerization mutations can be (i) in a first CH3 domain and in a second CH3 domain, and / or (ii) in a first CH1 domain and / or its paired CL domain and / or (iii) in a second CH1 domain and / or its paired CL domain.

[0009] In an aspect, the invention provides a multispecific molecule comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 3 (CH3 domain), and wherein (i) the first polypeptide and the second polypeptide each comprises a mutation at position P374 (Eu numbering) in its respective CH3 domain, and (ii) the first polypeptide and / or the second polypeptide comprises one or more heterodimerization mutations.

[0010] In some embodiments, the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 1 (CH1 domain).

[0011] In some embodiments, the molecule further comprises a third polypeptide and a fourth polypeptide, wherein the third polypeptide and the fourth polypeptide each comprises an immunoglobulin light chain constant (CL) domain, wherein each of said CL domains is paired with its cognate CH1 domain.

[0012] In some embodiments, the first, second, third and fourth polypeptide each comprises an immunoglobulin variable region, wherein the first polypeptide and the third polypeptide associate to form a first antigen-binding site, and the second polypeptide and the fourth polypeptide associate to form a second antigen-binding site.Page 4 of 370 307304067v1

[0013] In some embodiments, the CH1 domain of the first polypeptide is paired with the CL domain of the third polypeptide and the CH1 domain of the second polypeptide is paired with the CL domain of the fourth polypeptide, and wherein (i) at least one of the first polypeptide and the second polypeptide comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain and / or at least one of the third polypeptide and the fourth polypeptide comprises a mutation at position P141 (Eu numbering) in its respective CL domain, and (ii) one or more of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.

[0014] In some embodiments, the first polypeptide and the second polypeptide each comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain.

[0015] In some embodiments, each of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.

[0016] In some embodiments, one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide.

[0017] In some embodiments, the exchanged one or more heavy chain domains and / or one or more light chain domains comprise an immunoglobulin variable region.

[0018] In some embodiments, each of the exchanged heavy chain domains and / or light chain domains comprises an immunoglobulin variable region.

[0019] In some embodiments, the mutation at position P374 (Eu numbering) is an amino acid substitution P374X.

[0020] In some embodiments, X in the amino acid substitution P374X can be any amino acid. In some embodiments, X in the amino acid substitution P374X is selected from A, G, and Q.

[0021] In some embodiments, the mutation at position P151 (Eu numbering) is an amino acid substitution P151X.Page 5 of 370 307304067v1

[0022] In some embodiments, X in the amino acid substitution P151X can be any amino acid.

[0023] In some embodiments, X in the amino acid substitution P151X is A or G.

[0024] In some embodiments, said one or more heterodimerization mutations are selected from charge steering mutations, knob into hole mutations, complementary mutations, and any combinations thereof.

[0025] In some embodiments, the one or more heterodimerization mutations are at position(s) selected from S364, E357, T366, L368, K370, F405, Y407, and K409, according to Eu numbering, and any combinations thereof.

[0026] In some embodiments, the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain; 2) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain;Page 6 of 370 307304067v110) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain; 11) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain; 13) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain; 17) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain;Page 7 of 370 307304067v125) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 26) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 28) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 29) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 30) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain; 32) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain;Page 8 of 370 307304067v140) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; 41) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 43) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 44) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 45) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 47) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or 48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain.

[0027] In some embodiments, the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 2) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 3) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 4) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain;Page 9 of 370 307304067v15) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 6) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 7) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 8) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 9) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 10) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 11) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; 12) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain; 13) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; or 16) S364D and K409D in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain.

[0028] In some embodiments, the molecule comprises the following heterodimerization mutations (Eu numbering): 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain;Page 10 of 370 307304067v13) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain; 5) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 9) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 17) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain;Page 11 of 370 307304067v118) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain; 20) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain; 24) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain;Page 12 of 370 307304067v133) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 35) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain; 39) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain;Page 13 of 370 307304067v148) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain; 50) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; 54) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain; 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

[0029] In some embodiments, the multispecific molecule comprises:Page 14 of 370 307304067v11) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in the second polypeptide; 2) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 3) V185D in the first polypeptide CH1 domain and S174K in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 4) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and V185K in the second polypeptide CH1 domain and S174D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 5) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 6) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 7) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide; 8) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the first polypeptide; 9) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain, wherein P151A is present in the first polypeptide;Page 15 of 370 307304067v110) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and V185D in the second polypeptide CH1 domain and S174K in its paired CL domain, wherein P151A is present in the first polypeptide; 11) V185K in the first polypeptide CH1 domain and S174D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 12) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 13) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; or 14) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide.

[0030] In another aspect, the invention provides a multispecific molecule comprising first, second, third and fourth polypeptides, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 1 (CH1 domain), and the third polypeptide and the fourth polypeptide each comprises an immunoglobulin light chain constant (CL) domain, wherein the CH1 domain of the first polypeptide is paired with the CL domain of the third polypeptide and the CH1 domain of the second polypeptide is paired with the CL domain of the fourth polypeptide, and wherein (i) at least one of the first polypeptide and the second polypeptide each comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain and / or at least one of the third polypeptide and the fourth polypeptide comprises a mutation at position P141 (Eu numbering) in its respective CL domain, and (ii) one or more of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.Page 16 of 370 307304067v1

[0031] In some embodiments, the first polypeptide and the second polypeptide each comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain.

[0032] In some embodiments, each of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.

[0033] In some embodiments, the first, second, third and fourth polypeptide each comprises an immunoglobulin variable region, wherein the first polypeptide and the third polypeptide associate to form a first antigen-binding site, and the second polypeptide and the fourth polypeptide associate to form a second antigen-binding site.

[0034] In some embodiments, one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide.

[0035] In some embodiments, the exchanged one or more heavy chain domains and / or one or more light chain domains comprises an immunoglobulin variable region.

[0036] In some embodiments, each of the exchanged heavy chain domains and / or light chain domains comprises an immunoglobulin variable region.

[0037] In some embodiments, the mutation at position P151 (Eu numbering) is an amino acid substitution P151X.

[0038] In some embodiments, X in the amino acid substitution P151X can be any amino acid.

[0039] In some embodiments, X in the amino acid substitution P151X is A or G.

[0040] In some embodiments, each of the first polypeptide and the second polypeptide further comprises an immunoglobulin heavy chain constant domain 3 (CH3 domain) and (i) a mutation at position P374 (Eu numbering) in its respective CH3 domain; and (ii) the first polypeptide and / or the second polypeptide comprises one or more heterodimerization mutations.Page 17 of 370 307304067v1

[0041] In some embodiments, the mutation at position P374 (Eu numbering) is an amino acid substitution P374X.

[0042] In some embodiments, X in the amino acid substitution P374X can be any amino acid.

[0043] In some embodiments, X in the amino acid substitution P374X is selected from A, G, and Q.

[0044] In some embodiments, said one or more heterodimerization mutations are selected from charge steering mutations, complementary mutations, and any combinations thereof.

[0045] In some embodiments, said one or more heterodimerization mutations are at position(s) selected from S364, E357, T366, L368, K370, F405, Y407, and K409, according to Eu numbering, and any combinations thereof.

[0046] In some embodiments, the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain; 5) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain;Page 18 of 370 307304067v19) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 17) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain; 20) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain;Page 19 of 370 307304067v124) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 35) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain;Page 20 of 370 307304067v139) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain; 50) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; orPage 21 of 370 307304067v154) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain. 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

[0047] In some embodiments, the multispecific molecule comprises: 1) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in the second polypeptide; 2) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 3) V185D in the first polypeptide CH1 domain and S174K in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 4) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; V185K in the second polypeptide CH1 domain and S174D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide;Page 22 of 370 307304067v15) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 6) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 7) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide; 8) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the first polypeptide; 9) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain, wherein P151A is present in the first polypeptide; 10) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and V185D in the second polypeptide CH1 domain and S174K in its paired CL domain, wherein P151A is present in the first polypeptide; 11) V185K in the first polypeptide CH1 domain and S174D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 12) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 13) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; or 14) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain withPage 23 of 370 307304067v1variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide.

[0048] In some embodiments, the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain; 2) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain; 10) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain; 11) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain; 13) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain;Page 24 of 370 307304067v114) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain; 17) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 25) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 26) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 28) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain;Page 25 of 370 307304067v129) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 30) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain; 32) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain; 40) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; 41) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 43) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain;Page 26 of 370 307304067v144) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 45) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 47) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or 48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain.

[0049] In some embodiments, the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 2) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 3) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 4) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 5) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 6) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 7) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 8) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 9) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain;Page 27 of 370 307304067v110) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 11) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; 12) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain; 13) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; or 16) S364D and K409D in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain.

[0050] In some embodiments, the immunoglobulin variable region of the first, second, third and / or fourth polypeptide comprises a mutation at position Q38 and / or Q39 (Chothia numbering).

[0051] In some embodiments, the mutations at position Q38 and / or Q39 (Chothia numbering) is an amino acid substitution Q38X and / or Q39X.

[0052] In some embodiments, the mutations at position Q38 and / or Q39 (Chothia numbering) is an amino acid substitution Q38E and / or Q39K.

[0053] In some embodiments, the CH3 domains of the first and second polypeptides are from an IgG, IgA, IgD, IgE, or IgM.

[0054] In some embodiments, the CH3 domains of the first and second polypeptides are from an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD1, or IgD2.

[0055] In some embodiments, the CH1 domains of the first and second polypeptides are from an IgG, IgA, IgD, IgE, or IgM.Page 28 of 370 307304067v1

[0056] In some embodiments, the CH1 domains of the first and second polypeptides are from an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD1, or IgD2.

[0057] In some embodiments, the second and / or fourth polypeptide is a kappa light chain, a lambda light chain, or a fusion of kappa and lambda light chain.

[0058] In some embodiments, the CL is a kappa light chain or a lambda light chain.

[0059] In some embodiments, the fusion of kappa and lambda light chain comprises a lambda variable region fused onto a kappa constant domain or a kappa variable region fused onto a lambda constant domain.

[0060] In some embodiments, the P151 mutation is in the CH1 domain and the heterodimerization mutations are in the kappa light chain.

[0061] In some embodiments, the P151 mutation is in the CH1 domain and the heterodimerization mutations are in the lambda light chain.

[0062] In some embodiments, the exchanged one or more light chain domains and one or more of the third and fourth polypeptides are kappa light chains.

[0063] In some embodiments, the exchanged one or more light chain domains and one or more of the third and fourth polypeptides are lambda light chains.

[0064] In some embodiments, the exchanged one or more immunoglobulin variable regions of light chain domains are lambda light chains and one or more of the third and fourth polypeptides are kappa light chains.

[0065] In some embodiments, the exchanged one or more immunoglobulin variable regions of light chain domains are kappa light chain and one or more of the third and fourth polypeptides are lambda light chain.

[0066] In some embodiments, said multispecific molecule is a bispecific antibody (bsAb).

[0067] In a related aspect, the invention provides an isolated polynucleotide encoding the multispecific molecule of any of the foregoing embodiments.Page 29 of 370 307304067v1

[0068] In a related aspect, the invention provides a vector comprising the isolated polynucleotide as described herein.

[0069] In a related aspect, the invention provides a host cell comprising the vector as described herein or one or more recombinant polynucleotides encoding the multispecific molecule of any of the foregoing embodiments. In some embodiments, the host cell further comprises an antiapoptotic.

[0070] In a related aspect, the invention provides a composition, comprising the multispecific molecule of any of the foregoing embodiments, the isolated polynucleotide as described herein, the vector as described herein, or the host cell as described herein.

[0071] In a related aspect, the invention provides a method of manufacturing one or more multispecific molecules of any of the foregoing embodiments, comprising: a) incubating the host cell as described herein under conditions allowing for expression of said one or more multispecific molecules; and b) recovering said one or more multispecific molecules from the host cell culture. In some embodiments, the method further comprises using an antiapoptotic.

[0072] In some embodiments, two or more multispecific molecules of any of the foregoing embodiments are manufactured in the host cell.

[0073] In a related aspect, the invention provides a method of producing one or more multispecific molecules in a cell or tissue in a subject in need thereof, comprising administering to the subject the isolated polynucleotide as described herein, the vector as described herein, or the host cell as described herein. In some embodiments, the method further comprises using an antiapoptotic.

[0074] In some embodiments, two or more multispecific molecules of any of the foregoing embodiments are produced in the subject.

[0075] In a related aspect, the invention provides a method of screening a multispecific molecule of any of the foregoing embodiments using high throughput screening, comprising:Page 30 of 370 307304067v1i) providing the isolated polynucleotide as described herein or the vector as described herein to a culture comprising one or more mammalian cells; ii) expressing the multispecific molecule as described herein; iii) collecting and filtering a supernatant of the culture; iv) purifying the multispecific molecule; and v) screening the multispecific molecule.

[0076] In a related aspect, the invention provides a method of delivering an isolated polynucleotide as described herein to a subject in need thereof, comprising: i) encapsulating the isolated polynucleotide in a lipid nanoparticle; and ii) delivering the lipid nanoparticle to the subject.

[0077] In some embodiments, the isolated polynucleotide is mRNA.

[0078] In an aspect, the invention provides a multispecific molecule comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 3 (CH3 domain), and wherein (i) the first polypeptide and the second polypeptide each comprises a means for altering protein folding at position P374 (Eu numbering) in its respective CH3 domain, and (ii) the first polypeptide and / or the second polypeptide comprises one or more dimerization means.

[0079] In some embodiments, the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 1 (CH1 domain).

[0080] In some embodiments, the molecule further comprises a third polypeptide and a fourth polypeptide, wherein the third polypeptide and the fourth polypeptide each comprises an immunoglobulin light chain constant (CL) domain, wherein each of said CL domains is paired with its cognate CH1 domain.

[0081] In some embodiments, the first, second, third and fourth polypeptide each comprises an immunoglobulin variable region, wherein the first polypeptide and the third polypeptide associate to form a first antigen-binding site, and the second polypeptide and the fourth polypeptide associate to form a second antigen-binding site.Page 31 of 370 307304067v1

[0082] In some embodiments, the CH1 domain of the first polypeptide is paired with the CL domain of the third polypeptide and the CH1 domain of the second polypeptide is paired with the CL domain of the fourth polypeptide, and wherein (i) at least one of the first polypeptide and the second polypeptide comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain and / or at least one of the third polypeptide and the fourth polypeptide comprises a mutation at position P141 (Eu numbering) in its respective CL domain, and (ii) one or more of the first, second, third, or fourth polypeptide comprises one or more dimerization means.

[0083] In some embodiments, the first polypeptide and the second polypeptide each comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain.

[0084] In some embodiments, each of the first, second, third, or fourth polypeptide comprises one or more dimerization means.

[0085] In some embodiments, one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide.

[0086] In some embodiments, the exchanged one or more heavy chain domains and / or one or more light chain domains comprise an immunoglobulin variable region.

[0087] In some embodiments, each of the exchanged heavy chain domains and / or light chain domains comprises an immunoglobulin variable region.

[0088] In some embodiments, the mutation at position P374 (Eu numbering) is an amino acid substitution P374X.

[0089] In some embodiments, X in the amino acid substitution P374X can be any amino acid. In some embodiments, X in the amino acid substitution P374X is selected from A, G, and Q.

[0090] In some embodiments, the mutation at position P151 (Eu numbering) is an amino acid substitution P151X.

[0091] In some embodiments, X in the amino acid substitution P151X can be any amino acid.Page 32 of 370 307304067v1

[0092] In some embodiments, X in the amino acid substitution P151X is A or G.

[0093] In some embodiments, said one or more dimerization means are selected from charge steering mutations, knob into hole mutations, complementary mutations, and any combinations thereof.

[0094] In some embodiments, said one or more dimerization means are at position(s) selected from S364, E357, T366, L368, K370, F405, Y407, and K409, according to Eu numbering, and any combinations thereof.

[0095] In some embodiments, the multispecific molecule comprises the following dimerization means (Eu numbering): 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain; 2) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain; 10) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain;Page 33 of 370 307304067v111) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain; 13) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain; 17) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 25) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain;Page 34 of 370 307304067v126) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 28) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 29) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 30) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain; 32) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain; 40) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain;Page 35 of 370 307304067v141) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 43) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 44) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 45) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 47) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or 48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain.

[0096] In some embodiments, the multispecific molecule comprises the following dimerization means (Eu numbering): 1) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 2) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 3) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 4) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 5) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain;Page 36 of 370 307304067v16) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 7) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 8) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 9) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 10) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 11) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; 12) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain; 13) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; or 16) S364D and K409D in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain.

[0097] In some embodiments, the molecule comprises the following dimerization means (Eu numbering): 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain;Page 37 of 370 307304067v14) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain; 5) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 9) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no dimerization means in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no dimerization means in its paired CL domain; 17) no dimerization means in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no dimerization means in the first or second polypeptide CH1 domain; and T178W in its paired CL domain;Page 38 of 370 307304067v119) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain; 20) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain; 24) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain;Page 39 of 370 307304067v134) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 35) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain; 39) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain;Page 40 of 370 307304067v149) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain; 50) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; 54) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain; 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

[0098] In some embodiments, the multispecific molecule comprises: 1) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in the second polypeptide;Page 41 of 370 307304067v12) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain; and no dimerization means in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 3) V185D in the first polypeptide CH1 domain and S174K in its paired CL domain; and no dimerization means in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 4) V173A and S181Y in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; and V185K in the second polypeptide CH1 domain and S174D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 5) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 6) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 7) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide; 8) V173A and S181W in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the first polypeptide; 9) no dimerization means in the first polypeptide CH1 domain and T178Y in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain, wherein P151A is present in the first polypeptide; 10) no dimerization means in the first polypeptide CH1 domain and T178Y in its paired CL domain; and V185D in the second polypeptide CH1 domain and S174K in its paired CL domain, wherein P151A is present in the first polypeptide; 11) V185K in the first polypeptide CH1 domain and S174D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in each of the first and second polypeptide;Page 42 of 370 307304067v112) V173A and S181Y in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 13) V173A and S181W in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; or 14) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide.

[0099] In another aspect, the invention provides a multispecific molecule comprising first, second, third and fourth polypeptides, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 1 (CH1 domain), and the third polypeptide and the fourth polypeptide each comprises an immunoglobulin light chain constant (CL) domain, wherein the CH1 domain of the first polypeptide is paired with the CL domain of the third polypeptide and the CH1 domain of the second polypeptide is paired with the CL domain of the fourth polypeptide, and wherein (i) at least one of the first polypeptide and the second polypeptide each comprises a means for altering protein folding at position P151 (Eu numbering) in its respective CH1 domain and / or at least one of the third polypeptide and the fourth polypeptide comprises a mutation at position P141 (Eu numbering) in its respective CL domain, and (ii) one or more of the first, second, third, or fourth polypeptide comprises one or more dimerization means.

[0100] In some embodiments, the first polypeptide and the second polypeptide each comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain.

[0101] In some embodiments, each of the first, second, third, or fourth polypeptide comprises one or more dimerization means.

