Domain pair

Abstract

The present invention relates to novel CH3 domain pairs.

Description

[0001] Attorney Docket No: 209070-016003 / PCT

[0002] Domain Pair

[0003] Cross-Reference to Related Applications

[0004] This application claims the benefit of priority of U. S. Provisional Application No. 63 / 744,834, filed January 13, 2025, and U. S. Provisional Application No. 63 / 922,976, filed on November 21, 2025, the entire contents of which are each incorporated herein by reference.

[0005] Background

[0006] Means for driving heterodimerization of proteins and protein domains, including CH3 domains, has received considerable attention over recent years. Despite advances, the generation of heterodimeric products remains challenging. Mispairing can lead heterogeneous products, reduced yields, and the need for extensive purification processes to isolate the target product. In the context of CH3 domains, current technologies, such as knob-into-hole (KIH) amino acid substitutions, charge-pair engineering, and domain-swapping techniques, have resulted in advances but face significant limitations in adaptability, scalability, and efficiency.

[0007] In addition to reduced heterogeneity, the stability of protein / protein domain heterodimers is a critical consideration fortheir development as therapeutics and diagnostic tools. Improving stability reduces the risk of the heterodimers losing efficacy and triggering unwanted immune responses, as well as being beneficial for manufacturability and shelf-life. In the context of CH3 domains, limited tools for improving stability are currently available, with most focusing on modifications to the CH3-CH3 domain interface, such as the introduction of KIH amino acid substitutions, as mentioned above.

[0008] Summary

[0009] The present invention relates to novel CH3 domain pairs. These domain pairs comprise two CH3 domains with specific amino acid substitutions that enable dimerization of the CH3 domains and / or modify the interface between the CH3 domain pair and an optional variable domain pair.

[0010] In one aspect, the domain pair comprises two CH3 domains with specific amino acid substitutions that enable dimerization of the CH3 domains. These CH3 domains have been shown to heterodimerize with high fidelity.

[0011] Thus, in a first aspect, the present invention provides a CH3 domain pair, wherein:

[0012] a) in one CH3 domain of the CH3 domain pair:

[0013] 1

[0014] ACTIVE 718034029V1i) amino acid 368 is substituted with valine (V) or isoleucine (I),

[0015] ii) amino acid 394 is substituted with methionine (M), and

[0016] iii) amino acid 407 is substituted with glycine (G) or alanine (A);

[0017] b) in the other CH3 domain of the CH3 domain pair:

[0018] I) amino acid 366 is substituted with valine (V),

[0019] ii) amino acid 397 is substituted with leucine (L),

[0020] iii) amino acid 405 is substituted with alanine (A), and

[0021] iv) amino acid 409 is substituted with tyrosine (Y) or phenylalanine (F); and when amino acid 407 is substituted with glycine (G), then amino acid 409 is substituted with tyrosine (Y), and when amino acid amino acid 407 is substituted with alanine (A), then amino acid 409 is substituted with phenylalanine (F), and

[0022] wherein the amino acids are numbered according to the Eu index.

[0023] In some embodiments, the present invention provides a CH3 domain pair, wherein:

[0024] a) one CH3 domain of the CH3 domain pair comprises substitutions 368V or 3681, 394M, and 407G; and

[0025] b) the other CH3 domain of the CH3 domain pair comprises substitutions 366V, 397L, 405A, and 409Y;

[0026] wherein the amino acids are numbered according to the Eu index.

[0027] In an alternative embodiment, the present invention provides a CH3 domain pair, wherein:

[0028] a) one CH3 domain of the CH3 domain pair comprises substitutions 368V or 3681, 394M, and 407A; and

[0029] b) the other CH3 domain of the CH3 domain pair comprises substitutions 366V, 397L, 405A, and 409F;

[0030] wherein the amino acids are numbered according to the Eu index.

[0031] In some embodiments, the present invention provides a CH3 domain pair, wherein:

[0032] a) one CH3 domain of the CH3 domain pair comprises substitutions L368V or 3681, T394M, and Y407G; and

[0033] b) the other CH3 domain of the CH3 domain pair comprises substitutions T366V, V397L or M397L, F405A, and K409Y or R409Y;

[0034] wherein the amino acids are numbered according to the Eu index.