[0102] In some embodiments, the first, second, third and fourth polypeptide each comprises an immunoglobulin variable region, wherein the first polypeptide and the thirdPage 43 of 370 307304067v1polypeptide associate to form a first antigen-binding site, and the second polypeptide and the fourth polypeptide associate to form a second antigen-binding site.

[0103] In some embodiments, one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide.

[0104] In some embodiments, the exchanged one or more heavy chain domains and / or one or more light chain domains comprises an immunoglobulin variable region.

[0105] In some embodiments, each of the exchanged heavy chain domains and / or light chain domains comprises an immunoglobulin variable region.

[0106] In some embodiments, the mutation at position P151 (Eu numbering) is an amino acid substitution P151X.

[0107] In some embodiments, X in the amino acid substitution P151X can be any amino acid.

[0108] In some embodiments, X in the amino acid substitution P151X is A or G.

[0109] In some embodiments, each of the first polypeptide and the second polypeptide further comprises an immunoglobulin heavy chain constant domain 3 (CH3 domain) and (i) a mutation at position P374 (Eu numbering) in its respective CH3 domain; and (ii) the first polypeptide and / or the second polypeptide comprises one or more dimerization means.

[0110] In some embodiments, the mutation at position P374 (Eu numbering) is an amino acid substitution P374X.

[0111] In some embodiments, X in the amino acid substitution P374X can be any amino acid.

[0112] In some embodiments, X in the amino acid substitution P374X is selected from A, G, and Q.Page 44 of 370 307304067v1

[0113] In some embodiments, said one or more dimerization means are selected from charge steering mutations, complementary mutations, and any combinations thereof.

[0114] In some embodiments, said one or more dimerization means are at position(s) selected from S364, E357, T366, L368, K370, F405, Y407, and K409, according to Eu numbering, and any combinations thereof.

[0115] In some embodiments, the molecule comprises the following dimerization means (Eu numbering): 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain; 5) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 9) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain;Page 45 of 370 307304067v112) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no dimerization means in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no dimerization means in its paired CL domain; 17) no dimerization means in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no dimerization means in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain; 20) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain; 24) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain;Page 46 of 370 307304067v127) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 35) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain; 39) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain;Page 47 of 370 307304067v142) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain; 50) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; 54) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain; 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain;Page 48 of 370 307304067v157) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

[0116] In some embodiments, the multispecific molecule comprises: 1) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in the second polypeptide; 2) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain; and no dimerization means in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 3) V185D in the first polypeptide CH1 domain and S174K in its paired CL domain; and no dimerization means in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 4) V173A and S181Y in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; V185K in the second polypeptide CH1 domain and S174D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 5) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 6) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in each of the first and second polypeptide;Page 49 of 370 307304067v17) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide; 8) V173A and S181W in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the first polypeptide; 9) no dimerization means in the first polypeptide CH1 domain and T178Y in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain, wherein P151A is present in the first polypeptide; 10) no dimerization means in the first polypeptide CH1 domain and T178Y in its paired CL domain; and V185D in the second polypeptide CH1 domain and S174K in its paired CL domain, wherein P151A is present in the first polypeptide; 11) V185K in the first polypeptide CH1 domain and S174D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no dimerization means in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 12) V173A and S181Y in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 13) V173A and S181W in the first polypeptide CH1 domain and no dimerization means in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; or 14) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide.

[0117] In some embodiments, the multispecific molecule comprises the following dimerization means (Eu numbering): 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain;Page 50 of 370 307304067v12) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain; 10) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain; 11) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain; 13) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain;Page 51 of 370 307304067v117) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 25) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 26) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 28) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 29) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 30) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain;Page 52 of 370 307304067v132) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain; 40) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; 41) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 43) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 44) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 45) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain;Page 53 of 370 307304067v147) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or 48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain.

[0118] In some embodiments, the multispecific molecule comprises the following dimerization means (Eu numbering): 1) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 2) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 3) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 4) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 5) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 6) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 7) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 8) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 9) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 10) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 11) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; 12) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain;Page 54 of 370 307304067v113) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; or 16) S364D and K409D in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain.

[0119] In some embodiments, the immunoglobulin variable region of the first, second, third and / or fourth polypeptide comprises a mutation at position Q38 and / or Q39 (Chothia numbering).

[0120] In some embodiments, the mutations at position Q38 and / or Q39 (Chothia numbering) is an amino acid substitution Q38X and / or Q39X.

[0121] In some embodiments, the mutations at position Q38 and / or Q39 (Chothia numbering) is an amino acid substitution Q38E and / or Q39K.

[0122] In some embodiments, the CH3 domains of the first and second polypeptides are from an IgG, IgA, IgD, IgE, or IgM.

[0123] In some embodiments, the CH3 domains of the first and second polypeptides are from an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD1, or IgD2.

[0124] In some embodiments, the CH1 domains of the first and second polypeptides are from an IgG, IgA, IgD, IgE, or IgM.

[0125] In some embodiments, the CH1 domains of the first and second polypeptides are from an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD1, or IgD2.

[0126] In some embodiments, the second and / or fourth polypeptide is a kappa light chain, a lambda light chain, or a fusion of kappa and lambda light chain.

[0127] In some embodiments, the CL is a kappa light chain or a lambda light chain.Page 55 of 370 307304067v1

[0128] In some embodiments, the fusion of kappa and lambda light chain comprises a lambda variable region fused onto a kappa constant domain or a kappa variable region fused onto a lambda constant domain.

[0129] In some embodiments, the P151 mutation is in the CH1 domain and the dimerization means are in the kappa light chain.

[0130] In some embodiments, the P151 mutation is in the CH1 domain and the dimerization means are in the lambda light chain.

[0131] In some embodiments, the exchanged one or more light chain domains and one or more of the third and fourth polypeptides are kappa light chains.

[0132] In some embodiments, the exchanged one or more light chain domains and one or more of the third and fourth polypeptides are lambda light chains.

[0133] In some embodiments, the exchanged one or more immunoglobulin variable regions of light chain domains are lambda light chains and one or more of the third and fourth polypeptides are kappa light chains.

[0134] In some embodiments, the exchanged one or more immunoglobulin variable regions of light chain domains are kappa light chains and one or more of the third and fourth polypeptides are lambda light chains.

[0135] In some embodiments, said molecule is a bispecific antibody.

[0136] In another aspect, the invention provides an isolated polynucleotide encoding the multispecific molecule of any of the foregoing embodiments.

[0137] In another aspect, the invention provides a vector comprising the isolated polynucleotide as described herein.

[0138] In another aspect, the invention provides a host cell comprising the vector as described herein or one or more recombinant polynucleotides encoding the multispecificPage 56 of 370 307304067v1molecule of any of the foregoing embodiments. In some embodiments, the host cell further comprises an antiapoptotic.

[0139] In another aspect, the invention provides a composition comprising the multispecific molecule of any of the foregoing embodiments, the isolated polynucleotide as described herein, the vector as described herein, or the host cell as described herein.

[0140] In another aspect, the invention provides a method of manufacturing one or more multispecific molecules of any of the foregoing embodiments, comprising: a) incubating the host cell as described herein under conditions allowing for expression of said one or more multispecific molecules; and b) recovering said one or more multispecific molecules from the host cell culture. In some embodiments, the method further comprises using an antiapoptotic.

[0141] In some embodiments, two or more multispecific molecules are manufactured in the host cell.

[0142] In another aspect, the invention provides a method of producing one or more multispecific molecules of any of the foregoing embodiments in a cell or tissue in a subject in need thereof, comprising administering to the subject the isolated polynucleotide as described herein, the vector as described herein, or the host cell as described herein. In some embodiments, the method further comprises using an antiapoptotic.

[0143] In some embodiments, two or more multispecific molecules of any of the foregoing embodiments are produced in the subject.

[0144] In another aspect, the invention provides a method of screening a multispecific molecule of any of the foregoing embodiments using high throughput screening, comprising: i) providing the isolated polynucleotide as described herein or the vector as described herein to a culture comprising one or more mammalian cells; ii) expressing the multispecific molecule as described herein; iii) collecting and filtering a supernatant of the culture; iv) purifying the multispecific molecule; and v) screening the multispecific molecule.Page 57 of 370 307304067v1

[0145] In another aspect, the invention provides a method of delivering an isolated polynucleotide as described herein to a subject in need thereof, comprising: i) encapsulating the isolated polynucleotide in a lipid nanoparticle; and ii) delivering the lipid nanoparticle to the subject.

[0146] In some embodiments, the isolated polynucleotide is mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] The accompanying Figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0148] FIGS.1A-1B depict exemplary mutations in the CH1-CL interface. FIG.1A illustrates an antibody in which the heavy chains are colored black, and light chains white. Variable domains are patterned. The CH1-CL interface is highlighted by a dashed box. FIG.1B shows structural view of the native CH1-CL interaction sites selected for mutagenesis. The CH1 domain is colored black, and the CL domain is white. Residue numbers are indicated according to Eu numbering throughout. Distances between residues selected for mutagenesis are indicated by dashed lines. The bold asterisks shown in either black or white in the panels of FIG.1B represent the sites of the oxygen of the amino acid residue.

[0149] FIG.2 depicts exemplary mutations at the CH1-CL interface which enhance pairing of HC with cognate LC. Monoclonal antibodies (mAbs) harboring point mutations in the CH1-CL interface expressed at varying levels. Bispecific antibodies (bsAbs) are shown having either: 1. CH1-CL mutations on HC1 / LC1, 2. HC1 / LC1 and HC2 / LC2 Fabs, 3. variable domain exchange on HC1 / LC1 with point mutations on HC2 / LC2, or 4. variable domain exchange and point mutations on HC1 / LC1 and point mutations on HC2 / LC2 Fab.

[0150] FIGS.3A-3C depict mutation of P151A in the CH1 domain, impacting HC / LC pairing. FIG.3A shows expression of mAbs featuring mutations to influence HC-LC pairing and additional mutation of the cis proline CH1 P151A or CL P141A. FIG.3B shows expression half-antibodies harboring either the ++ / -- (CH1L145K / S181K / CLS131D / T178Dor CH1++ / CL--)Page 58 of 370 307304067v1mutations or the -- / ++ (CH1L145D / S181D / CLS131K / T178K or CH1-- / CL++) mutations along with mutation of other prolines in the CH1 domain. FIG.3C shows human IgG1 mAbs harboring the indicated mutations were expressed, purified, and evaluated by SDS-PAGE. FIG.3A includes the following mAbs, among others described herein: RSVFB143 (SEQ ID NOs: 1004, 1016, 1068, 1080); RSVFB142 (SEQ ID NOs: 1005, 1017, 1069, 1081); RSVFB139 (SEQ ID NOs: 1006, 1018, 1070, 1082); RSVFB155 (SEQ ID NOs: 1007, 1019, 1071, 1083); RSVFB156 (SEQ ID NOs: 1008, 1020, 1072, 1084); RSVFB153 (SEQ ID NOs: 1010, 1022, 1074, 1086); RSVFB149 (SEQ ID NOs: 1011, 1023, 1075, 1087); RSVFB148 (SEQ ID NOs: 1012, 1024, 1076, 1088); RSVFB147 (SEQ ID NOs: 1013, 1025, 1077, 1089); and RSVFB136 (SEQ ID NOs: 1014, 1026, 1078, 1090), which are representative and non-limiting.

[0151] FIGS.4A-4C depict bsAbs featuring P151A with charge-steering mutations pairing HC-LC. FIG.4A shows design of the bsAb with exchanged, or “swapped” variable domains on Fab1, and point mutations on Fab2 for enhanced HC-LC pairing. SDS-PAGE gels shows the purity of bsAbs. FIG.4B shows that HC-LC pairing efficiency was assessed by mass spectrometry and % efficiency was graphed. Descriptions of the antibodies and species expressed are described in the table. The table also includes further description of the pairing efficiency as measured by mass spectrometry. FIG.4C shows that titers were measured using surface biolayer interferometry (Octet), and HC-LC pairing efficiency was assessed by mass spectrometry and % efficiency was graphed. Descriptions of the antibodies and species expressed are described in the table. FIG.4B includes the following bsAbs, among others described herein: GUC3B11 (SEQ ID NOs: 1030, 1040, 1050, 1060, 1094, 1104, 1114) and GUC3B14 (SEQ ID NOs: 1033, 1043, 1053, 1063, 1097, 1107, 1117), which are representative and non- limiting.

[0152] FIGS.5A-5B depict an exemplary sequence comparison of C1-type Ig domains and structure comparison of CH1-CL heterodimer with CH3 homodimer. FIG.5A shows a sequence alignment of C1-type Ig domains from the human proteome. Conserved residues are shown within the rectangles, and the position of the IgG CH1 P151 is indicated by an arrow, which is the focus of embodiments described herein. The sequences in the alignment are in the following order of SEQ ID NOs: 6477-6562. SEQ ID NO: 6514 is duplicated in the figure are follow one another. FIG.5B shows a structural comparison of the CH1-CL heterodimer with thePage 59 of 370 307304067v1CH3 homodimer (PDB ID 3AVE for Fc and PDB ID 3NA9 for CH1-CL). The CH1 domain is colored black; CL, white, and CH3 domains black and white. Structures were aligned on all residues.

[0153] FIGS.6A-6E demonstrate the application of pairing-induced folding to heterodimerization between CH3 domains. FIG.6A shows a bsAb featuring a VHH-Fc fusion on HC1 paired with a Fab HC2 + LC. FIGS.6B-6C show the residues used to promote HC-LC steering translated to structurally equivalent sites in the CH3 domain. FIG.6D shows the results of bsAbs having the indicated mutations regarding their abilities to promote heterodimer formation and to prevent secretion of mAb and half-antibody contaminants. Each potential species is indicated on the SDS-PAGE gel image. FIG.6E shows the results of evaluating bsAbs for their monodispersity by analytical size-exclusion chromatography. Shown is a comparison bsAbs having the format shown in panel 6A, and having either a wildtype or mutated Fc domain. FIG.6D includes the following bsAbs, among others described herein: RFPIB21 (SEQ ID NOs: 236, 484, 732, 980, 1346, 1594); RFPIB20 (SEQ ID NOs: 237, 485, 733, 981, 1347, 1595); RFPIB16 (SEQ ID NOs: 238, 486, 734, 982, 1348, 1596); RFPIB15 (SEQ ID NOs: 239, 487, 735, 983, 1349, 1597); and RFPIB18 (SEQ ID NOs: 240, 488, 736, 984, 1350, 1598), which are representative and non-limiting. The bold asterisks shown in either black or white in the panels of the figures represent the sites of the oxygen of the amino acid residue. A double asterisk in either black or white in the panels represent the sites of the nitrogen of the amino acid residue. Distances between residues are indicated by dashed lines.

[0154] FIGS.7A-7C depict the effects of different mutations at P374. FIG.7A shows bsAbs harboring the ++ mutations (L368K / F405K) on HC1 and the – mutations (S364D / K409D) on HC2 and wherein both HCs featured mutation of P374 to the indicated aa residue were analyzed by SDS-PAGE for their abilities to prevent secretion of homodimer or half antibody species. FIG.7B shows the expression titers of the bsAbs were measured. FIG.7C shows the thermal stability of selected bsAbs was evaluated by Differential Scanning Calorimetry (DSC), and melting temperatures are indicated in the table. FIG.7A includes the following bsAbs, among others described herein: RFPIB62 (SEQ ID NOs: 241, 489, 737, 985, 1351, 1599); RFPIB61 (SEQ ID NOs: 242, 490, 738, 986, 1352, 1600); RFPIB60 (SEQ ID NOs: 243, 491, 739, 987, 1353, 1601); RFPIB59 (SEQ ID NOs: 244, 492, 740, 988, 1354, 1602); RFPIB57Page 60 of 370 307304067v1(SEQ ID NOs: 245, 493, 741, 989, 1355, 1603); RFPIB56 (SEQ ID NOs: 246, 494, 742, 990, 1356, 1604); RFPIB55 (SEQ ID NOs: 247, 495, 743, 991, 1357, 1605); RFPIB54 (SEQ ID NOs: 248, 496, 744, 992, 1358, 1606); RFPIB53 (SEQ ID NOs: 249, 497, 745, 993, 1359, 1607); RFPIB52 (SEQ ID NOs: 250, 498, 746, 994, 1360, 1608); RFPIB51 (SEQ ID NOs: 251, 499, 747, 995, 1361, 1609); RFPIB50 (SEQ ID NOs: 252, 500, 748, 996, 1362, 1610); RFPIB49 (SEQ ID NOs: 253, 501, 749, 997, 1363, 1611); RFPIB48 (SEQ ID NOs: 254, 502, 750, 998, 1364, 1612); RFPIB47 (SEQ ID NOs: 255, 503, 751, 999, 1365, 1613); RFPIB46 (SEQ ID NOs: 256, 504, 752, 1000, 1366, 1614); and RFPIB45 (SEQ ID NOs: 257, 505, 753, 1001, 1367, 1615), which are representative and non-limiting.

[0155] FIGS.8A-8D show biophysical characterizations of proline-mutated heavy chain bsAbs. FIG.8A shows a description of bsAbs evaluated for their biophysical properties. FIG.8B shows the binding affinities of the bsAbs to FcRn using Surface Plasmon Resonance (SPR). The on-rates were measured at pH 6.0 and off-rates were measured at pH 7.4. FIG.8C shows the evaluation of the stability of the bsAbs incubated at 37 ºC either in HEPES-buffered saline (HBS) or human serum (Hu Serum). Antibodies were incubated at 1 mg / mL and evaluated by analytical SEC (aSEC) and the proportions of either high or low molecular weight impurities were quantified by A280. FIG.8D shows the non-specific binding properties of bsAbs against the molecules indicated in the graph. CNTO607 was used as a positive control for non-specific binding and CNTO5825 was used as a negative control.

[0156] FIGS.9A-9C depict the determination of the pairing efficiency of proline-mutated CH3 domain bsAbs. FIG.9A shows SDS-PAGE analysis of protein A-purified supernatants from cells expressing bsAbs featuring either wt Fc or (++) x (--) Fc. FIG.9B shows an analysis of the (++PA) x (--PA) or (++PG) x (--PG) Fc variants. FIG.9C shows an analysis of KIH or proline-mutated KIH Fc bsAbs.

[0157] FIGS.10A-10B depict the optimization of the charge complementarity mutants in the CH3 domain. The top panels of FIGS.10A-10B show SDS-PAGE analysis of bsAbs having different permutations of mutations of L368K / D / E, F405K / D / E, S364K / D / E, and K409D / E. All bsAbs featured the mutations indicated in the tables (bottom panel of FIGS.10A-10B) in addition to mutation of P374G on both HCs except RFPIB14 (SEQ ID NOS 11, 259, 507, 755,Page 61 of 370 307304067v11121, and 1369) (wt Fc) (RFPIB23 (SEQ ID NOS 13, 261, 509, 757, 1123, and 1371) ((++) x (-- )), and RFPIB102 (SEQ ID NOS 258, 506, 754, 1002, 1368, and 1616) (P374A Fc).

[0158] FIGS.11A-11D depict HC heterodimerization by adding P374A to the described mutations. FIGS.11A-11B show SDS-PAGE analysis of bsAbs harboring the CH3 mutations are shown in the tables. Each design was evaluated with or without addition of the P374A mutation. FIGS.11C-11D show that selected bsAbs were evaluated for efficiency of heterodimer formation using a DNA ratio experiment similar to that shown in Figure 9. Mutation abbreviations are consistent with the tables in FIG.9A. Abbreviations are described in the figure.

[0159] FIGS.12A-12K depict heterodimeric CH3 domain bsAbs. SDS-PAGE analysis of bsAbs harboring the CH3 mutations are shown in the tables. The charge mutations L368K / F405K are indicated as (++). Variants having only L368K are indicated by (+.). Variants having only F405K are indicated by (.+). The charge mutations S364D / K409D are indicated as (--). Variants having only S364D are indicated by (- .). Variants having only K409D are indicated by (.-).

[0160] FIGS.13A-13I depict HC-LC pairing designs. A-I, bsIgG formats evaluated for efficiency of HC-LC pairing. All bsIgG featured the hole mutations (T366S / L368A / Y407V) along with the RF mutations (H435R / Y436F) on HC 1 (black) and the knob mutation (T366W) on HC2 (gray). Light chains are colored white. Variable domains are patterned. BsIgG featured either one or two point mutations indicated by asterisks (*), exchanged variable domains (curved arrow), or the P151A mutation (PA). Positions of the modifications are indicated on the images.

[0161] FIGS.14A-14B depict optimal designs for HC-LC pairing. FIG.14A shows SEC chromatograms of bsIgG identified as having > 90 % monodispersity by SEC and > 90 % target species by LC-MS. Protein names are indicated on the graphs, and antibody designs are indicated in Table 9. FIG.14B shows LC-MS analysis of the optimal bsIgG designs. NR indicates samples were non-reduced. Max. intensities were calculated based on ion counts.

[0162] FIGS.15A-15D depict designs to enhance HC-LC pairing efficiency for a v- region pairs harboring a lambda LC. FIGs.15A-15C show design of the bsAbs. Heavy chains are colored black and gray, and light chains are colored white. Variable regions are patterned.Page 62 of 370 307304067v1Asterisks (*) indicate point mutations. Isotype of CL domains are indicated as kappa (k) or lambda (λ). “PA” indicates the CH1P151A mutation. FIG.15D shows HC-LC pairing efficiency was assessed by mass spectrometry and % efficiency was graphed. Descriptions of the antibodies and species expressed are described in the table.

[0163] FIGS.16A-16G depict biophysical analysis of the structural impact of mutations in the CH3, CH1, and CL domains. FIGs.16A-16E show CD spectra of individual domains having the mutations indicated on the spectra. Constructs are indicated in the graphs. FIGs. 16F-16G show a comparison of the 2D-15N-HSQC spectra of wild-type vs P374A CH3 domains. Chemical shift perturbations are shown in FIG.16G and were calculated according to described methods. FIG 16 also shows an alignment of SEQ ID NO: 6563 and SEQ ID NO: 6564. FIGs. 16A-16E include the following Abs, among others described herein: RFPIW63 (CH3 Knob-PA; SEQ ID NOs: 1665, 1675); RFPIW61 (CH3 Knob; SEQ ID NOs: 1666, 1676); RFPIW60 (CH3 Hole; SEQ ID NOs: 1667, 1677); RFPIW58 (CH3 Hole-PA; SEQ ID NOs: 1668, 1678); RFPIW45 (CH3 PA; SEQ ID NOs: 1669, 1679); RFPIW21 (CH3 WT; SEQ ID NOs: 1670, 1680); CH1 WT (SEQ ID NO: 1671); CH1 P151A (SEQ ID NO: 1672); CL WT (SEQ ID NO: 1673); and CL P141A (SEQ ID NO: 1674).

[0164] FIGS.17A-17C depict the crystal structure of the KIH P374A Fc. FIG.17A shows a comparison of the P374A Fc (RFPIB319 (SEQ ID NOS 1777, 1778, 1779, and 1780)) to other IgG1 Fc structures. RFPIB319 was aligned over all atoms against four IgG1 Fc structures (PDB IDs 3AVE, 5HY9, 4NQS, and 5DI8). The r.m.s.d. values are shown on the figure. Core glycans are shown for RFPIB319. FIG.17B shows that the A-B loop of the P374A Fc (RFPIB319, gray) has a local structure similar to wild-type IgG1 (black). The alignment is over the displayed residues between RFPIB319 and PDB ID 3AVE. FIG.17C shows 2Fo-Fc electron density map of the region of RFPIB319 displayed in panel B shown at 2 s / n.

[0165] FIGS.18A-18F depict comparison of CH1-CL designs to described mutation sets. FIG.18A shows summary of the bsAbs evaluated and characterizations. All bsAbs featured hole PA mutations (CH3T366S / L368A / Y407V / P374A) on HC1 and knob PA (CH3T366W / P374A) on HC2. The designs of the HC-LC pairing mutations are indicated. The samples indicated as Design A and ProAla featured exchange of either VL and VH (Design A-VHVL and ProAla),Page 63 of 370 307304067v1CH1 and CL (Design A-CH1CL), or exchange of the VH-CH1 with VL-CL (Design A Fab). Titers were measured using surface biolayer interferometry (Octet) using protein A sensors. The % pairing efficiency was measured by subunit-analysis LC-MS. FIG.18B shows analytical SEC analysis of the bsAbs. Samples are indicated. Absorbance at 280 nM was normalized to the main peak (monomer), and the % monomer was reported. FIGs.18C-18D show LC-MS analysis of select bsAbs with pairing efficiency > 85 %. Samples were analyzed either intact (FIG.18C) or after cleavage above the hinge (FIG.18D) (Fabdello, Genovis). FIGs.18E-18F show tables describing the relative ion intensities of different species identified after protein A purification.

[0166] FIGS.19A-19C depict secretion analysis. BsAbs having the format described in Figure 6A were expressed at the HC1:HC2 DNA ratios shown in the figure. FIG.19A shows culture supernate was flowed through protein A affinity resin and eluates were analyzed by SDS- PAGE. FIG.19B shows culture supernate was flowed through protein G affinity resin and eluates were analyzed by SDS-PAGE. FIG.19C shows culture supernatants were analyzed by SDS-PAGE without purification.

[0167] FIGS.20A-20D depict results of characterization of exemplary bsIgGs. FIG.20A shows a format of bsIgGs of Example 24. FIG.20A shows exemplary format of bsIgG, wherein Fab 1 is featured and an exchanged variable domain. Additional mutations on each polypeptide are indicated in FIG.20A. FIGS.20B-20D show antibody titers, SEC monodispersity, and % target species of bsIgGs of Example 24 having the format shown in the exemplary format in panel of FIG.20A. Individual values are shown as open circles and median value is indicated by the bar.

[0168] FIGS.21A-21C depict results of the functional evaluation of exemplary bsIgGs. FIGS.21A-21C show specific binding to targets in accordance with the designs of the present invention, including T cell binding, SK-BR-3 cell binding, and % cytotoxicity of the exemplary bsIgGs of Example 24 having the format shown in the format in panel A of FIG.20. DETAILED DESCRIPTION DefinitionsPage 64 of 370 307304067v1

[0169] It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0170] Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0171] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0172] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0173] The conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0174] The transitional terms “comprising,” “consisting essentially of,” and “consisting of” are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of” excludes any element, step, or ingredient not specified in the claim; and (iii)Page 65 of 370 307304067v1“consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.”

[0175] “Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms “subject” and “patient” are used interchangeably herein.

[0176] “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result, or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.

[0177] “Specific binding” or “specifically binds” or “specifically binding” or “binds” refer to an antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the antibody binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (KD) of about 5x10-8M or less, for example about 1x10-9M or less, about 1x10-10M or less, about 1x10-11M or less, or about 1x10-12M or less, typically with the KD that is at least one hundred-fold less than its KD for binding to a non- specific antigen (e.g., BSA, casein). The dissociation constant may be measured using known protocols. Antibodies that bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes (chimpanzee, chimp). While a monospecific antibody binds one antigen or one epitope, a bispecific antibody binds two distinct antigens or two distinct epitopes.

[0178] “Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies (mAbs) including murine, human, humanized and chimericPage 66 of 370 307304067v1monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific (bsAbs), trispecific, tetraspecific etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The position of any amino acid in an antibody as described herein in any numbering scheme can be determined by one of ordinary skill in the art with reference to the positions as described herein using, e.g., the Eu, Kabat, IMGT, and / or Chothia numbering schemes.

[0179] “Complementarity determining regions” (CDR) are antibody regions that bind an antigen. CDRs may be defined using various delineations such as Kabat (Wu et al. (1970) J Exp Med 132: 211-50) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77) and AbM (Martin and Thornton (1996) J Bmol Biol 263: 800-15). The correspondence between the various delineations and variable region numbering are described (see e.g., Lefranc et al. (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun, (2001) J Mol Biol 309:657-70; International ImMunoGeneTics (IMGT) database; Web resources, imgt_org). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The term “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Kabat, Chothia, IMGT or AbM, unless otherwise explicitly stated in the specification.Page 67 of 370 307304067v1

[0180] Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0181] “Antigen binding fragment” refers to a portion of an immunoglobulin molecule that binds an antigen. Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include the VH, the VL, the VH and the VL, Fab, F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3- FR4 portions, the HCDR1, the HCDR2 and / or the HCDR3 and the LCDR1, the LCDR2 and / or the LCDR3. VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Patent Publ. Nos. WO1998 / 44001, WO1988 / 01649, WO1994 / 13804 and WO1992 / 01047.

[0182] “Monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C- terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation. Monoclonal antibodies typically bind one antigenic epitope. A bispecific monoclonal antibody binds two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent.

[0183] “Bispecific” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific antibody may have cross-reactivityPage 68 of 370 307304067v1to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0184] “Heterodimerization mutations” refers to mutations which favor heterodimerization of the first polypeptide and the second polypeptide over homodimerization of two first polypeptides and two second polypeptides or generation of half-antibodies.

[0185] Provided herein are multispecific molecules having enhanced pairing; for example multispecific molecules comprising one or more polypeptides each comprising a heavy chain constant domain 3 (CH3), and optionally a variable domain (e.g., variable light chain domain (VL) or variable heavy chain domain (VH)) that binds a target antigen, and, in lieu of a wild-type antibody light chain constant domain (CL) and antibody heavy chain constant domain 1 (CH1), have variant CL and / or CH1 and / or CH3 domains as described herein. In specific embodiments, one or more of the CH3 domains and / or CH1 domains and / or CL domains comprise one or more mutations in certain proline residues and / or one or more heterodimerization mutations to enhance (i) pairing of the CH3 domains of each polypeptide; (ii) pairing of the first CH1 domain and first CL domain; and / or (iii) pairing of the second CH1 domain and second CL domain. The one or more heterodimerization mutations favor heterodimerization of a first polypeptide and a second polypeptide over homodimerization of two first polypeptides and two second polypeptides.

[0186] In specific embodiments, a multispecific molecule binds to two or more target antigens. In specific embodiments, a multispecific molecule comprises two or more polypeptides, wherein the polypeptides form paratopes that bind to one or more target antigens. In specific embodiments, a pair of polypeptides each comprise (i) a Fc domain comprising a CH2 and a CH3 domain, and (ii) a variable domain and / or a CL and / or CH1 domain, wherein said variable domains form a paratope that binds to a target antigen.

[0187] The inventors, in view of the teachings in the specification, appreciated that bispecific antibodies (bsAbs) can provide novel mechanisms of action such as immune cell redirection, dual antigen targeting, dual epitope targeting to enhance ADC internalization, and others. However, many challenges in production and screening of bsAbs persist up to now.Page 69 of 370 307304067v1Efforts to manufacture bsAbs have largely focused on protein-based delivery. A major aim for bsAb technology has been delivery via nucleic acids, using the patient’s own cells as a production facility. The inventors address, inter alia, the two major challenges for nucleic acid- based delivery of bsAbs: A) production of potentially toxic off-target species and B) spatio- temporal control of antibody production. IgG-like bsAbs, featuring two antigen-binding Fab domains, each harboring a unique light chain (LC), and heterodimeric heavy chain (HC) subunits, have advantages over fragment-based bsAbs such as single chain fragment variable (scFv) based bsAbs. However, a hurdle to nucleic acid delivery of bsAbs is ensuring correct pairing of heterodimeric CH3 regions and pairing of heavy chains with their cognate light chains, which is addressed herein.

[0188] The method provided herein demonstrates that, inter alia, mutation of CH1P151 (referred to as P151 throughout, according to Eu numbering) in combination with specific mutations to enhance HC-LC pairing overcomes the inability of the antibody to form its intermediate that could induce full folding of the CH1 domain and allow antibody secretion. Additionally, mutation of the CH3P374 (referred to as P374 throughout, according to Eu numbering), paired with charge steering mutations, or equivalents thereof, to induce formation of the folding intermediate, allow production of highly pure multispecific antibodies, such as for example and not limitation, bispecific antibodies (bsAbs). Cells that expressed these four- polypeptide bsAbs failed to secrete off-target antibody homodimers or antibodies with mis- paired LCs.

[0189] This invention provides, inter alia, production of therapeutics from a single-step purification and provides nucleic-acid based in vivo delivery of highly pure, IgG-like bsAbs to treat previously undruggable diseases. Also provided herein are IgG-like bsAbs for use in therapy. Specifically provided herein are IgG-like bsAbs for use in the treatment of previously undruggable diseases.

[0190] Described herein is an exemplary platform biotechnology in which the CH3 domain of a Fc region were substituted with variant CH3 mutations having proline mutations and one or more heterodimerization mutations in the CH3-CH3 interface which direct pairing between CH3 domains and the CH1 and CL domains of a Fab region were substituted withPage 70 of 370 307304067v1variant CH1 and CL domains having proline mutations in the CH1 domain or CL domain and one or more heterodimerization mutations in the CH1-CL interface which direct pairing between HCs and cognate LCs. This platform is useful for directing appropriate heavy chain-light chain pairing in multiparatopic or multispecific (e.g., bispecific (bsAb)) antibodies, since the variant CH3 domains preferentially dimerize with each other and the variant CH1 and CL domains preferentially dimerize with each other, but not with the CH1 and CL domains of the native Fab. See FIG.1A-1B. Proline Mutations

[0191] The multispecific molecules disclosed herein can include one or more proline mutations in certain regions of the polypeptides to mediate folding of the polypeptides. The proline mutations work in tandem with the one or more heterodimerization mutations disclosed herein to enhance heterodimerization of the polypeptides to produce multispecific molecules.

[0192] In some embodiments, the multispecific molecules comprise a first polypeptide and a second polypeptide, each comprising an immunoglobulin heavy chain constant domain 3 (CH3 domain), wherein each of the first polypeptide and the second polypeptide comprises a P374 mutation (Eu numbering) in its respective CH3 domain. In some embodiments, the mutation is selected from any amino acid. In some embodiments, the mutation is selected from A, G, and Q. In some embodiments, the first and / or second polypeptide further comprise one or more heterodimerization mutations.

[0193] In some embodiments, the multispecific molecules comprise a first polypeptide and a second polypeptide, each comprising an immunoglobulin heavy chain constant domain 1 (CH1 domain), wherein at least one of the first polypeptide and the second polypeptide comprises a P151 mutation (Eu numbering) in its respective CH1 domain. In some embodiments, the mutation is selected from any amino acid. In some embodiments, the mutation is selected from A and G. In some embodiments, the first and / or second polypeptide further comprise one or more heterodimerization mutations.

[0194] In some embodiments, the multispecific molecules comprise a third polypeptide and a fourth polypeptide, each comprising an immunoglobulin light chain constant domain 1 (CLPage 71 of 370 307304067v1domain), wherein at least one of the third polypeptide and the fourth polypeptide comprises a P141 mutation (Eu numbering) in its respective CL domain. In some embodiments, the mutation is selected from any amino acid. In some embodiments, the mutation is selected from A and G. In some embodiments, the third and / or fourth polypeptide further comprise one or more heterodimerization mutations.

[0195] In some embodiments, the multispecific molecules comprise a P374 mutation (Eu numbering) in each of the first polypeptide and the second polypeptide, and a P151 mutation in at least one of the first polypeptide and the second polypeptide. In some embodiments, the P374 mutation is selected from A, G, and Q. In some embodiments, the P151 mutation is selected from A and G. In some embodiments, the first and / or second polypeptide further comprise one or more heterodimerization mutations. Heterodimerization Mutations

[0196] The multispecific molecules disclosed herein can include one or more heterodimerization mutations in either the first polypeptide or the second polypeptide that favor heterodimerization of the first polypeptide and the second polypeptide over homodimerization of two first polypeptides and two second polypeptides.

[0197] In some embodiments, the one or more heterodimerization mutations are located in the CH3 domains of the first polypeptide and / or the second polypeptide and enhance heterodimerization of the CH3 domains of the first polypeptide and the second polypeptide. In some embodiments, the one or more heterodimerization mutations are located in the CH3 domains of each of the first polypeptide and the second polypeptide and enhance heterodimerization of the CH3 domains of the first polypeptide and the second polypeptide.

[0198] In some embodiments, the one or more heterodimerization mutations are located in the CH1 domains of the first polypeptide and / or the second polypeptide and / or the paired CL domains of the first polypeptide and / or the second polypeptide and enhance heterodimerization of the CH1 domain and its paired CL domain of the first polypeptide and the second polypeptide. In some embodiments, the one or more heterodimerization mutations are located in the CH1 domains and the paired CL domains of each of the first polypeptide and the second polypeptidePage 72 of 370 307304067v1and enhance heterodimerization of the CH1 domain and its paired CL domain of the first polypeptide and the second polypeptide.

[0199] In some embodiments, the one or more heterodimerization mutations are located (i) in the CH3 domains of the first polypeptide and / or the second polypeptide and (ii) in the CH1 domains of the first polypeptide and / or the second polypeptide and / or the paired CL domains of the first polypeptide and / or the second polypeptide. In some embodiments, the one or more heterodimerization mutations can be (i) in a first CH3 domain and in a second CH3 domain, and / or (ii) in a first CH1 domain and / or its paired CL domain and / or (iii) in a second CH1 domain and / or its paired CL domain. In some embodiments, the one or more heterodimerization mutations can be in (i) in a first CH3 domain and in a second CH3 domain, and (ii) in a first CH1 domain and its paired CL domain, and (iii) in a second CH1 domain and its paired CL domain.

[0200] Multispecific molecules according to the disclosure may also be generated using designs such as the knob-in-hole or knobs-into-holes (Genentech), CrossMAbs (Roche), electrostatically-matched (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), and Biclonic (Merus) strategies.

[0201] In the “knob-in-hole” strategy (see, e.g., Intl. Publ. No. WO 2006 / 028936), select amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob”. Nonlimiting exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366W / T366S, L368A, and Y407V; T366S, L368A, and Y407V / T366W (Eu numbering).Page 73 of 370 307304067v1

[0202] CrossMAb technology, in addition to utilizing the “knob-in-hole” strategy to promoter Fab arm exchange utilizes CH1 / CL domain swaps in one half arm to ensure correct light chain pairing of the resulting bispecific antibody (see, e.g., U.S. Patent No.8,242,247).