[0035] In an alternative embodiment, the present invention provides a CH3 domain pair, wherein:

[0036] a) one CH3 domain of the CH3 domain pair comprises substitutions L368V or L368I, T394M, and Y407A; and

[0037] 2

[0038] ACTIVE 718034029V 1b) the other CH3 domain of the CH3 domain pair comprises substitutions T366V, V397L or M397L, F405A, and K409F or R409F;

[0039] wherein the amino acids are numbered according to the Eu index.

[0040] In some embodiments, the present invention provides a CH3 domain pair, wherein:

[0041] a) a CH3 domain of the CH3 domain pair comprises substitutions L368V or L368I, T394M, and Y407G;

[0042] b) the other CH3 domain of the CH3 domain pair comprises substitutions T366V, V397L, F405A, and K409Y; and

[0043] wherein the amino acids are numbered according to the Eu index.

[0044] In an alternative embodiment, the present invention provides a CH3 domain pair, wherein: a) a CH3 domain of the CH3 domain pair comprises substitutions L368V or L368I, T394M, and Y407A;

[0045] b) the other CH3 domain of the CH3 domain pair comprises substitutions T366V, V397L, F405A, and K409F; and

[0046] wherein the amino acids are numbered according to the Eu index.

[0047] In one embodiment, a CH3 domain of the CH3 domain pair of the invention comprises substitution 368V, for example substitution L368V.

[0048] In an alternative embodiment, a CH3 domain of the CH3 domain pair of the invention comprises substitution 3681, for example substitution L368I.

[0049] In some embodiments, the CH3 domains further comprise amino acid substitutions that generate a disulfide bridge between the CH3 domains. The substitutions are preferably engineered cysteine residues. Suitable engineered cysteines suitable for generating a disulfide bridge between two CH3 domains are known in the art. For example, one CH3 domain of the CH3 domain pair may further comprise substitution S354C, and the other CH3 domain of the CH3 domain pair may further comprise substitution Y349C. In some embodiments, one CH3 domain of the CH3 domain pair further comprises substitution K392C or N392C, preferably K392C, and the other CH3 domain of the CH3 domain pair may further comprises substitution D399C.

[0050] Thus, in some embodiments, the present invention provides a CH3 domain pair, wherein: a) a CH3 domain of the CH3 domain pair comprises substitutions L368V or L368I, T394M, D399C, and Y407G; and

[0051] 3

[0052] ACTIVE 718034029V 1b) the other CH3 domain of the CH3 domain pair comprises substitutions T366V, K392C, V397L, F405A, and K409Y;

[0053] wherein the amino acids are numbered according to the Eu index.

[0054] An lgG1 CH3 domain comprising substitutions L368V, T394M, D399C, and Y407G is also referred to as “CH311 ”, or “t1 ”, herein, while an IgG 1 CH3 domain comprising substitutions T366V, K392C, V397L, F405A, and K409Y is referred to as “CH3 t2”, or “t2”, herein. A CH3 domain pair comprising domains CH311 and CH312, is also referred to as a “CH311 -t2 domain pair”, “CH3 t1-t2” or “t1 -t2” herein.

[0055] In an alternative embodiment, the present invention provides a CH3 domain pair, wherein: a) a CH3 domain of the CH3 domain pair comprises substitutions L368V or L368I, T394M, D399C, and Y407A;

[0056] b) the other CH3 domain of the CH3 domain pair comprises substitutions T366V, K392C, V397L, F405A, and K409F; and

[0057] wherein the amino acids are numbered according to the Eu index.

[0058] In some embodiments, the CH3 domain pair comprises a specific combination of CH3 domains. In some embodiments, one CHS domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 1 and the other CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 2. In some embodiments, one CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 3 and the other CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 4. In some embodiments, one CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 5, and the other CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 6. In some embodiments, one CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 7 and the other CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 8. In some embodiments, one CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 9 and the other CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 10. In some embodiments, one CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 11 and the other CH3 domain of the CH3 domain pair of the invention has the sequence set forth in SEQ ID NO: 12.