[0203] Other strategies such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface may be used, as described in US Patent Publ. No. US2010 / 0015133; US Patent Publ. No. US2009 / 0182127; US Patent Publ. No. US2010 / 028637 or US Patent Publ. No. US2011 / 0123532. In other strategies, steric hindrance and / or hydrophobic regions may be used to promote heterodimerization. In other strategies, heterodimerization may be promoted by the following exemplary nonlimiting substitutions (expressed as modified positions in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W as described in U.S. Patent Publ. No. US2012 / 0149876 or U.S. Patent Publ. No. US2013 / 0195849.

[0204] Multispecific molecules according to the disclosure may also comprise heterodimerization mutations at F170, S183 and / or V185 (Eu numbering) in the CH1 domain of the first and / or second polypeptide and at L135 (Eu numbering) in the paired CL domain. In some embodiments, the CH1 domain of the first and / or second polypeptide may comprise F126C, F170V, S183I, V185L, and C220S (Eu numbering) heterodimerization mutations, and the paired CL domain may comprise E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (Eu numbering) heterodimerization mutations.

[0205] In specific embodiments, a method of producing a multispecific molecule, the method comprising: providing a first polypeptides and / or second polypeptide and its respective light chain (LC), wherein one or both of the first polypeptides and second polypeptide comprises a constant heavy chain domain 1 (CH1), and its respective LC comprises a constant light chain (CL) domain, wherein the CH1 domain of the one or both of the first polypeptides and second polypeptide comprises a heterodimerization mutation at F170, S183 and / or V185, and the CLPage 74 of 370 307304067v1domain of the LC chain comprises a heterodimerization mutation at L135 (Eu numbering). In some embodiments, the fragment comprises an antigen binding fragment.

[0206] In some embodiments, a method of producing a multispecific molecule, the method comprising: providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and second (LC2) light chain, wherein each HC1 and HC2 comprises a heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), wherein the HC1 CH1 comprises a heterodimerization mutation at F170, S183 and / or V185, and the LC1 CL comprises a heterodimerization mutation at L135 (Eu numbering). In some embodiments, the fragment comprises an antigen binding fragment.

[0207] In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a Q39K substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a Q38D substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a Q39K substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a Q38D substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering). In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a Q39D substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a Q38K substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a Q39D substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a Q38K substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering).Page 75 of 370 307304067v1

[0208] In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44K substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a G100D substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a G44K substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G100D substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering). In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44D substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a G100K substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a G44D substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G100K substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering).

[0209] In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44C substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a G100C substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a G44C substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G100C substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering). In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44C substitution (Chothia numbering) and a third and / or fourth polypeptidePage 76 of 370 307304067v1comprising a VL comprising a G100C substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a G44C substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G100C substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering).

[0210] In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44K substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a G101D substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a G44K substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G101D substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering). In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44D substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a G101K substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a G44D substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G101K substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering).

[0211] In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44K substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a G101D substitution (Chothia numbering). In some embodiments, the multispecificPage 77 of 370 307304067v1molecule comprises a first and / or second polypeptide comprising a VH comprising a G44K substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G101D substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering). In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a G44D substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a G101K substitution (Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a G44D substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a G101K substitution (Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L heterodimerization mutation (Eu numbering) and a CL domain of the third and / or fourth polypeptide comprises a L135F heterodimerization mutation (Eu numbering). In some embodiments, the heterodimerization mutations may be used as described in PCT Pat. Appl. No. PCT / US2024 / 032454 which is incorporated herein by reference in its entirety.

[0212] In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a Q39K substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a Q38E substitution (according to Chothia numbering). In some embodiments, the multispecific molecule comprises a first and / or second polypeptide comprising a VH comprising a Q39K substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a Q39E substitution (according to Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L (according to Eu numbering) heterodimerization mutation and a CL domain of the third and / or fourth polypeptide comprises a L135F (according to Eu numbering) heterodimerization mutation. In some embodiments, the multispecific molecule according to the present disclosure comprises a first and / or second polypeptide comprising a VH comprising a Q38E substitution (Chothia numbering) and a third and / or fourth polypeptide comprising a VL comprising a Q39K substitution (according to Chothia numbering). In some embodiments, the multispecificPage 78 of 370 307304067v1molecule comprises a first and / or second polypeptide comprising a VH comprising a Q38E substitution (Chothia numbering), a third and / or fourth polypeptide comprising a VL comprising a Q39K substitution (according to Chothia numbering), wherein a CH1 domain of the first and / or second polypeptide comprises a F170V, S183I, and / or V185L (according to Eu numbering) heterodimerization mutation and a CL domain of the third and / or fourth polypeptide comprises a L135F (according to Eu numbering) heterodimerization mutation.

[0213] Additional nonlimiting heterodimerization mutations in the CH3 domains include the following exemplary nonlimiting substitutions (expressed as modified position(s) in the first CH3 domain of the first heavy chain and modified position(s) in the second CH3 domain of the second heavy chain according to Eu numbering): 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain; 2) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain; 10) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain;Page 79 of 370 307304067v111) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain; 13) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain; 17) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 25) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain;Page 80 of 370 307304067v126) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 28) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 29) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 30) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain; 32) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain; 40) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain;Page 81 of 370 307304067v141) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 43) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 44) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 45) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 47) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or 48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain.

[0214] Additional nonlimiting heterodimerization mutations in the CH1 domains and paired CL domains include the following exemplary nonlimiting substitutions (expressed as modified position(s) in the CH1 domain of the first polypeptide or the second polypeptide and modified position(s) in the paired CL domain according to Eu numbering): 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain;Page 82 of 370 307304067v15) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 9) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 17) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain;Page 83 of 370 307304067v120) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain; 24) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain;Page 84 of 370 307304067v135) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain; 39) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain;Page 85 of 370 307304067v150) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; 54) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain; 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

[0215] SEEDbody technology may be utilized to generate bispecific antibodies of the invention. SEEDbodies have, in their constant domains, select IgG residues substituted with IgA residues to promote heterodimerization as described in U.S. Patent No. US20070287170.

[0216] Mutations are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.Page 86 of 370 307304067v1Domain Swap or Domain Exchange

[0217] In certain aspects, a multispecific molecule has one or more heavy chain domains exchanged for domains on the light chain and / or one or more domains of the light chain exchanged for domains of the heavy chain. In some embodiments, a multispecific molecule comprises at least two polypeptides comprising an immunoglobulin variable region, wherein the variable region of the two or more polypeptides are exchanged. In some embodiments, the bispecific molecule comprising first, second, third and fourth polypeptides comprises one or more heavy chain domains exchanged for domains on the light chain and / or one or more domains of the light chain exchanged for domains of the heavy chain on each polypeptide.

[0218] In some embodiments, the multispecific molecule comprising polypeptides with exchanged domains has a kappa light chain. In some embodiments, the multispecific molecule comprising polypeptides with exchanged domains has a lambda light chain. In some embodiments, the multispecific molecule comprises polypeptides with exchanged domain wherein the exchanged variable region of the light chain domain on the heavy chain is of a lambda chain and the constant domain present in the light chain is of a kappa chain. In some embodiments, the multispecific molecule comprises polypeptides with exchanged domains wherein the exchanged variable region of the light chain domain on the heavy chain is of a kappa chain and constant domain present in the light chain is of a lambda chain.

[0219] In some embodiments, a multispecific molecule has one or more heavy chain domains exchanged for domains on the light chain and / or one or more domains of the light chain exchanged for domains of the heavy chain in addition to heterodimerization mutations as described herein. In some embodiments, a multispecific molecule has one or more heavy chain domains exchanged for domains on the light chain and / or one or more domains of the light chain exchanged for domains of the heavy chain in addition to a proline mutation(s). In some embodiments, a multispecific molecule has one or more heavy chain domains exchanged for domains on the light chain and / or one or more domains of the light chain exchanged for domains of the heavy chain in addition to one or more heterodimerization mutations and a proline mutation(s) as described herein. In some embodiments, a multispecific molecule has one or more heavy chain domains exchanged for domains on the light chain and / or one or morePage 87 of 370 307304067v1domains of the light chain exchanged for domains of the heavy chain in addition to mutations in variable regions as described herein. The multispecific molecule comprising domain exchange(s) can be developed as described in Int. Pat. Pub. No. WO 2006 / 093794 which is incorporated herein by reference in its entirety. Multispecific molecules

[0220] In certain aspects, a multispecific molecule comprises polypeptides that bind to one or more target antigens, or “paratopes” that bind to one or more target antigens. In specific embodiments, the polypeptide of a multispecific molecule is an antigen-binding polypeptide. In specific embodiments, a polypeptide of a multispecific molecule comprises a variable domain that binds to a target antigen and a constant domain. In specific embodiments, a polypeptide of a multispecific molecule comprises at least one variant CH3 and / or CL and / or CH1 domains as described herein. In specific embodiments, the at least one variant CH3 domain binds to another CH3 domain, optionally a variant CH3 domain, of a multispecific molecule as described herein to form a dimer. In specific embodiments, the at least one variant CL and / or CH1 domains binds to another CL and / or CH1 domain, optionally a variant CL and / or CH1 domain, of a multispecific molecule as described herein to form a dimer. In a preferred embodiment, a polypeptide of a multispecific molecule comprises (i) a constant domain comprising a CH2 domain and a CH3 domain, optionally at least one variant CH3 domain; and (ii) a variable domain and at least one variant CL and / or CH1 domain.

[0221] In specific embodiments, a multispecific molecule comprises an antigen-binding polypeptide as described herein. In specific embodiments, the multispecific molecule comprises two, three, four, five, six, seven, or eight antigen-binding polypeptides. In specific embodiments, each of the two, three, four, five, six, seven, or eight antigen-binding polypeptides comprises a constant domain and a variable domain that binds to a target antigen. In specific embodiments, each of the two, three, four, five, six, seven, or eight antigen-binding polypeptides comprises at least one variant CH3 domain. In specific embodiments, each of the two, three, four, five, six, seven, or eight antigen-binding polypeptides comprises at least one variant CL and / or CH1 domain. In specific embodiments, each of the two, three, four, five, six, seven, or eight antigen-binding polypeptides comprises a constant domain with at least one variant CH3Page 88 of 370 307304067v1domain and a variable domain that binds to a target antigen and at least one variant CL and / or CH1 domains. In specific embodiments, a variant CH3 domain of an antigen-binding polypeptide binds to another CH3 domain, optionally a variant CH3 domain, of another antigen- binding polypeptide to form a dimer (in particular, heterodimers, e.g., six polypeptides can form up to three dimers with each other). In specific embodiments, a variant CL and / or CH1 domain of an antigen-binding polypeptide binds to another CL and / or CH1 domain, optionally a variant CL and / or CH1 domain, of another antigen-binding polypeptide to form a dimer (in particular, heterodimers, e.g., six polypeptides can form up to three dimers with each other). In a preferred embodiment, a multispecific molecule comprises two polypeptides, wherein each polypeptide comprises a constant domain, a variable domain and at least one variant CH3 and / or CL and / or CH1 domain, wherein the variable domains form a variable region that binds to a target antigen and the CL and / or CH1 domains, optionally at least one variant CL and / or CH1 domain, bind to each other to form dimers, preferably heterodimers, and the CH3 regions bind to each other to form dimers, preferably heterodimers. In a preferred embodiment, a multispecific molecule comprises four polypeptides, wherein each polypeptide comprises a constant domain, a variable domain and at least one variant CL and / or CH1 and / or CH3 domain, wherein the variable domains form two variable regions that each bind to a target antigen, the CL and / or CH1 domains, optionally at least one variant CL and / or CH1 domain, bind to each other to form two dimers, and the CH3 regions bind to each other to form dimers, preferably heterodimers. In a preferred embodiment, a multispecific molecule comprises six polypeptides, wherein each polypeptide comprises a constant domain, a variable domain, and at least one variant CL and / or CH1 and / or CH3 domains, wherein the variable domains form three variable regions that each bind to a target antigen, the CL and / or CH1 domains, optionally at least one variant CL and / or CH1 domain, bind to each other to form three dimers, and the CH3 regions bind to each other to form dimers, preferably heterodimers.

[0222] In specific embodiments, a polypeptide of a multispecific molecule comprises a light chain polypeptide comprising a variable domain, and a heavy chain polypeptide comprising a variable domain. In specific embodiments, the variable domains of the light and heavy chains form a variable region comprising a paratope that binds to a target antigen. In specific embodiments, a light chain polypeptide is a first light chain polypeptide (CL1), second light chain polypeptide (LC2), third light chain polypeptide (LC3), fourth light chain polypeptidePage 89 of 370 307304067v1(LC4), or further light chain polypeptide of an multispecific molecule (e.g., LC5+). In specific embodiments, a heavy chain polypeptide is the first heavy chain polypeptide (HC1), second heavy chain polypeptide (HC2), third heavy chain polypeptide (HC3), fourth heavy chain polypeptide (HC4), or further heavy chain polypeptide of an multispecific molecule (e.g., LC5+).

[0223] In specific embodiments, a light chain polypeptide comprises an immunoglobulin or antibody light chain or one or more fragments thereof. In specific embodiments, a light chain polypeptide comprises an immunoglobulin or antibody kappa (κ) chain, lambda (λ) chain, sigma (σ) chain, iota (ι) chain, or one or more fragments thereof. In specific embodiments, a light chain polypeptide is an intact immunoglobulin light chain comprising a light chain variable domain (VL) and light chain constant domain (CL). In a preferred embodiment, the light chain constant domain is replaced fully or partially with a variant CL domain as described herein. In specific embodiments, a light chain polypeptide may contain, but is not required to contain, an immunoglobulin light chain constant region or a fragment thereof. In specific embodiments, a light chain polypeptide is not an intact immunoglobulin light chain. In specific embodiments, a light chain polypeptide does not comprise a wild-type constant domain (CL), or a fragment thereof, of an immunoglobulin light chain. In specific embodiments, a light chain polypeptide comprises a heterodimerization mutation of a light chain constant domain. In specific embodiments, the heterodimerization mutation of a light chain constant domain binds to a CH1 domain to form a dimer. In specific embodiments, the heterodimerization mutation of a light chain constant domain mediates dimerization between the light chain and the heavy chain. In specific embodiments, a light chain polypeptide comprises a fusion of kappa and lambda light chains. In specific embodiments, the fusion of kappa and lambda light chains comprises a lambda variable region fused onto a kappa constant domain or a kappa variable region fused onto a lambda constant domain. In specific embodiments, the fusion of kappa and lambda light chains comprises a lambda variable region fused onto a kappa constant domain. In specific embodiments, the fusion of kappa and lambda light chains comprises a kappa variable region fused onto a lambda constant domain.

[0224] In specific embodiments, a heavy chain polypeptide comprises an immunoglobulin or antibody heavy chain, or one or more fragments thereof. In specific embodiments, a heavy chain polypeptide comprises an immunoglobulin or antibody gamma (γ)Page 90 of 370 307304067v1chain, delta (δ) chain, alpha (α) chain, mu (μ) chain, epsilon (ε) chain, or one or more fragments thereof. In specific embodiments, a heavy chain polypeptide is an intact immunoglobulin heavy chain comprising a heavy chain variable domain (VH), a heavy chain constant domain 1 (CH1), a hinge region, a heavy chain constant domain 2 (CH2), and a heavy chain constant domain 3 (CH3). In a preferred embodiment, the heavy chain constant domain 3 (CH3) is replaced fully or partially with a variant CH3 domain as described herein. In a preferred embodiment, the heavy chain constant domain 1 (CH1) is replaced fully or partially with a variant CH1 domain as described herein. In a preferred embodiment, the light chain constant domain 1 (CL) is replaced fully or partially with a variant CL domain as described herein. In a preferred embodiment, the CH3 and / or CH1 and / or CL domains are replaced fully or partially with variant CH3 and / or CH1 and / or CL domains as described herein. In specific embodiments, a heavy chain polypeptide can but not need contain an immunoglobulin heavy chain constant domain 1 (CH1) or a fragment thereof. In specific embodiments, a heavy chain polypeptide can but not need contain an immunoglobulin heavy chain constant domain 2 (CH2) or a fragment thereof. In specific embodiments, a heavy chain polypeptide can but not need contain an immunoglobulin heavy chain constant domain 3 (CH3) or a fragment thereof. In specific embodiments, a heavy chain polypeptide can but not need contain an immunoglobulin heavy chain constant domain 2 (CH2) or a fragment thereof and an immunoglobulin heavy chain constant domain 3 (CH3). In specific embodiments, a heavy chain polypeptide is not an intact immunoglobulin heavy chain. In specific embodiments, a heavy chain polypeptide does not comprise a wild-type immunoglobulin heavy chain constant domain 3 (CH3) or a fragment thereof. In specific embodiments, a heavy chain polypeptide does not comprise a wild-type immunoglobulin heavy chain constant domain 1 (CH1) or a fragment thereof. In specific embodiments, a heavy chain polypeptide comprises a proline mutation and / or one or more heterodimerization mutations of a heavy chain constant domain 3 (CH3). In specific embodiments, the heterodimerization mutation of CH3 binds to a second CH3 domain, optionally a variant CH3 domain comprising a proline mutation and / or one or more heterodimerization mutations, to form a dimer. In specific embodiments, the one or more heterodimerization mutations of a heavy chain constant domain mediates heterodimerization between the two heavy chains. In specific embodiments, a heavy chain polypeptide comprises a fragment of a heavy chain constant domain (CH3). In specific embodiments, a heavy chain polypeptide comprises a proline mutation and / or one or morePage 91 of 370 307304067v1heterodimerization mutations of a heavy chain constant domain 1 (CH1). In specific embodiments, the one or more heterodimerization mutations of a heavy chain constant domain 1 (CH1) binds to a light chain constant domain (CL), optionally a variant CL domain comprising a proline mutation and / or one or more heterodimerization mutations, to form a heterodimer. In specific embodiments, the one or more heterodimerization mutations of a CH1 domain mediates heterodimerization between the heavy chain and the light chain. In specific embodiments, a heavy chain polypeptide comprises a fragment of a CH1 domain.

[0225] In specific embodiments, a light chain polypeptide as described herein is a first light chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a first VL and a first CL, wherein the first VL is a first immunoglobulin light chain variable domain, and the first CL is a first immunoglobulin light chain constant domain or variant thereof as described herein. In specific embodiments, a heavy chain polypeptide is a first heavy chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a first VH and a first CH1, wherein the first VH is a first immunoglobulin heavy chain variable domain, and the first CH1 is a first human immunoglobulin gamma isotype 1 (IgG1) heavy chain constant domain 1 or variant thereof as described herein. In specific embodiments, the first VL and first VH form a first paratope that binds to a first target antigen. In specific embodiments, the first VL and first VH form a first variable region that comprises a first paratope that binds to a first target antigen.

[0226] In specific embodiments, a light chain polypeptide as described herein is a second light chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a second VL and a second CL, wherein the second VL is a second immunoglobulin light chain variable domain, and the second CL is a second immunoglobulin light chain constant domain or variant thereof as described herein. In specific embodiments, a heavy chain polypeptide is a second heavy chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a second VH and a second CH1, wherein the second VH is a second immunoglobulin heavy chain variable domain, and the second CH1 is a second human immunoglobulin gamma isotype 1 (IgG1) heavy chain constant domain 1 or variant thereof as described herein. In specific embodiments, the second VL and second VH form a second paratope that binds to a second target antigen. In specific embodiments, the second VL and second VH form a second variable region that comprises a second paratope that binds to a second target antigen.Page 92 of 370 307304067v1

[0227] In specific embodiments, a light chain polypeptide as described herein is a third light chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a third VL and a third CL, wherein the third VL is a third immunoglobulin light chain variable domain, and the third CL is a third immunoglobulin light chain constant domain or variant thereof as described herein. In specific embodiments, a heavy chain polypeptide is a third heavy chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a third VH and a third CH1, wherein the third VH is a third immunoglobulin heavy chain variable domain, and the third CH1 is a third human immunoglobulin gamma isotype 1 (IgG1) heavy chain constant domain 1 or variant thereof as described herein. In specific embodiments, the third VL and third VH form a third paratope that binds to a third target antigen. In specific embodiments, the third VL and third VH form a third variable region that comprises a third paratope that binds to a third target antigen.

[0228] In specific embodiments, a light chain polypeptide as described herein is a fourth light chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a fourth VL and a fourth CL, wherein the fourth VL is a fourth immunoglobulin light chain variable domain, and the fourth CL is a fourth immunoglobulin light chain constant domain or variant thereof as described herein. In specific embodiments, a heavy chain polypeptide is a fourth heavy chain polypeptide comprising, in amino-terminus to carboxy-terminus order: a fourth VH and a fourth CH1, wherein the fourth VH is a fourth immunoglobulin heavy chain variable domain, and the fourth CH1 is a fourth human immunoglobulin gamma isotype 1 (IgG1) heavy chain constant domain 1 or variant thereof as described herein. In specific embodiments, the fourth VL and fourth VH form a fourth paratope that binds to a fourth target antigen. In specific embodiments, the fourth VL and fourth VH form a fourth variable region that comprises a fourth paratope that binds to a fourth target antigen.

[0229] In specific embodiments, the first heavy chain polypeptide further comprises an immunoglobulin constant domain. In specific embodiments, the first heavy chain polypeptide further comprises a heavy chain constant domain 2 (CH2) of an antibody or a fragment thereof, optionally wherein the CH2 domain is fused to the C-terminus of the first heavy chain constant domain 1 (CH1) or first heavy chain spacer region. In specific embodiments, the first heavy chain polypeptide further comprises a heavy chain constant domain 3 (CH3) of an antibody or aPage 93 of 370 307304067v1fragment thereof, optionally wherein the CH3 domain is fused to the C-terminus of the first heavy chain CH1 domain, C-terminus of the first heavy chain spacer region, or C-terminus of the CH2 domain when present. In specific embodiments, the first heavy chain polypeptide further comprises a heavy chain fourth constant (CH4) domain of an antibody or a fragment thereof, optionally wherein the CH4 domain is fused to the C-terminus of the first heavy chain CH1 domain, C-terminus of the first heavy chain spacer region, C-terminus of the CH2 domain when present, or C-terminus of the CH3 domain when present. CH3 Domains and Variants

[0230] In certain aspects, a polypeptide of a multispecific molecule comprises a immunoglobulin constant heavy chain region comprising a heavy chain constant domain 2 (CH2) and a heavy chain constant domain 3 (CH3) or variant CH3 domain that forms a dimer with a CH3 domain or variant CH3 domain of another polypeptide of the multispecific molecule. In specific embodiments, a polypeptide of a multispecific molecule as described herein comprises a means for dimerization with another polypeptide of the multispecific molecule. In specific embodiments, the means for dimerization is a CH3 domain or variant CH3 domain, as described herein. In specific embodiments, the variant CH3 domain comprises a proline mutation (e.g., P374A, P374G, or P374Q) (Eu numbering) and / or one or more heterodimerization mutations.

[0231] In specific embodiments, a variant CH3 domain replaces a CH3 domain. In specific embodiments, a variant CH3 replaces fully or partially the dimerization sequence of a CH3 domain.

[0232] In specific embodiments, a CH3 domain of a multispecific molecule is modified as described herein. CH3 Domains

[0233] In specific embodiments, a CH3 domain as described herein comprises an IgG1 heavy chain constant domain 3 (CH3). In specific embodiments, a CH3 domain is a human IgG1 heavy chain constant domain 3 (CH3). In specific embodiments, a CH3 domain is a wild-type human IgG1 heavy chain constant domain 1 (CH3), or a fragment thereof. In specific embodiments, a CH3 domain comprises or consists of the amino acid sequence of the IgG1Page 94 of 370 307304067v1heavy chain constant domain 1 (CH3) of immunoglobulin heavy constant gamma 1 as set forth in UniProt Accession Number P01857-1, or a fragment thereof (e.g., the CH3 domain sequence without CH1 and CH2 domain sequences). The amino acid sequence of the immunoglobulin heavy constant gamma 1 (CH1) as set forth in UniProt Accession Number P01857-1 is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1). The amino acid sequence of the CH3 domain is GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2).

[0234] In specific embodiments, the CH3 domain is a variant CH3 domain comprising an amino acid sequence that differs from the wild-type CH3 domain of SEQ ID NO:2. In specific embodiments, a variant CH3 domain comprises at least one amino acid substitution, deletion, or insertion relative to the wild-type CH3 domain in at least one position. As described herein, the at least one position of a CH3 domain can be identified according to Eu numbering, which is the same as the CH3 amino acid numbering in TABLES 1A-1C, below. In specific embodiments, a variant CH3 domain differs from the wild-type CH3 domain of SEQ ID NO:2 in substitutions, deletions, or insertions of one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids. In specific embodiments, a variant CH3 domain differs from the wild-type CH3 domain of SEQ ID NO:2 only by having no more than ten amino acid substitutions. In specific embodiments, a variant CH3 domain differs from the wild-type CH3 domain of SEQ ID NO:2 by having an amino acid substitution at P374, according to Eu numbering, alone or in combination with one or more other mutations as specified herein. In specific embodiments, a variant CH3 domain differs from the wild-type CH3 domain of SEQ ID NO:2 by having one or more amino acid substitutions at positions S364, E357, T366, L368, K370, F405, Y407, and / or K409, according to Eu numbering, and any combinations thereof.Page 95 of 370 307304067v1

[0235] In specific embodiments, the CH3 domain comprises or consists of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2, optionally further comprising one, two, three, four, five, six, seven, eight, nine, or ten or more single amino acid substitutions at the following positions, with positions numbered according to Eu numbering: 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain; 2) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain; 10) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain; 11) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain;Page 96 of 370 307304067v113) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain; 17) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 25) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 26) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain;Page 97 of 370 307304067v128) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 29) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 30) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain; 32) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain; 40) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; 41) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain;Page 98 of 370 307304067v143) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 44) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 45) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 47) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or 48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain. CL and CH1 Domains and Variants

[0236] In certain aspects, a polypeptide of a multispecific molecule comprises an immunoglobulin constant light chain (CL) domain or variant CL domain that forms a dimer with an immunoglobulin heavy chain constant domain 1 (CH1) domain or variant CH1 domain of another polypeptide of the multispecific molecule. In specific embodiments, a CL domain is a CL domain or variant CL domain (e.g., kappa or lambda) as described herein. In specific embodiments, a CH1 domain is a CH1 domain or variant CH domain (e.g., IgG1 heavy chain constant domain 1 (CH1) as described herein. In specific embodiments, a polypeptide of a multispecific molecule as described herein comprises a means for dimerization with another polypeptide of the multispecific molecule. In specific embodiments, the means for dimerization is a CL domain or variant CL domain (e.g., kappa or lambda), or a CH1 domain or a variant CH1 domain (e.g., IgG1 heavy chain constant domain 1), as described herein. In specific embodiments, the variant CH1 domain comprises a proline mutation (e.g., P151A or P151G) (Eu numbering) and / or one or more heterodimerization mutations. In specific embodiments, the CL domain comprises a proline mutation (e.g., P141A) (Eu numbering) and / or one or more heterodimerization mutations.Page 99 of 370 307304067v1

[0237] In specific embodiments, a variant CL domain replaces a CL domain. In specific embodiments, a variant CL replaces fully or partially the dimerization sequence of a light chain constant (CL) domain. In specific embodiments, a variant CH1 domain replaces fully or partially a heavy chain constant domain 1 (CH1). In specific embodiments, a variant CH1 domain replaces a CH1 domain. In specific embodiments, a heavy chain variant CH1 domain replaces fully or partially the dimerization sequence of a heavy chain constant domain 1 (CH1).

[0238] In specific embodiments, a CH1 or CL domain of a multispecific molecule, and preferably both, are modified as described herein. In specific embodiments, a first CH1 domain or first CL domain, or a second CH1 domain or second CL domain of a multispecific molecule, and preferably all, are modified as described herein. CH1 Domains

[0239] In specific embodiments, a CH1 domain as described herein comprises an IgG1 heavy chain constant domain 1 (CH1). In specific embodiments, a CH1 domain is a human IgG1 heavy chain constant domain 1 (CH1). In specific embodiments, a CH1 domain is a wild-type human IgG1 heavy chain constant domain 1 (CH1), or a fragment thereof. In specific embodiments, a CH1 domain comprises or consists of the amino acid sequence of the IgG1 heavy chain constant domain 1 (CH1) of immunoglobulin heavy constant gamma 1 as set forth in UniProt Accession Number P01857-1, or a fragment thereof (e.g., the CH1 domain sequence without CH2 and CH3 domain sequences). The amino acid sequence of the immunoglobulin heavy constant gamma 1 (CH) as set forth in UniProt Accession Number P01857-1 is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1). In specific embodiments, the CH1 domain comprises or consists of the amino acid sequence of: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:3).Page 100 of 370 307304067v1

[0240] In specific embodiments, the CH1 domain is a variant CH1 domain comprising an amino acid sequence that differs from the wild-type CH1 domain of SEQ ID NO:3. In specific embodiments, a variant CH1 domain comprises at least one amino acid substitution, deletion, or insertion relative to the wild-type CH1 domain in at least one position. As described herein, the at least one position of a CH1 domain can be identified according to Eu numbering, which is the same as the CH1 amino acid numbering in TABLES 2A-2D, below. In specific embodiments, a variant CH1 domain differs from the wild-type CH1 domain of SEQ ID NO:1 or SEQ ID NO:3 in substitutions, deletions, or insertions of one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids. In specific embodiments, a variant CH1 domain differs from the wild- type CH1 domain of SEQ ID NO:1 or SEQ ID NO:3 only by having no more than ten amino acid substitutions. In specific embodiments, a variant CH1 domain differs from the wild-type CH1 domain of SEQ ID NO:1 or SEQ ID NO:3 by having a substitution at P151. In specific embodiments, a variant CH1 domain differs from the wild-type CH1 domain of SEQ ID NO:1 or SEQ ID NO: 3 by having a substitution at P151 and one or more other mutations as described herein.

[0241] In specific embodiments, the CH1 domain comprises or consists of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3, optionally further comprising one, two, three, four, five, six, seven, eight, nine or ten or more single amino acid substitutions at the following positions, with positions numbered according to Eu numbering: 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain;Page 101 of 370 307304067v15) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 9) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 17) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain;Page 102 of 370 307304067v120) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain; 24) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain;Page 103 of 370 307304067v135) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain; 39) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain;Page 104 of 370 307304067v150) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; 54) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain (Eu numbering); 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

[0242] In specific embodiments, the multispecific molecule comprises A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain, and / or L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain, in combination with a substitution at P151, and wherein one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide. In specific embodiments, thePage 105 of 370 307304067v1multispecific molecule comprises (i) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide, or (ii) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide. CL(kappa) Domains

[0243] In specific embodiments, a CL domain as described herein comprises an immunoglobulin kappa light chain constant domain (CL(kappa)). In specific embodiments, a CL domain is a human immunoglobulin kappa light chain constant domain (CL(kappa)). In specific embodiments, a CL domain is a wild-type human immunoglobulin kappa light chain constant domain (CL(kappa)), or a fragment thereof. In specific embodiments, a CL domain comprises or consists of the amino acid sequence of the immunoglobulin kappa light chain constant domain (CL(kappa)) as set forth in UniProt Accession Number P01857, or a fragment thereof (e.g., the Ig-like annotated domain). The amino acid sequence of the immunoglobulin kappa light chain constant domain (CL(kappa)) as set forth in UniProt Accession Number P01857 is: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:4).

[0244] In specific embodiments, the CL domain is a variant CL domain comprising an amino acid sequence that differs from the wild-type CL(kappa) domain of SEQ ID NO:4. In specific embodiments, a variant CL domain comprises at least one amino acid substitution, deletion, or insertion relative to the wild-type CL(kappa) domain of SEQ ID NO:4 in at least one position. As described herein, at least one position of a CL domain (i.e., CL(kappa)) can be identified according to Eu numbering, which is the same as the CL(kappa) amino acid numbering in TABLES 3A-3E, below. In specific embodiments, a variant CL domain differs from the wild-type CL(kappa) domain of SEQ ID NO:4 in a substitution, deletion, or insertion of one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids. In a specific embodiments, a variant CL domain differs from the wild-type CL(kappa) domain ofPage 106 of 370 307304067v1SEQ ID NO:4 only by having no more than ten amino acid substitutions. In specific embodiments, a variant CL domain differs from the wild-type CL(kappa) domain of SEQ ID NO:4 by having a substitution at P141. In specific embodiments, a variant CL domain differs from the wild-type CL(kappa) domain of SEQ ID NO:4 by having another mutation as described herein. In specific embodiments, a variant CL domain differs from the wild-type CL(kappa) domain of SEQ ID NO:5 by having a substitution at P141 and one or more other mutations as described herein.

[0245] In specific embodiments, the variant CL domain comprises or consists of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4, optionally further comprising one or two single amino acid substitutions at the following positions, with positions numbered according to Eu numbering: 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain; 5) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain;Page 107 of 370 307304067v19) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 17) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain; 20) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain;Page 108 of 370 307304067v124) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 35) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain;Page 109 of 370 307304067v139) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain; 50) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain;Page 110 of 370 307304067v154) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain; 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

[0246] In specific embodiments, the multispecific molecule comprises A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain, and / or L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain, in combination with a substitution at P151, and wherein one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide. In specific embodiments, the multispecific molecule comprises (i) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide, or (ii) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide.Page 111 of 370 307304067v1CL(lambda) Domains

[0247] In specific embodiments, a CL domain as described herein comprises an immunoglobulin lambda light chain constant domain (CL(lambda)). In specific embodiments, a CL domain is a human immunoglobulin lambda light chain constant domain (CL(lambda)). In specific embodiments, a CL domain is a wild-type human immunoglobulin lambda light chain constant domain (CL(lambda)), or a fragment thereof. In specific embodiments, a CL domain comprises or consists of the amino acid sequence of the immunoglobulin lambda light chain constant domain 1 (CL(lambda)) as set forth in UniProt Accession Number P0CG04, or a fragment thereof (e.g., the Ig-like annotated domain). The amino acid sequence of the immunoglobulin lambda light chain constant domain 1 (CL(lambda)) as set forth in UniProt Accession Number P0CG04 is: GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:5).

[0248] In specific embodiments, the CL domain is a variant CL domain comprising an amino acid sequence that differs from the wild-type CL(lambda) domain of SEQ ID NO:5. In specific embodiments, a variant CL domain comprises at least one amino acid substitution, deletion, or insertion relative to the wild-type CL(lambda) domain of SEQ ID NO:5 in at least one position. As described herein, the at least one position of a CL domain (i.e., CL(lambda)) can be identified according to Eu numbering, which is the same as the CL(lambda) amino acid numbering in TABLE 4, below. In specific embodiments, a variant CL domain differs from the wild-type CL(lambda) domain of SEQ ID NO:5 in a substitution, deletion, or insertion of one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids. In a specific embodiments, a variant CL domain differs from wild-type CL(lambda) domain of SEQ ID NO:5 only by having no more than ten amino acid substitutions. In specific embodiments, a variant CL domain differs from the wild-type CL(lambda) domain of SEQ ID NO:5 by having a substitution at P141. In specific embodiments, a variant CL domain differs from the wild-type CL(lambda) domain of SEQ ID NO:5 by having another mutation as described herein. In specific embodiments, a variant CL domain differs from the wild-type CL(lambda) domain of SEQ ID NO:5 by having a substitution at P141 and one or more other mutations as described herein.Page 112 of 370 307304067v1

[0249] In specific embodiments, the variant CL domain comprises or consists of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5, optionally further comprising one, two, three, four, five, or six or more single amino acid substitutions at positions corresponding to the kappa chain of SEQ ID NO:5, with positions numbered according to Eu numbering. Variant CH1-CL Pairing Sets

[0250] In certain aspects, a multispecific molecule as described herein comprises a light chain polypeptide comprising an immunoglobulin light chain constant (CL) domain (e.g., kappa or lambda) and a heavy chain polypeptide comprising an immunoglobulin heavy chain constant domain 1 (CH1) domain, wherein the CL and CH1 domains bind together to form a dimer and at least one of the CL domain and CH1 domain is a variant CL domain or variant CH1 domain as described herein. Such a pair of CL and CH1 domains is referred to herein as a variant CH1-CL pairing set. In specific embodiments, a multispecific molecule as described herein comprises two or more variant CH1-CL pairing sets. In specific embodiments, a bispecific molecule as described herein comprises two variant CH1-CL pairing sets.

[0251] For clarity, the Eu, KABAT, and IMGT positions of several mutations disclosed in the instant application are presented in TABLE 1 below. A person of ordinary skill in the art would be able to determine the position of the mutations in any numbering scheme based on the information below. TABLE 1: Correspondence between Eu, Kabat, and IMGT numbering schemes. Domain Eu Numbering Kabat Numbering IMGT Numbering A141 139 24 L145 143 28 CH1 V173 177 56 S181 186 64 F116 116 9 CL(kappa) S131 131 24Page 113 of 370 307304067v1Domain Eu Numbering Kabat Numbering IMGT Numbering T178 178 71 T116 116 10 CL(lambda) T131 131 25 Y178 178 71 Spacers and tags

[0252] In certain aspects, a spacer region of a polypeptide of a multispecific molecule is fused to the C-terminus of its CL or CH1 domain. In specific embodiments, a light chain spacer region is fused to the C-terminus of a light chain constant domain (CL) of an multispecific molecule as described herein. In specific embodiments, a heavy chain spacer region is fused to the C-terminus of a heavy chain constant domain 1 (CH1) of a multispecific molecule as described herein.