[0059] In some embodiments, a CH3 domain pair of the invention is suitable for use as a heterodimerization domain. For example, one or more moieties may be attached to a CH3 domain pair of the invention. Attachment of one or more moieties to a CH3 domain pair of the 4

[0060] ACTIVE 718034029V 1invention can result in a complex, e.g. a polypeptide complex. Exemplary complexes are shown schematically in FIG. 1E. In some embodiments, the CH3 domain pair of the invention may function to bring together two desired moieties, whereby each moiety may be attached to one of the CH3 domains of the CH3 domain pair of the invention. Heterodimerization between the CH3 domains of a CH3 domain pair of the invention then may bring the two desired moieties into proximity with one another. Exemplary moieties that may be attached to a CH3 domain pair of the invention include additional binding or recognition moieties, such as antigen-binding domains, antibody fragments, and alternative binding domains or scaffolds, including VHHs, nanobodies, scFvs, Fabs, single-domain antibodies (sdAbs), anticalins, affibodies, adnectins, and other non-lg-fold binders or compact protein scaffolds; immune-modulating moieties, including cytokines, growth factors, chemokines, and checkpoint-pathway ligands or decoys; non-immune-modulating moieties, including functional proteins such as enzymes, receptor ectodomains, targeted toxins, and other catalytic or binding proteins; small-molecule moieties, including therapeutic drugs and payloads; immune-stimulatory moieties; radiochemistry and imaging moieties, including diagnostic or therapeutic radionuclides, fluorophores, and other imaging tags; nucleic-acid moieties, including oligonucleotides; targeted protein degradation moieties; and pharmacokinetic or biophysical modifiers and other auxiliary moieties, such as PEGylation and related polymers, albumin-binding or FcRn-tuning half-life extenders, cell¬ penetrating or endosomal-escape peptides, masking or unmasking peptides, and other moieties that modify solubility, stability, biodistribution, or systemic exposure.

[0061] A moiety may be attached to a CH3 domain of a CH3 domain pair of the invention at the N-terminus or the C-terminus. Where one, two or more moieties are attached to a CH3 domain pair of the invention, the moieties may be attached to the N-terminus and / or the C-terminus of the CH3 domains (see, e.g., FIG. 1E). Where more than one antigen-binding moiety is attached to one or more of the CH3 domains of a CH3 domain pair of the invention, the resulting polypeptide complex may be multivalent and / or multispecific.

[0062] A moiety may be attached to a CH3 domain of the CH3 domain pair of the invention either directly, for example through any suitable chemical bond or through a linker, such as an amino acid linker.

[0063] A CH3 domain pair of the invention may be included in an antibody as described herein to drive heterodimerization between two chains of the antibody, such as two different heavy chains, or a heavy chain and a light chain. For example, the CH3 domains of a CH3 domain pair of the invention may be incorporated into the antibody chains at the appropriate position to drive the desired chain heterodimerization.

[0064] 5

[0065] ACTIVE 718034029V 1In a second aspect, the invention relates to a CH3 domain pair containing amino acid substitutions that alter the face of the CH3 domain pair that, when the CH3 domain pair is present in an antibody, is oriented toward the domain pair N-terminal to it in the antibody structure. These amino acid substitutions are particularly suited for use in antibodies where the domain pair N-terminal to the CH3 domain pair is a variable domain pair. Such amino acid substitutions can optimize the developability of a heterodimeric protein, such as an antibody, to accommodate a variety of variable domains. Moreover, such amino acid substitutions combine multiple interface modification strategies. In addition, to providing advantages where the domain pair N-terminal to the CH3 domain pair is a variable domain pair, it is to be understood that the CH3 domain pairs described below may also be employed as heterodimerization domains wherein one or more moieties are attached to a CH3 domain pair of the invention, as described above.