[0253] In specific embodiments, a light chain spacer region comprises a hinge region. In specific embodiments, a light chain spacer region connects to a hinge region. In specific embodiments, the heavy chain spacer region comprises a hinge region. In specific embodiments, a heavy chain spacer region connects to a hinge region. In specific embodiments, the hinge region is an antibody hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region).

[0254] In specific embodiments, the light chain spacer region or heavy chain spacer region comprises or consists of an amino acid sequence of from 1-5 amino acids in length. In specific embodiments, the light chain spacer region or heavy chain spacer region comprises or consists of an amino acid sequence selected from the group consisting of G, and EPKSC (SEQ ID NO:6).

[0255] In specific embodiments, the spacer region of a polypeptide of a multispecific molecule connects to another moiety (e.g., another protein). In specific embodiments, the spacer region of a polypeptide of a multispecific molecule connects to a C-terminal tag, optionally wherein the C-terminal tag is an affinity tag or purification tag. In specific embodiments, the C- terminal tag is a 6x His tag (SEQ ID NO: 7), a streptavidin tag (e.g., a Strep-tag II tag), a FLAG tag, or a MYC tag. In specific embodiments, a 6x His tag is a 6x poly-histidine (i.e., His-His- His-His-His-His (SEQ ID NO:7). In specific embodiments, the C-terminal tag comprises orPage 114 of 370 307304067v1consists of the amino acid sequence of HHHHHH (SEQ ID NO:7). In specific embodiments, the C-terminal tag comprises or consists of the amino acid sequence of WSHPQFEK (SEQ ID NO:8). In specific embodiments, the C-terminal tag comprises or consists of the amino acid sequence of DYKDDDDK (SEQ ID NO:9). In specific embodiments, the C-terminal tag comprises or consists of the amino acid sequence of EQKLISEEDL (SEQ ID NO:10). Target Antigens

[0256] In specific embodiments, a paratope of a multispecific molecule as described herein binds to an epitope of a target antigen. In specific embodiments, the paratope is formed by the variable region, preferably composed of, for example, a light chain variable domain associated with a heavy chain variable domain, of a multispecific molecule as described herein. In specific embodiments, the paratope is formed by two variable domains (e.g., a light chain variable domain and a heavy chain variable domain) of a multispecific molecule as described herein.

[0257] In specific embodiments, a target antigen is an antigen associated with a disease or disorder in a subject. In specific embodiments, a target antigen is an antigen associated with a pathology or pathogen, such as cancer(s) in a subject. In specific embodiments, a target antigen is an antigen specific for a cancer in a subject. In specific embodiments, a target antigen is an antigen of a pathogen, such as a viral pathogen(s), such as for example respiratory syncytial virus (RSV). Antigens associated with pathology or pathogens are known in the art (see, for example “An expanded universe of cancer targets”, Cell 184, March 4, 2021, Elsevier Inc.); “Target- Pathogen: a structural bioinformatic approach to prioritize drug targets in pathogens”, Nucleic Acids Res.2018 Jan 4; 46: D413–D418; “Identification of Attractive Drug Targets in Neglected- Disease Pathogens Using an In Silico Approach”, PLOS, August 24, 2010.

[0258] In specific embodiments, a target antigen is a tumor associated antigen (TAA), which is an antigen that is overexpressed in tumor cells (preferably of a cancer) relative to non- tumor cells in the subject. In specific embodiments, a first paratope of a multispecific molecule as described herein binds a first tumor associated antigen (TAA1), a second paratope of the multispecific molecule, if present, binds a second tumor associated antigen (TAA2), a third paratope of the multispecific molecule, if present, binds a third tumor associated antigenPage 115 of 370 307304067v1(TAA3), and a fourth paratope of the multispecific molecule, if present, binds a fourth tumor associated antigen (TAA4).

[0259] In specific embodiments, a target antigen is a tumor specific antigen (TSA), which is unique to tumor cells, or expressed only on tumor cells in the subject. In specific embodiments, a first paratope of a multispecific molecule as described herein binds a first tumor specific antigen (TSA1), a second paratope of the multispecific molecule, if present, binds a second tumor specific antigen (TSA2), a third paratope of the multispecific molecule, if present, binds a third tumor specific antigen (TSA3), and a fourth paratope of the multispecific molecule, if present, binds a fourth tumor specific antigen (TSA4).

[0260] In specific embodiments, a target antigen is selected from interleukin 13 (IL-13), thymic stromal lymphopoietin (TSLP), and interleukin 22 (IL-22). In specific embodiments, the multispecific molecule targets at least two antigens. In specific embodiments, the multispecific molecule targets IL-13 and TSLP. In specific embodiments, the multispecific molecule targets IL-13 and Il-22. Variable Regions, Variable Domains, And Paratopes

[0261] In one aspect, the multispecific molecules of the present invention bind to a target antigen (i.e., a paratope of the multispecific molecule binds an epitope of the target antigen). In specific embodiments, a multispecific molecule as described herein comprises one or more variable regions that form one or more paratopes that bind to one or more target antigens. In specific embodiments, a variable region comprises two variable domains (e.g., two variable domains from two polypeptides as described herein that bind a target antigen). In specific embodiments, a variable region comprises a light chain variable domain and a heavy chain variable domain of an antibody that binds to a target antigen. In specific embodiments, a variable region comprises a light chain variable domain and a heavy chain variable domain of one arm of an antibody that binds to a target antigen.

[0262] In specific embodiments, a polypeptide of a multispecific molecule as described herein comprises a means for binding with a target antigen. In a specific embodiment, the means for binding is a variable domain (e.g., a light chain variable domain or a heavy chain variablePage 116 of 370 307304067v1domain). In specific embodiments, the means for binding of two polypeptide chains of a multispecific molecule as described herein form a paratope.

[0263] In specific embodiments, the variable region of a multispecific molecule as described herein forms a paratope that binds to a target antigen (e.g., an epitope of the target antigen). In specific embodiments, the variable domains of two polypeptide chains of a multispecific molecule as described herein form a paratope that binds to a target antigen (e.g., an epitope of the target antigen). In specific embodiments, the light chain complementary determining region 1 (LCDR1), chain complementary determining region 2 (LCDR2), light chain complementary determining region 3 (LCDR3), heavy chain complementary determining region 1 (HCDR1), heavy chain complementary determining region 2 (HCDR2), and heavy chain complementary determining region 3 (HCDR3) form a paratope that binds to a target antigen (e.g., an epitope of the target antigen).

[0264] In specific embodiments, a paratope as described herein binds to a target antigen. In specific embodiments, a paratope as described herein binds to an epitope of a target antigen. In specific embodiments, different paratopes of the same multispecific molecule (e.g., a first paratope, a second paratope, a third paratope, or a fourth paratope) respectively bind to different target antigens.

[0265] In specific embodiments, a multispecific molecule comprises two variable regions that bind to two epitopes. In specific embodiments, a multispecific molecule comprises two variable regions that bind to two target antigens. In specific embodiments, a multispecific molecule comprises two variable regions that bind to two different epitopes, optionally wherein the two different epitopes are from different target antigens.

[0266] In specific embodiments, a multispecific molecule comprises three variable regions that bind to three epitopes. In specific embodiments, a multispecific molecule comprises three variable regions that bind to three target antigens. In specific embodiments, a multispecific molecule comprises three variable regions that bind to three different epitopes, optionally wherein each of the three different epitopes are from different target antigens.Page 117 of 370 307304067v1

[0267] In specific embodiments, a multispecific molecule comprises four variable regions that bind to four epitopes. In specific embodiments, a multispecific molecule comprises four variable regions that bind to four target antigens. In specific embodiments, a multispecific molecule comprises four variable regions that bind to four different epitopes, optionally wherein each of the four different epitopes are from different target antigens.

[0268] In specific embodiments, a variable domain of a polypeptide as described herein comprises an antibody light chain variable domain or a fragment thereof. In specific embodiments, a variable domain or a fragment thereof comprises the amino acid sequence of an antibody light chain framework region 1 (LFR1), light chain complementary determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementary determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementary determining region 3 (LCDR3), light chain framework region 4 (LFR4), or any combination thereof that binds to a target antigen or forms a paratope that binds to a target antigen.

[0269] In specific embodiments, a variable domain of a polypeptide as described herein comprises an antibody heavy chain variable domain or a fragment thereof. In specific embodiments, a variable domain or a fragment thereof comprises the amino acid sequence of an antibody heavy chain framework region 1 (HFR1), heavy chain complementary determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementary determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementary determining region 3 (HCDR3), heavy chain framework region 4 (HFR4), or any combination thereof that binds to a target antigen or forms a paratope that binds to a target antigen. Multispecific Molecules

[0270] In one aspect, the multispecific molecules of the present invention comprise dimers of two polypeptides, each comprising a variable domain and a variant CL or CH1 domain as described herein, wherein said variable domains form a paratope, and said variant CL and CH1 domains bind to each other to form a dimer. Full or partial replacement of the dimerization interface between a light chain constant domain (CL) and heavy chain constant domain 1 (CH1) provides a means for selective assembly of cognate antibody chains. For example, a biparatopicPage 118 of 370 307304067v1antibody has one arm (e.g., a light chain polypeptide and a heavy chain polypeptide) modified with a variant CL and CH1 domains as described herein, and its other arm is not modified; or has both arms modified with different variant CL and CH1 domains as described herein. In another example, a triparatopic antibody has one arm (e.g., a light chain polypeptide and a heavy chain polypeptide) modified with the variant CL and CH1 domains as described herein, one arm (e.g., a light chain polypeptide and a heavy chain polypeptide) modified with different variant CL and CH1 domains as described herein, and its other arm is not modified.

[0271] In specific embodiments, a multispecific molecule is an immunoglobulin or antigen-binding fragment thereof. In specific embodiments, a multispecific molecule is an antibody or antigen-binding fragment thereof. In specific embodiments, a multispecific molecule is an immunoglobulin fragment or antibody fragment comprising a variable region and at least one constant domain and that binds a target antigen. In specific embodiments, the immunoglobulin or antibody fragment is a Fab, Fab’, F(ab’)2, or bispecific Fab. In specific embodiments, the antibody is a biparatopic antibody, a bispecific antibody, a triparatopic antibody, a trispecific antibody, a tetraparatopic antibody, tetraspecific antibody, a multiparatopic antibody, or a multispecific antibody. In specific embodiments, a multispecific molecule is an intact immunoglobulin or antibody. In specific embodiments, a multispecific molecule is not an intact immunoglobulin or antibody. In specific embodiments, a multispecific molecule is any fragment of an antigen-binding molecule as described herein that binds to a target antigen.

[0272] In some embodiments, the format of the multispecific molecule as illustrated herein confers minor to no generation of populations of half antibodies or homodimeric impurities. In some embodiments, the format of the multispecific molecule as illustrated herein confers prevention of the secretion of impurities such as, but not limited to, half antibodies or homodimeric antibodies. In some embodiments, the format of the multispecific molecule as illustrated herein confers prevention of secretion of heavy chains mis-paired to light chains. In some embodiments, the format of the multispecific molecule as illustrated herein confers a low to an undetectable percentage of aggregate and / or fragment formation.Page 119 of 370 307304067v1

[0273] In some embodiments, the format of the multispecific molecule as illustrated herein confers more than about 90% monomer formation. In some embodiments, the format of the multispecific molecule as illustrated herein confers monomer formation in the range of about 90% to about 100%. In some embodiments, the format of the multispecific molecule as illustrated herein confers monomer formation in the range of about 90% to about 95%. In some embodiments, the format of the multispecific molecule as illustrated herein confers monomer formation in the range of about 95% to about 100%.

[0274] In some embodiments, the format of the multispecific molecule as illustrated herein confers more than about 90% monomer formation upon incubation at 37°C in human serum or a buffer solution up to at least 7 days. In some embodiments, the format of the multispecific molecule as illustrated herein confers monomer formation in the range of about 90% to about 100% upon incubation at 37°C in human serum or a buffer solution up to at least 7 days. In some embodiments, the format of the multispecific molecules as illustrated herein confers monomer formation in the range of about 90% to about 95% upon incubation at 37°C in human serum or a buffer solution up to at least 7 days. In some embodiments, the format of the multispecific molecules as illustrated herein confers monomer formation in the range of about 95% to about 100% upon incubation at 37°C in human serum or a buffer solution up to at least 7 days.

[0275] In some embodiments, the format of the multispecific molecule as illustrated herein enhances the correct pairing of heavy chains and light chains. In some embodiments, the correct pairing of heavy chains and light chains of the multispecific molecule is more than about 90% of the total paired molecules. In some embodiments, the correct pairing of heavy chains and light chains of the multispecific molecule ranges from about 90% to about 100% of the total paired molecules. In some embodiments, the correct pairing of heavy chains and light chains of the multispecific molecule ranges from about 90% to about 95% of the total paired molecules. In some embodiments, the correct pairing of heavy chains and light chains of the multispecific molecule ranges from about 95% to about 100% of the total paired molecules.

[0276] In some embodiments, the format of the multispecific molecule as illustrated herein confers efficient heterodimer formation. In some embodiments, the efficient heterodimerPage 120 of 370 307304067v1formation is more than about 90% of the total population of paired molecules. In some embodiments, the efficient heterodimer formation ranges from about 90% to about 100% of the total population of paired molecules. In some embodiments, the efficient heterodimer formation ranges from about 90% to about 95% of the total population of paired molecules. In some embodiments, the efficient heterodimer formation ranges from about 95% to about 100% of the total population of paired molecules.

[0277] In some embodiments, the format of the multispecific molecule as illustrated herein increases the yield of the multispecific molecule as compared to the control molecule. The control molecule comprises the same amino acid sequence as the multispecific molecule but without (i) a substitution at P151, P374, and / or P141; (ii) heterodimerization mutations; and / or (iii) variable domain exchange(s) as illustrated herein.

[0278] In some embodiments, the multispecific molecule has a high specificity towards a target antigen(s). High specificity may include, but is not limited to, no or limited cross- reactivity and no or limited unwanted binding to other proteins.

[0279] In some embodiments, the multispecific molecule has more than about 70% monodispersity as analyzed by size exclusion chromatography (SEC). In some embodiments, the multispecific molecule has more than about 80% monodispersity as analyzed by size exclusion chromatography (SEC). In some embodiments, the multispecific molecule has more than about 85% monodispersity as analyzed by size exclusion chromatography (SEC). In some embodiments, the multispecific molecule has more than about 90% monodispersity as analyzed by size exclusion chromatography (SEC). In some embodiments, the multispecific molecule has more than about 95% monodispersity as analyzed by size exclusion chromatography (SEC). In some embodiments, the multispecific molecule has about 100% monodispersity as analyzed by size exclusion chromatography (SEC). Fab-Based Antigen-Binding Molecules

[0280] In specific embodiments, a multispecific molecule is a Fab. In specific embodiments, the Fab comprises an antibody fragment having a variable region that binds to aPage 121 of 370 307304067v1target antigen and comprises a light chain and a fragment of a heavy chain bridged by a disulfide bond. In specific embodiments, the “pseudo Fab” (pFab) comprises a variable region that binds to a target antigen and two variant CL and CH1 domains as described herein. In specific embodiments, a multispecific molecule is a Fab’. In specific embodiments, the Fab’ comprises an antibody fragment having a single variable region that binds to a target antigen, comprising a Fab and an additional portion of the heavy chain through the hinge region. In specific embodiments, the pFab’ comprises a variable region that binds to a target antigen, two variant CL and CH1 domains as described herein, and a heavy chain hinge region. In specific embodiments, a multispecific molecule is a F(ab’)2. In specific embodiments, the F(ab’)2comprises two Fab’ molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains. In specific embodiments, the Fab’ molecules of the F(ab’)2 may be directed toward the same or different epitopes. In specific embodiments, the pF(ab’)2 comprises two variable regions that each bind to a target antigen, two antigen-binding domains, two or more variant CL and CH1 domains as described herein, and a heavy chain hinge region. In specific embodiments, a multispecific molecule is a bispecific Fab. In specific embodiments, the bispecific Fab comprises a Fab molecule having two variable regions that each bind to a target antigen, each of which may be directed to a different epitope).

[0281] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab-like antibody fragments can be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab’)2fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. The antibody fragment may also be a “linear antibody,” for example, as described in the references cited above. Such linear antibodies may be monospecific or multi- specific, such as bispecific.Page 122 of 370 307304067v1

[0282] Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen-binding site that bind to a target antigen. The immunoglobulin molecules provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule that contains an antigen-binding site that binds to a target antigen. In a specific embodiment, an antibody provided herein is an IgG antibody, such as an IgG1 antibody, IgG2 antibody or IgG4 antibody (e.g., IgG4 nullbody and variants of IgG4 antibodies). In a preferred embodiment, the IgG antibody is an IgG1 antibody.

[0283] In specific embodiments, a multispecific molecule is a Fab, Fab’, F(ab’)2, or bispecific Fab that comprises a variable region derived from an antibody specific for one or more target antigens. In specific embodiments, an antigen-binding molecule comprises a variable region that is derived from an antibody specific for one or more target antigens, and variant CL and CH1 domain as described herein. Multispecific Antibody-Based Antigen-Binding Molecules

[0284] A multispecific molecule of the present disclosure may be derived from an antibody by full or partial replacement of the dimerization interface between a light chain constant domain and a heavy chain constant domain 1 (CH1) of an antibody with the variant CL and CH1 domains as described herein. Monoclonal Antibodies

[0285] In specific embodiments, the multispecific molecule provided herein comprises a monoclonal antibody or a fragment thereof. Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., 1975, Nature 256:495-97, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).

[0286] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized with a target antigen as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to an epitope of the target antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro.Page 123 of 370 307304067v1After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

[0287] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, which, in specific embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0288] Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Exemplary myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63- Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, 1984, Immunol.133:3001-05; and Brodeur et al., 1987, Monoclonal Antibody Production Techniques and Applications 51-63).

[0289] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the target antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as RIA or ELISA. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., 1980, Anal. Biochem.107:220-39.

[0290] Once hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, forPage 124 of 370 307304067v1example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal, for example, by i.p. injection of the cells into mice.

[0291] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0292] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., 1993, Curr. Opinion in Immunol.5:256-62 and Plückthun, 1992, Immunol. Revs.130:151- 88.

[0293] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols (O’Brien and Aitken eds., 2002). In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177- 186; Kettleborough et al., 1994, Eur. J. Immunol.24:952-958; Persic et al., 1997, Gene 187:9- 18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Application No. PCT / GB91 / O1134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and WO97 / 13844; and U.S. Patent Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.Page 125 of 370 307304067v1

[0294] In principle, synthetic antibody clones are selected by screening phage libraries containing phages that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened against the desired target antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution.

[0295] Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non- covalently, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol.12:433-55.

[0296] Repertoires of VH and VL genes can be separately cloned by PCR and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., supra. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., 1993, EMBO J 12:725-34. Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by Hoogenboom and Winter, 1992, J. Mol. Biol.227:381-88.