[0066] Accordingly, in some embodiments, the invention provides a CH3 domain pair, wherein: a) the one CHS domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of: (i) 341A; (ii) 373F; (iii) 398Q; and (iv) 404Y; and / or b) the other CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of: (i) 341A; (ii) 373F; and (iii) 404Y, wherein the positions are numbered according to the Eu index. Such amino acid substitutions can be combined with other amino acid substitutions described herein or CH3 substitutions that are known in the art, in particular amino acid substitutions that drive dimerization of CH3 domains.

[0067] In another embodiment, the invention provides a CHS domain pair, wherein: the one CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of: (i) G341A; (ii) Y373F; (iii) L398Q; and (iv) F404Y; and / or b) the other CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of: (i) G341A; (ii) Y373F; and (iii) F404Y.

[0068] In another embodiment, the present invention provides a CH3 domain pair, wherein:

[0069] a) the first CH3 domain of the CH3 domain pair comprises:

[0070] (i) amino acid substitutions L398Q and F404Y; and optionally

[0071] (ii) amino acid substitution Y373F; and / or

[0072] b) the second CH3 domain of the CH3 domain pair comprises:

[0073] (i) amino acid substitution G341A; and / or

[0074] (ii) amino acid substitutions Y373F and F404Y.

[0075] In another embodiments, the present invention provides a CH3 domain pair, wherein:

[0076] a) the first CH3 domain of the CH3 domain pair comprises:

[0077] 6

[0078] ACTIVE 718034029V 1(i) amino acid substitutions L398Q and F404Y; and optionaliy

[0079] (ii) amino acid substitution Y373F; and

[0080] b) the second CH3 domain of the CH3 domain pair comprises:

[0081] (i) amino acid substitutions Y373F and F404Y; and optionally

[0082] (ii) amino acid substitution G341A.

[0083] In another embodiment, the present invention provides a CH3 domain pair, wherein:

[0084] a) the first CH3 domain of the CH3 domain pair comprises:

[0085] (i) amino acid substitutions L398Q and F404Y; and optionally

[0086] (ii) amino acid substitution Y373F; and

[0087] b) the second CH3 domain of the CH3 domain pair comprises:

[0088] (i) amino acid substitution G341A; and

[0089] (ii) amino acid substitutions Y373F and F404Y.

[0090] In some embodiments, certain amino acid substitutions, when combined together, modify the face of the CH3 domain pair, resulting in certain shared properties. Such amino acid substitution combinations can manifest in measurable properties of a heterodimeric protein, e.g. an antibody, into which the CH3 domain pair has been incorporated, such as an increase in stability, reflected in an increase in Tm, as compared to the heterodimeric protein without the combination of amino acid substitutions.

[0091] Accordingly, in some embodiments, the disclosure provides a CH3 domain pair, wherein:

[0092] (A)

[0093] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;

[0094] (B)

[0095] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L398Q and F404Y; and

[0096] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F and F404Y;

[0097] (C)

[0098] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and

[0099] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;

[0100] (D)

[0101] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y;

[0102] 7

[0103] ACTIVE 718034029V 1(E)

[0104] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and

[0105] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitution G341A; or

[0106] (F)

[0107] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and

[0108] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y.

[0109] in another embodiment, the present invention provides a CH3 domain pair, wherein:

[0110] (A)

[0111] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F and F404Y;

[0112] (B)

[0113] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;

[0114] (C)

[0115] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L398Q and F404Y; and

[0116] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;

[0117] (D)

[0118] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L398Q and F404Y; and

[0119] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F and F404Y; OR

[0120] (F)

[0121] (a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution Y373F, L398Q and F404Y; and

[0122] (b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

[0123] In some embodiments, the present invention provides a CH3 domain pair, wherein:

[0124] a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L398Q and F404Y; and

[0125] 8

[0126] ACTIVE 718034029V 1b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

[0127] in some embodiments, the present invention provides a CH3 domain pair, wherein:

[0128] a) the first CHS domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and

[0129] b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

[0130] In some embodiments, a CH3 domain pair described herein includes only one CHS domain of the pair being engineered to include one or more amino acid substitutions that modify the variable domain interface, while the other CH3 domain remains unmodified or retains the wild¬ type sequence with respect to the interface substitutions or only includes amino acid substitutions that enable heterodimerization of CH3 domains. For example, a first CH3 domain of the pair may include substitutions at one or more positions, such as 341, 373, 398, and / or 404, to modify the CH3 interface with a variable domain, while the second CH3 domain of the pair is a wild-type CH3 domain or includes substitutions at one or more positions of 366, 368, 394, 397, 405, 407, and / or 409 that enable heterodimerization of CH3 domains. This approach allows for the introduction of desired characteristics into a heterodimeric protein or protein complex with minimal alteration to the overall structure, and may be advantageous in certain contexts where preservation of certain features is desired on one chain.

[0131] Accordingly, in some embodiments, the interface substitutions described above can be used either alone or in combination with amino acid substitutions that enable heterodimerization of CH3 domains. Such amino acid substitutions include the amino acid substitutions of the invention as set out above and elsewhere herein. Alternatively, the interface amino acid substitutions may be combined with known amino acid substitutions that enable heterodimerization of CH3 domains as described below.

[0132] The interface substitutions described herein may be present on one, or both CH3 domains of the CH3 domain pair. For example, the interface substitutions may be present only in the second CH3 domain.

[0133] Thus, in one embodiment, the present invention provides a CH3 domain pair, wherein:

[0134] (i) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 1, and the second CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 43, 44, 45, 46, 47, 48, 49, 50, 51, 67, 68, 69, 70, 71, 72, 73, 74, and 75;

[0135] 9

[0136] ACTIVE 718034029V 1(ii) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 3, and the second CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 43, 44, 45, 46, 47, 48, 49, 50, 51, 67, 68, 69, 70, 71, 72, 73, 74, and 75;

[0137] (iii) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 5, and the second CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 43, 44, 45, 46, 47, 48, 49, 50, 51, 67, 68, 69, 70, 71, 72, 73, 74, and 75;

[0138] (iv) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 7, and the second CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 43, 44, 45, 46, 47, 48, 49, 50, 51, 67, 68, 69, 70, 71, 72, 73, 74, and 75;

[0139] the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 9, and the second CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 43, 44, 45, 46, 47, 48, 49, 50, 51, 67, 68, 69, 70, 71, 72, 73, 74, and 75; or the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 11, and the second CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 43, 44, 45, 46, 47, 48, 49, 50, 51, 67, 68, 69, 70, 71, 72, 73, 74, and 75.

[0140] Alternatively, the interface substitutions may be present only in the first CH3 domain.

[0141] Thus, in one embodiment, the present invention provides a CH3 domain pair, wherein:

[0142] (i) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 2;

[0143] (ii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 4;

[0144] (iii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 6;

[0145] (iv) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 8;

[0146] (v) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 10; or

[0147] (vi) the first CHS domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 12.

[0148] 10

[0149] ACTIVE 718034029V 1Alternatively, the interface substitutions may be present in both CH3 domains.

[0150] Thus, in one embodiment, the present invention provides a CH3 domain pair, wherein:

[0151] (i) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 31, 34, 37, and 40, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43;

[0152] (ii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 31, 34, 37, and 40, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 46;

[0153] (iii) the first CH3 domain of the CHS domain pair has a sequence selected from any one of SEQ ID NOS: 28, 31, 34, 37, and 40, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 49;

[0154] (iv) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 44;

[0155] (v) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 47;

[0156] (vi) the first CHS domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 50;

[0157] (vii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 45;

[0158] (viii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 48;

[0159] (ix) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 51;

[0160] (x) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 67;

[0161] (xi) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 70;

[0162] (xii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the second CH3 domain of the CH3 domain pair 11

[0163] ACTIVE 718034029V 1has a sequence set forth in SEQ ID NO: 73;

[0164] (xiii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 68;

[0165] (xiv) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 71;

[0166] (xv) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 74;

[0167] (xvi) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 69;

[0168] (xvii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 72; or

[0169] (xviii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 75.