[0297] Screening of the libraries can be accomplished by various techniques known in the art. For example, a target antigen can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, conjugated to biotin for capture with Strep-tag II-coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., 1990, Proteins 8:309-14 and WO 92 / 09690, and by use of a low coating density of antigen as described in Marks et al., 1992, Biotechnol.10:779-83.Page 126 of 370 307304067v1

[0298] Antibodies can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length antibody clone using VH and / or VL sequences (e.g., the Fv sequences), or various CDR sequences from VH and VL sequences, from the phage clone of interest and suitable constant region (e.g., Fc) sequences described in Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0299] Antibodies described herein can also, for example, include chimeric antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos.5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0300] Antibodies or antigen-binding fragments produced using techniques such as those described herein can be isolated using standard, well known techniques. For example, antibodies or antigen-binding fragments can be suitably separated from, e.g., culture medium, ascites fluid, serum, cell lysate, synthesis reaction material or the like by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. In specific embodiments, an isolated or purified antibody is substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. Humanized Antibodies

[0301] In specific embodiments, the multispecific molecule provided herein comprises a humanized antibody (e.g., deimmunized or composite human antibody) or a fragment thereof. In specific embodiments, a humanized antibody comprises human framework region and / or human constant region sequences. In specific embodiments, a humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, includingPage 127 of 370 307304067v1IgG1, IgG2, IgG3 and IgG4 (e.g., variants of IgG4 and IgG4 nullbody). In specific embodiments, a humanized antibody comprises kappa or lambda light chain constant sequences.

[0302] Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International publication No. WO 91 / 09967; and U.S. Patent Nos.5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805- 814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Patent No.5,565,332), and other techniques disclosed in, e.g., U.S. Pat. No.6,407,213, U.S. Pat. No.5,766,886, WO 93 / 17105, Tan et al., J. Immunol.169:111925 (2002), Caldas et al., Protein Eng.13(5):353-60 (2000), Morea et al., Methods 20(3):26779 (2000), Baca et al., J. Biol. Chem.272(16):10678-84 (1997), Roguska et al., Protein Eng.9(10):895904 (1996), Couto et al., Cancer Res.55 (23 Supp):5973s- 5977s (1995), Couto et al., Cancer Res.55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol.235(3):959-73 (1994). See also U.S. Patent Pub. No. US 2005 / 0042664 A1 (Feb.24, 2005), each of which is incorporated by reference herein in its entirety.

[0303] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.

[0304] In specific embodiments, humanized antibodies are constructed by CDR grafting, wherein the amino acid sequences of the six CDRs of a parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the “specificity determining residues,” or SDRs (Padlan et al., 1995, FASEB J.9:133-39). In thePage 128 of 370 307304067v1technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods 36:25-34).

[0305] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. For example, according to the so-called “best-fit” method, the sequence of the variable domain of a non- human (e.g., rodent) antibody is screened against the entire library of known human variable- domain sequences. The human sequence that is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol.151:2296-308; and Chothia et al., 1987, J. Mol. Biol.196:901-17). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol. 151:2623-32). In specific cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII). In another method, human germline genes are used as the source of the framework regions.

[0306] In an alternative paradigm based on comparison of CDRs, called superhumanization, FR homology is irrelevant. The method comprises comparison of the non- human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., 2002, J. Immunol.169:1119-25).

[0307] It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selectedPage 129 of 370 307304067v1candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng.13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol.234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen- binding.

[0308] Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes, and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (Lazar et al., 2007, Mol. Immunol.44:1986-98).

[0309] In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those that are based upon the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol.23:1105-16; Dufner et al., 2006, Trends Biotechnol.24:523-29; Feldhaus et al., 2003, Nat. Biotechnol.21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel.17:847-60).

[0310] In the FR library approach, a collection of residue variants are introduced at specific positions in the FR followed by screening of the library to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol.224:487-99), or from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem.272:10678-84).Page 130 of 370 307304067v1

[0311] In FR shuffling, whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’Acqua et al., 2005, Methods 36:43-60). The libraries may be screened for binding in a two-step process, first humanizing VL, followed by VH. Alternatively, a one-step FR shuffling process may be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol.44:3049-60).

[0312] The “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non- human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96% homologous to human germline gene antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute’s Third Annual PEGS, The Protein Engineering Summit, 2007).

[0313] The “human engineering” method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk,” “moderate risk,” or “high risk” residues. The classification is performed using a global risk / reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding. The particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable region with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderatePage 131 of 370 307304067v1risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail in Studnicka et al., 1994, Protein Engineering 7:805-14; U.S. Pat. Nos.5,766,886; 5,770,196; 5,821,123; and 5,869,619; and PCT Publication WO 93 / 11794.

[0314] A composite human antibody can be generated using, for example, Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To generate composite human antibodies, variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody. Such antibodies can comprise human constant region sequences, e.g., human light chain and / or heavy chain constant regions.

[0315] In specific embodiments, an antigen-binding molecule comprises a deimmunized antibody whose T-cell epitopes have been removed. Methods for making deimmunized antibodies have been described. (See, e.g., Jones et al., Methods Mol Biol.2009;525:405-23, xiv, and De Groot et al., Cell. Immunol.244:148-153(2006)). Deimmunized antibodies comprise T-cell epitope-depleted variable regions and human constant regions. Briefly, VH and VL of an antibody are cloned and T-cell epitopes are subsequently identified by testing overlapping peptides derived from the VH and VL of the antibody in a T cell proliferation assay. T cell epitopes are identified via in silico methods to identify peptide binding to human MHC class II. Mutations are introduced in the VH and VL to abrogate binding to human MHC class II. Mutated VH and VL are then utilized to generate the deimmunized antibody. Human Antibodies

[0316] In specific embodiments, the multispecific molecule provided herein comprises a fully human anti-human antibody or a fragment thereof. Fully human antibodies may be produced by any method known in the art. Human antibodies provided herein can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal antibodies of the present disclosure can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodiesPage 132 of 370 307304067v1have been described, for example, by Kozbor, 1984, J. Immunol.133:3001-05; Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Boerner et al., 1991, J. Immunol.147:86-95.

[0317] It is also possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice that express human antibody repertoires have been used to generate high-affinity human sequence monoclonal antibodies against a wide variety of potential drug targets (see, e.g., Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol.8(4):455-58; U.S. Pat. Nos.6,075,181 and 6,150,584; and Lonberg et al., 2005, Nature Biotechnol.23:1117-25).

[0318] Alternatively, the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (e.g., such B lymphocytes may be recovered from an individual or may have been immunized in vitro) (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and U.S. Pat. No.5,750,373).

[0319] Gene shuffling can also be used to derive human antibodies from non-human, for example, rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting” or “guided selection,” either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain / human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain / human chain chimeric scFv or Fab wherein the human chain restores the antigen-binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone (e.g., the epitope guides (imprints) the choice of the human chain partner). When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see, e.g., PCT WO 93 / 06213; and Osbourn et al., 2005, Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin. Examples ofPage 133 of 370 307304067v1guided selection to humanize mouse antibodies towards cell surface antigens include the folate- binding protein present on ovarian cancer cells (see, e.g., Figini et al., 1998, Cancer Res.58:991- 96) and CD147, which is highly expressed on hepatocellular carcinoma (see, e.g., Bao et al., 2005, Cancer Biol. Ther.4:1374-80).

[0320] A potential disadvantage of the guided selection approach is that shuffling of one antibody chain while keeping the other constant could result in epitope drift. In order to maintain the epitope recognized by the non-human antibody, CDR retention can be applied (see, e.g., Klimka et al., 2000, Br. J. Cancer.83:252-60; and Beiboer et al., 2000, J. Mol. Biol. 296:833-49). In this method, the non-human VH CDR3 is commonly retained, as this CDR may be at the center of the antigen-binding site and may be the most important region of the antibody for antigen recognition. In specific instances, however, VH CDR3 and VL CDR3, as well as VH CDR2, VL CDR2, and VL CDR1 of the non-human antibody may be retained. Multispecific Antibodies

[0321] Multispecific antibodies such as bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In specific embodiments, the multispecific antibodies provided herein are bispecific antibodies. In specific embodiments, bispecific antibodies are mouse, chimeric, human, or humanized antibodies. In specific embodiments, one of the binding specificities is for one target and / or target antigen and the other is for another target and / or target antigen. In specific embodiments, bispecific antibodies may bind to two different epitopes of the same target and / or target antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibody).

[0322] Methods for making multispecific antibodies are known in the art, such as, by co- expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details of generating multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).Page 134 of 370 307304067v1Fc engineering

[0323] It may be desirable to modify an antibody provided herein by Fc engineering. In specific embodiments, the modification to the Fc region of the antibody results in the decrease or elimination of an effector function of the antibody. In specific embodiments, the effector function is antibody-dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In specific embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP and CDC. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.

[0324] In specific embodiments, the modification to the Fc region of the antibody results in the enhancement of an effector function of the antibody. In specific embodiments, the effector function is ADCC, ADCP, and / or CDC. In specific embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP and CDC. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. In specific embodiments, Knobs-in-holes (KIH) technology was used to engineer the antibody.

[0325] To increase the serum half-life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment), for example, as described in U.S. Pat. No.5,739,277. Term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.Page 135 of 370 307304067v1Antibody Variants

[0326] In specific embodiments, amino acid sequence modification(s) of the antibodies or antigen-binding fragments provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the antibodies described herein, it is contemplated that antibody variants can be prepared. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art would appreciate that amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

[0327] In specific embodiments, antibodies provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the antibody. The antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.

[0328] Variations may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 aminoPage 136 of 370 307304067v1acids. In specific embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution made at one or more predicted non- essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0329] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

[0330] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.Page 137 of 370 307304067v1

[0331] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Alternatively, conservative (e.g., within an amino acid group with similar properties and / or side chains) substitutions may be made, so as to maintain or not significantly change the properties. Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed.1975)): (1) non- polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[0332] Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0333] Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites. Accordingly, in one embodiment, an antibody or antigen-binding fragment thereof that binds to a target epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an antibody described herein, such as for example, the antibodies described in the Sequence Listing submitted herewith. In another embodiment, an antibody or antigen-binding fragment thereof that binds to a target antigen comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an antibody described herein, such as for example, the antibodies described in the Sequence Listing submitted herewith.Page 138 of 370 307304067v1

[0334] The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J.237:1-7; and Zoller et al., 1982, Nucl. Acids Res.10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315- 23), or other known techniques can be performed on the cloned DNA to produce the antigen- binding molecule variant DNA.

[0335] Any cysteine residue not involved in maintaining the proper conformation of the antibody provided herein also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (e.g., where the antibody is an antibody fragment such as an Fv fragment).

[0336] In specific embodiments, an antibody molecule of the present disclosure is a “de- immunized” antibody. A “de-immunized” antibody is an antibody derived from a humanized or chimeric antibody, which has one or more alterations in its amino acid sequence resulting in a reduction of immunogenicity of the antibody, compared to the respective original non-de- immunized antibody. One of the procedures for generating such antibody mutants involves the identification and removal of T-cell epitopes of the antibody molecule. In a first step, the immunogenicity of the antibody molecule can be determined by several methods, for example, by in vitro determination of T-cell epitopes or in silico prediction of such epitopes, as known in the art. Once the critical residues for T-cell epitope function have been identified, mutations can be made to remove immunogenicity and retain antibody activity. For review, see, for example, Jones et al., 2009, Methods in Molecular Biology 525:405-23. In vitro affinity maturation

[0337] In specific embodiments, antibody variants having an improved property such as affinity, stability, or expression level as compared to a parent antibody may be prepared by in vitro affinity maturation. Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Libraries of antibodies are displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association (e.g., covalently or non-covalently) with their encoding mRNA or DNA. Affinity selection of the displayedPage 139 of 370 307304067v1antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies. Two or three rounds of mutation and selection using display methods such as phage display usually results in antibody fragments with affinities in the low nanomolar range. Affinity matured antibodies can have nanomolar or even picomolar affinities for the target antigen.

[0338] Phage display is a widespread method for display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to the bacteriophage coat protein. Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning.” Phage bound to antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For review, see, for example, Hoogenboom, 2002, Methods. Mol. Biol.178:1-37; and Bradbury and Marks, 2004, J. Immunol. Methods 290:29-49.

[0339] In a yeast display system (see, e.g., Boder et al., 1997, Nat. Biotech.15:553–57; and Chao et al., 2006, Nat. Protocols 1:755-68), the antibody may be fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to ...

Claims

WHAT IS CLAIMED IS:

1. A multispecific molecule comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 3 (CH3 domain), and wherein (i) the first polypeptide and the second polypeptide each comprises a mutation at position P374 (Eu numbering) in its respective CH3 domain, and (ii) the first polypeptide and / or the second polypeptide comprises one or more heterodimerization mutations.

2. The multispecific molecule of claim 1, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 1 (CH1 domain).

3. The multispecific molecule of claim 2, wherein the molecule further comprises a third polypeptide and a fourth polypeptide, wherein the third polypeptide and the fourth polypeptide each comprises an immunoglobulin light chain constant (CL) domain, wherein each of said CL domains is paired with its cognate CH1 domain.

4. The multispecific molecule of claim 3, wherein the first, second, third and fourth polypeptide each comprises an immunoglobulin variable region, wherein the first polypeptide and the third polypeptide associate to form a first antigen-binding site, and the second polypeptide and the fourth polypeptide associate to form a second antigen-binding site.

5. The multispecific molecule of claim 3 or claim 4, wherein the CH1 domain of the first polypeptide is paired with the CL domain of the third polypeptide and the CH1 domain of the second polypeptide is paired with the CL domain of the fourth polypeptide, and wherein (i) at least one of the first polypeptide and the second polypeptide comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain and / or at least one of the third polypeptide and the fourth polypeptide comprises a mutation at position P141 (Eu numbering) in its respective CL domain, and (ii) one or more of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.

6. The multispecific molecule of claim 5, wherein the first polypeptide and the second polypeptide each comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain.Page 342 of 370 307304067v17. The multispecific molecule of any one of claims 3-6, wherein each of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.

8. The multispecific molecule of any one of claims 3-7, wherein one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide.

9. The multispecific molecule of claim 8, wherein the exchanged one or more heavy chain domains and / or one or more light chain domains comprise an immunoglobulin variable region.

10. The multispecific molecule of claim 9, wherein each of the exchanged heavy chain domains and / or light chain domains comprises an immunoglobulin variable region.

11. The multispecific molecule of any one of claims 1-10, wherein the mutation at position P374 (Eu numbering) is an amino acid substitution P374X.

12. The multispecific molecule of claim 11, wherein X in the amino acid substitution P374X can be any amino acid.

13. The multispecific molecule of claim 12, wherein X in the amino acid substitution P374X is selected from A, G, and Q.

14. The multispecific molecule of any one of claims 5-13, wherein the mutation at position P151 (Eu numbering) is an amino acid substitution P151X.

15. The multispecific molecule of claim 14, wherein X in the amino acid substitution P151X can be any amino acid.

16. The multispecific molecule of claim 14, wherein X in the amino acid substitution P151X is A or G.

17. The multispecific molecule of any one of claims 1-16, wherein said one or more heterodimerization mutations are selected from charge steering mutations, knob into hole mutations, complementary mutations, and any combinations thereof.Page 343 of 370 307304067v118. The multispecific molecule of any one of claims 1-16, wherein said one or more heterodimerization mutations are at position(s) selected from 364, 357, 366, 368, 370, 405, 407, 409, S364, E357, T366, L368, K370, F405, Y407, and K409, according to Eu numbering, and any combinations thereof.

19. The multispecific molecule of any one of claims 1-16, wherein the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain; 2) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain; 10) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain; 11) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain;Page 344 of 370 307304067v113) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain; 17) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 25) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 26) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain;Page 345 of 370 307304067v128) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 29) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 30) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain; 32) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain; 40) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; 41) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain;Page 346 of 370 307304067v143) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 44) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 45) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in the second polypeptide CH3 domain; 47) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or 48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain.

20. The multispecific molecule of any one of claims 1-19, wherein the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 2) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 3) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 4) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 5) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 6) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 7) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 8) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain;Page 347 of 370 307304067v19) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 10) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 11) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; 12) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain; 13) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; or 16) S364D and K409D in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain.

21. The multispecific molecule of any one of claims 5-20, wherein the molecule comprises the following heterodimerization mutations (Eu numbering): 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain; 5) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain;Page 348 of 370 307304067v17) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 9) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 10) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 17) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain; 20) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain;Page 349 of 370 307304067v122) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain; 24) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain; 25) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 35) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain;Page 350 of 370 307304067v137) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain; 39) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain; 40) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain; 50) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain;Page 351 of 370 307304067v152) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; 54) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain; 55) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

22. The multispecific molecule of any one of claims 5-21, wherein the multispecific molecule comprises: 1) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in the second polypeptide; 2) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide;Page 352 of 370 307304067v13) V185D in the first polypeptide CH1 domain and S174K in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 4) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and V185K in the second polypeptide CH1 domain and S174D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 5) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 6) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 7) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide; 8) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the first polypeptide; 9) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain, wherein P151A is present in the first polypeptide; 10) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and V185D in the second polypeptide CH1 domain and S174K in its paired CL domain, wherein P151A is present in the first polypeptide; 11) V185K in the first polypeptide CH1 domain and S174D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide;Page 353 of 370 307304067v112) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 13) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; or 14) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide.

23. A multispecific molecule comprising first, second, third and fourth polypeptides, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 1 (CH1 domain), and the third polypeptide and the fourth polypeptide each comprises an immunoglobulin light chain constant (CL) domain, wherein the CH1 domain of the first polypeptide is paired with the CL domain of the third polypeptide and the CH1 domain of the second polypeptide is paired with the CL domain of the fourth polypeptide, and wherein (i) at least one of the first polypeptide and the second polypeptide comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain and / or at least one of the third polypeptide and the fourth polypeptide comprises a mutation at position P141 (Eu numbering) in its respective CL domain, and (ii) one or more of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.

24. The multispecific molecule of claim 23, wherein the first polypeptide and the second polypeptide each comprises a mutation at position P151 (Eu numbering) in its respective CH1 domain.

25. The multispecific molecule of claim 23 or claim 24, wherein each of the first, second, third, or fourth polypeptide comprises one or more heterodimerization mutations.

26. The multispecific molecule of any one of claims 23-25, wherein the first, second, third and fourth polypeptide each comprises an immunoglobulin variable region, wherein the firstPage 354 of 370 307304067v1polypeptide and the third polypeptide associate to form a first antigen-binding site, and the second polypeptide and the fourth polypeptide associate to form a second antigen-binding site.

27. The multispecific molecule of any one of claims 23-26, wherein one or more heavy chain domains are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of the third polypeptide and the fourth polypeptide.

28. The multispecific molecule of claim 27, wherein the exchanged one or more heavy chain domains and / or one or more light chain domains comprises an immunoglobulin variable region.

29. The multispecific molecule of claim 28, wherein each of the exchanged heavy chain domains and / or light chain domains comprises an immunoglobulin variable region.

30. The multispecific molecule of any one of claims 23-29, wherein the mutation at position P151 (Eu numbering) is an amino acid substitution P151X.

31. The multispecific molecule of claim 30, wherein X in the amino acid substitution P151X can be any amino acid.

32. The multispecific molecule of claim 30, wherein X in the amino acid substitution P151X is A or G.

33. The multispecific molecule of any one of claims 23-32, wherein each of the first polypeptide and the second polypeptide further comprises an immunoglobulin heavy chain constant domain 3 (CH3 domain) and (i) a mutation at position P374 (Eu numbering) in its respective CH3 domain; and (ii) the first polypeptide and / or the second polypeptide comprises one or more heterodimerization mutations.

34. The multispecific molecule of claim 33, wherein the mutation at position P374 (Eu numbering) is an amino acid substitution P374X.

35. The multispecific molecule of claim 34, wherein X in the amino acid substitution P374X can be any amino acid.Page 355 of 370 307304067v136. The multispecific molecule of claim 34, wherein X in the amino acid substitution P374X is selected from A, G, and Q.

37. The multispecific molecule of any one of claims 23-36, wherein said one or more heterodimerization mutations are selected from charge steering mutations, knob into hole mutations, complementary mutations, and any combinations thereof.

38. The multispecific molecule of any one of claims 23-37, wherein said one or more heterodimerization mutations are at position(s) selected from S364, E357, T366, L368, K370, F405, Y407, K409, according to Eu numbering, and any combinations thereof.

39. The multispecific molecule of any one of claims 23-38, wherein the molecule comprises the following heterodimerization mutations (Eu numbering): 1) A141K in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 2) A141D in the first or second polypeptide CH1 domain; and F116K in its paired CL domain; 3) A141K in the first or second polypeptide CH1 domain; and L135D in its paired CL domain; 4) A141D in the first or second polypeptide CH1 domain; and L135K in its paired CL domain; 5) G143Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 6) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 7) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133Y in its paired CL domain; 8) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 9) L145K in the first or second polypeptide CH1 domain; and S131D in its paired CL domain;Page 356 of 370 307304067v110) L145D in the first or second polypeptide CH1 domain; and S131K in its paired CL domain; 11) S181K in the first or second polypeptide CH1 domain; and T178D in its paired CL domain; 12) S181D in the first or second polypeptide CH1 domain; and T178K in its paired CL domain; 13) V185K in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 14) V185D in the first or second polypeptide CH1 domain; and S174K in its paired CL domain; 15) V173A and S181Y in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 16) V173A and S181W in the first or second polypeptide CH1 domain; and no heterodimerization mutation in its paired CL domain; 17) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178Y in its paired CL domain; 18) no heterodimerization mutation in the first or second polypeptide CH1 domain; and T178W in its paired CL domain; 19) A141K and G143W in the first or second polypeptide CH1 domain; and F116D, V133S, L135A, and S176V in its paired CL domain; 20) A141K, G143S, L145K, and S183V in the first or second polypeptide CH1 domain; and F116D and V133W in its paired CL domain; 21) A141K and L145K in the first or second polypeptide CH1 domain; and F116D and S131D in its paired CL domain; 22) A141K and L145K in the first or second polypeptide CH1 domain; and L135D and S131D in its paired CL domain; 23) A141K and S181K in the first or second polypeptide CH1 domain; and F116D and T178D in its paired CL domain; 24) A141K and S181K in the first or second polypeptide CH1 domain; and L135D and T178D in its paired CL domain;Page 357 of 370 307304067v125) A141K and V185K in the first or second polypeptide CH1 domain; and F116D and S174D in its paired CL domain; 26) A141K and V185K in the first or second polypeptide CH1 domain; and L135D and S174D in its paired CL domain; 27) A141K, V173A, and S181Y in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 28) A141K, V173A, and S181W in the first or second polypeptide CH1 domain; and F116D in its paired CL domain; 29) A141K in the first or second polypeptide CH1 domain; and F116D and T178Y in its paired CL domain; 30) A141K in the first or second polypeptide CH1 domain; and F116D and T178W in its paired CL domain; 31) G143W and L145K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S131D in its paired CL domain; 32) G143W and S181K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178D in its paired CL domain; 33) G143W and V185K in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and S174D in its paired CL domain; 34) G143W, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 35) G143W, V173A, and S181W in the first or second polypeptide CH1 domain; and V133S, L135A, and S176V in its paired CL domain; 36) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178Y in its paired CL domain; 37) G143W in the first or second polypeptide CH1 domain; and V133S, L135A, S176V, and T178W in its paired CL domain; 38) G143S, L145A, S183V, and S181K in the first or second polypeptide CH1 domain; and V133W and T178D in its paired CL domain; 39) G143S, L145A, S183V, and V185K in the first or second polypeptide CH1 domain; and V133W and S174D in its paired CL domain;Page 358 of 370 307304067v140) G143S, L145A, S183V, V173A, and S181Y in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 41) G143S, L145A, S183V, V173A, and S181W in the first or second polypeptide CH1 domain; and V133W in its paired CL domain; 42) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178Y in its paired CL domain; 43) G143S, L145A, and S183V in the first or second polypeptide CH1 domain; and V133W and T178W in its paired CL domain; 44) L145K and S181K in the first or second polypeptide CH1 domain; and S131D and T178D in its paired CL domain; 45) L145K and V185K in the first or second polypeptide CH1 domain; and S131D and S174D in its paired CL domain; 46) L145K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 47) L145K, V173A, and S181W in the first or second polypeptide CH1 domain; and S131D in its paired CL domain; 48) L145K in the first or second polypeptide CH1 domain; and S131D and T178Y in its paired CL domain; 49) L145K in the first or second polypeptide CH1 domain; and S131D and T178W in its paired CL domain; 50) S181K and V185K in the first or second polypeptide CH1 domain; and T178D and S174D in its paired CL domain; 51) V185K, V173A, and S181Y in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 52) V185K, V173A, and S181W in the first or second polypeptide CH1 domain; and S174D in its paired CL domain; 53) V185K in the first or second polypeptide CH1 domain; and S174D and T178Y in its paired CL domain; 54) V185K in the first or second polypeptide CH1 domain; and S174D and T178W in its paired CL domain;Page 359 of 370 307304067v155) F170V, S182I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 56) F170V, S183I, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 57) F170I, S183L, and V185L in the first or second polypeptide CH1 domain; and L135F in its paired CL domain; 58) F170V, S182I, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F and E / Q124C in its paired CL domain; 59) F170I, S183L, V185L, and F126C in the first or second polypeptide CH1 domain; and L135F, and E / Q124C in its paired CL domain; 60) F170V, S182I, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain; and / or 61) F170I, S183L, V185L, F126C, and C220S in the first or second polypeptide CH1 domain; and L135F, E / Q124C, and C214S in its paired CL domain.

40. The multispecific molecule of any one of claims 23-39, wherein the multispecific molecule comprises: 1) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in the second polypeptide; 2) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 3) V185D in the first polypeptide CH1 domain and S174K in its paired CL domain; and no heterodimerization mutation in the second polypeptide CH1 domain and T178Y in its paired CL domain, wherein P151A is present in the second polypeptide; 4) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; V185K in the second polypeptide CH1 domain and S174D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 5) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide;Page 360 of 370 307304067v16) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and V173A and S181W in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 7) L145K in the first polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the first and third polypeptide; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the second polypeptide; 8) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in the first polypeptide; 9) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain, wherein P151A is present in the first polypeptide; 10) no heterodimerization mutation in the first polypeptide CH1 domain and T178Y in its paired CL domain; and V185D in the second polypeptide CH1 domain and S174K in its paired CL domain, wherein P151A is present in the first polypeptide; 11) V185K in the first polypeptide CH1 domain and S174D in its paired CL domain; and V173A and S181Y in the second polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 12) V173A and S181Y in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; 13) V173A and S181W in the first polypeptide CH1 domain and no heterodimerization mutation in its paired CL domain; and A141K in the second polypeptide CH1 domain and F116D in its paired CL domain, wherein P151A is present in each of the first and second polypeptide; or 14) A141K in the first polypeptide CH1 domain and F116D in its paired CL domain; and L145K in the second polypeptide CH1 domain and S131D in its paired CL domain with variable region exchange between the second and fourth polypeptide, wherein P151A is present in the first polypeptide.Page 361 of 370 307304067v141. The multispecific molecule of any one of claims 33-40, wherein the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) F405K in the first polypeptide CH3 domain and K409D / E in the second polypeptide CH3 domain; 2) K409D / E in the first polypeptide CH3 domain and F405K in the second polypeptide CH3 domain; 3) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D / E in the second polypeptide CH3 domain; 4) K439D / E in the first polypeptide CH3 domain and E356K in the second polypeptide CH3 domain; 5) F405K and K439D / E in the first polypeptide CH3 domain; and K409D / E and E356K in the second polypeptide CH3 domain; 6) L368K, F405K, and K439D / E in the first polypeptide CH3 domain; and S364D, K409D / E, and E356K in the second polypeptide CH3 domain; 7) E356K in the first polypeptide CH3 domain and K439D / E in the second polypeptide CH3 domain; 8) F405K and E356K in the first polypeptide CH3 domain; and K409D / E and K439D / E in the second polypeptide CH3 domain; 9) L368K, F405K, and E356K in the first polypeptide CH3 domain; and S364D, K409D / E and K439D / E in the second polypeptide CH3 domain; 10) S364D and K409D / E in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain; 11) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 12) F405K and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and K409D / E in the second polypeptide CH3 domain; 13) L368K, F405K, and T366W in the first polypeptide CH3 domain; and S364D, K409D / E, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain;Page 362 of 370 307304067v115) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and K409D / E in the second polypeptide CH3 domain; 16) F405K, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and S364D, T366W, K409D / E in the second polypeptide CH3 domain; 17) K409D / E and T366W in the first polypeptide CH3 domain; and T366S, L368A, Y407V, and F405K in the second polypeptide CH3 domain; 18) S364D, K409D / E, and T366W in the first polypeptide CH3 domain; and L368K, F405K, T366S, L368A, and Y407V in the second polypeptide CH3 domain; 19) K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W and F405K in the second polypeptide CH3 domain; 20) S364D, K409D / E, T366S, L368A, and Y407V in the first polypeptide CH3 domain; and L368K, F405K, and T366W in the second polypeptide CH3 domain; 21) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 22) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 23) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 24) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 25) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 26) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 27) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 28) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 29) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain;Page 363 of 370 307304067v130) E357N and F405K in the first polypeptide CH3 domain; and K370T and K409D / E in in the second polypeptide CH3 domain; 31) E357Q and F405K in the first polypeptide CH3 domain; and K370S and K409D / E in the second polypeptide CH3 domain; 32) E357K and F405K in the first polypeptide CH3 domain; and K370E and K409D / E in the second polypeptide CH3 domain; 33) K370T, L368K, and F405K in the first polypeptide CH3 domain; and E357N, S364D, and K409D / E in in the second polypeptide CH3 domain; 34) K370S, L368K, and F405K in the first polypeptide CH3 domain; and E357Q, S364D, and K409D / E in the second polypeptide CH3 domain; 35) K370E, L368K, and F405K in the first polypeptide CH3 domain; and E357K, S364D, and K409D / E in the second polypeptide CH3 domain; 36) E357N, L368K, and F405K in the first polypeptide CH3 domain; and K370T, S364D, and K409D / E in the second polypeptide CH3 domain; 37) E357Q, L368K, and F405K in the first polypeptide CH3 domain; and K370S, S364D, and K409D / E in the second polypeptide CH3 domain; 38) E357K, L368K, and F405K in the first polypeptide CH3 domain; and K370E, S364D, and K409D / E in the second polypeptide CH3 domain; 39) T350V, L351Y, F405A, and Y407V in the first polypeptide CH3 domain; and T350V, T366L, K392L, and T394W in the second polypeptide CH3 domain; 40) L368K, F405K, and D399K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; 41) F405K and D399K in the first polypeptide CH3 domain; and K409D in the second polypeptide CH3 domain; 42) L368K, F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and S364D, K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 43) F405K, T350V, T366L, K392L, and T394W in the first polypeptide CH3 domain; and K409D, T350V, L351Y, F405A, and Y407V in the second polypeptide CH3 domain; 44) L368K, F405K, K360E, and K409W in the first polypeptide CH3 domain; and S364D, K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain;Page 364 of 370 307304067v145) F405K, K360E, and K409W in the first polypeptide CH3 domain; and K409D, Q347R, D399V, and F405T in the second polypeptide CH3 domain; 46) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain; 47) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; or (48) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain.

42. The multispecific molecule of any one of claims 33-41, wherein the multispecific molecule comprises the following heterodimerization mutations (Eu numbering): 1) K370T in the first polypeptide CH3 domain; and E357N in the second polypeptide CH3 domain; 2) K370S in the first polypeptide CH3 domain; and E357Q in the second polypeptide CH3 domain; 3) K370E in the first polypeptide CH3 domain; and E357K in the second polypeptide CH3 domain; 4) K370T and F405K in the first polypeptide CH3 domain; and E357N and K409D / E in in the second polypeptide CH3 domain; 5) K370S and F405K in the first polypeptide CH3 domain; and E357Q and K409D / E in the second polypeptide CH3 domain; 6) K370E and F405K in the first polypeptide CH3 domain; and E357K and K409D / E in the second polypeptide CH3 domain; 7) E357N in the first polypeptide CH3 domain; and K370T in the second polypeptide CH3 domain; 8) E357Q in the first polypeptide CH3 domain; and K370S in the second polypeptide CH3 domain; 9) E357K in the first polypeptide CH3 domain; and K370E in the second polypeptide CH3 domain; 10) E357N and K409D / E in the first polypeptide CH3 domain; and K370T and F405K in in the second polypeptide CH3 domain;Page 365 of 370 307304067v111) E357Q and K409D / E in the first polypeptide CH3 domain; and K370S and F405K in the second polypeptide CH3 domain; 12) E357K and K409D / E in the first polypeptide CH3 domain; and K370E and F405K in the second polypeptide CH3 domain; 13) T366W in the first polypeptide CH3 domain; and T366S, L368A, and Y407V in the second polypeptide CH3 domain; 14) T366S, L368A, and Y407V in the first polypeptide CH3 domain; and T366W in the second polypeptide CH3 domain; 15) L368K and F405K in the first polypeptide CH3 domain; and S364D and K409D in the second polypeptide CH3 domain; or 16) S364D and K409D in the first polypeptide CH3 domain; and L368K and F405K in the second polypeptide CH3 domain.

43. The multispecific molecule of any one of claims 4-22 and 26-42, wherein the immunoglobulin variable region of the first, second, third and / or fourth polypeptide comprises a mutation at position Q38 and / or Q39 (Chothia numbering).

44. The multispecific molecule of claim 43, wherein the mutations at position Q38 and / or Q39 (Chothia numbering) is an amino acid substitution Q38X and / or Q39X.

45. The multispecific molecule of claim 43, wherein the mutations at position Q38 and / or Q39 (Chothia numbering) is an amino acid substitution Q38E and / or Q39K.

46. The multispecific molecule of any one of claims 1-22 and 33-45, wherein the CH3 domains of the first and second polypeptides are from an IgG, IgA, IgD, IgE, or IgM.

47. The multispecific molecule of claim 46, wherein the CH3 domains of the first and second polypeptides are from an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD1, or IgD2.

48. The multispecific molecule of any one of claims 5-47, wherein the CH1 domains of the first and second polypeptides are from an IgG, IgA, IgD, IgE, or IgM.

49. The multispecific molecule of claim 48, wherein the CH1 domains of the first and second polypeptides are from an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD1, or IgD2.Page 366 of 370 307304067v150. The multispecific molecule of any one of claims 3-49, wherein the second and / or fourth polypeptide is a kappa light chain, a lambda light chain, or a fusion of kappa and lambda light chain or a functional fragment thereof.

51. The multispecific molecule of any one of claims 3-50, wherein the CL is a kappa light chain or a lambda light chain.

52. The multispecific molecule of claim 50, wherein the fusion of kappa and lambda light chain comprises a lambda variable region fused onto a kappa constant domain or a kappa variable region fused onto a lambda constant domain.

53. The multispecific molecule of any one of claims 50-52, wherein the P151 mutation is in the CH1 domain and the heterodimerization mutations are in the kappa light chain.

54. The multispecific molecule of any one of claims 50-52, wherein the P151 mutation is in the CH1 domain and the heterodimerization mutations are in the lambda light chain.

55. The multispecific molecule of any one of claims 8-22 and 27-54, wherein the exchanged one or more light chain domains and one or more of the third and fourth polypeptides are kappa light chains.

56. The multispecific molecule of any one of claims 8-22 and 27-54, wherein the exchanged one or more light chain domains and one or more of the third and fourth polypeptides are lambda light chains.

57. The multispecific molecule of any one of claims 9-22 and 27-54, wherein the exchanged one or more immunoglobulin variable regions of light chain domains are lambda light chains and one or more of the third and fourth polypeptides are kappa light chains.

58. The multispecific molecule of any one of claims 9-22 and 27-54, wherein the exchanged one or more immunoglobulin variable regions of light chain domains are kappa light chains and one or more of the third and fourth polypeptides are lambda light chains.

59. The multispecific molecule of any one of claims 1-58, wherein said molecule is a bispecific antibody.Page 367 of 370 307304067v160. An isolated polynucleotide encoding the multispecific molecule of any one of claims 1- 59.

61. A vector comprising the isolated polynucleotide of claim 60.

62. A host cell comprising the vector of claim 61 or one or more recombinant polynucleotides encoding one or more of the multispecific molecule(s) of any one of claims 1- 59.

63. The host cell of claim 62, further comprising an antiapoptotic.

64. A composition, comprising the multispecific molecule of any one of claims 1-59, the isolated polynucleotide of claim 60, the vector of claim 61, or the host cell of claim 62 or claim 63.

65. A method of manufacturing one or more multispecific molecules, comprising: a) incubating the host cell of claim 62 or claim 63 under conditions allowing for expression of said one or more multispecific molecules; and b) recovering said one or more multispecific molecules from the host cell culture.

66. The method of claim 65, further comprising using an antiapoptotic.

67. The method of claim 65 or claim 66, wherein two or more multispecific molecules are manufactured in the host cell.

68. A method of producing one or more multispecific molecules in a cell or tissue in a subject in need thereof, comprising administering to the subject the isolated polynucleotide of claim 60, the vector of claim 61, or the host cell of claim 62 or claim 63.

69. The method of claim 68, further comprising using an antiapoptotic.

70. The method of claim 68 or claim 69, wherein two or more multispecific molecules are produced in the subject.

71. A method of screening a multispecific molecule of any one of claims 1 to 59 using high throughput screening, comprising:Page 368 of 370 307304067v1i) providing the isolated polynucleotide as described herein or the vector as described herein to a culture comprising one or more mammalian cells; ii) expressing the multispecific molecule as described herein; iii) collecting and filtering a supernatant of the culture; iv) purifying the multispecific molecule; and v) screening the multispecific molecule 72. A method of delivering an isolated polynucleotide of claim 60 to a subject in need thereof, comprising: i) encapsulating the isolated polynucleotide in a lipid nanoparticle; and ii) delivering the lipid nanoparticle to the subject.

73. The method of claim 72, wherein the isolated polynucleotide is mRNA.Page 369 of 370 307304067v1