[0170] In a preferred embodiment, the present invention provides a CH3 domain pair, wherein the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 28, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43.

[0171] In another preferred embodiment, the present invention provides a CH3 domain pair, wherein the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 31, and the second CHS domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43.

[0172] In a further preferred embodiment, the present invention provides a CH3 domain pair, wherein the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 34, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43.

[0173] As mentioned, above, the interface amino acid substitutions described herein may alternatively be combined with known amino acid substitutions that enable heterodimerization of domains, including CH3 domains. Known amino acid substitutions that enable heterodimerization of domains, include knob-into-hole (KIH) amino acid substitutions.

[0174] 12

[0175] ACTIVE 718034029V 1Thus, the CH3 domain pair of the invention may further comprise knob-into-hole (KIH) amino acid substitutions. In one embodiment, one CH3 domain of the CH3 domain pair may further comprise a knob amino acid substitution and the other CH3 domain of the second CH3 domain pair may further comprise a hole amino acid substitution.

[0176] Thus, in one embodiment, the interface amino acid substitutions described herein may be made in a first CH3 domain and second CH3 domain, wherein said first and second CH3 domain have the sequences set forth in SEQ ID NOS: 19 and 20, respectively.

[0177] Alternative the interface amino acid substitutions described herein may be combined with known amino acid substitutions that enable heterodimerization of CHS domains. Other known amino acid substitutions that enable heterodimerization of domains, include those listed below.

[0178] Thus, alternatively, the CH3 domain pair of the invention may comprise one CH3 domain of the CH3 domain pair which further comprises amino acid substitution Y349C and optionally amino acid substitution K370R; and the other CH3 domain of the CH3 domain pair further comprises amino acid substitutions S354C and E357W.

[0179] In another embodiment, the CHS domain pair of the invention may comprise a first CH3 domain of the CH3 domain pair which further comprises amino acid substitution Y349C and optionally amino acid substitution K370R; and a second CH3 domain of the CH3 domain pair which further comprises amino acid substitutions S354C and E357W.

[0180] In a further embodiment, the CH3 domain pair of the invention may comprise a first CH3 domain of the CH3 domain pair which further comprises amino acid substitution Y349C and K370R; and a second CH3 domain of the CH3 domain pair which further comprises amino acid substitutions S354C and E357W.

[0181] In one embodiment, the interface amino acid substitutions described herein may be made in a first CH3 domain and second CH3 domain, wherein said first and second CH3 domain have the sequences set forth in SEQ ID NOS: 13 and 14, respectively.

[0182] In a second aspect, the present invention provides a heterodimeric protein, or polypeptide complex, comprising:

[0183] a) a first polypeptide chain comprising a CH3 domain of a CH3 domain pair of the invention; and

[0184] b) a second polypeptide chain comprising the other CH3 domain of the CH3 domain pair of the invention.

[0185] 13

[0186] ACTIVE 718034029V 1The first and second polypeptide chains of the heterodimeric protein may further each comprise a CH2 domain.

[0187] Thus, in one embodiment, the heterodimeric protein, or polypeptide complex, comprises:

[0188] a) a first polypeptide chain comprising, in order, from the N-terminus to the C-terminus, a CH2 domain, and a CH3 domain of a CH3 domain pair of the invention; and

[0189] b) a second polypeptide chain comprising, in order, from the N-terminus to the C-terminus, a CH2 domain, and the other CH3 domain of the CH3 domain pair of the invention.

[0190] in some embodiments, provided herein is a complex comprising a CH3 domain pair according to the invention, wherein at least one moiety is attached to at least one CH3 domain of the CH3 domain pair. Such at least one moiety can be attached to the N-terminus or the C-terminus of such at least one CH3 domain. In some embodiments, at least two moieties are attached to the CH3 domain pair. In some embodiments, when such at least two moieties are attached, the moieties are attached to the N- and / or C-terminus of the CH3 domains of the CH3 domain pair.

[0191] In some embodiments, the at least one moiety is an antigen-binding moiety, enzyme, cytokine, or receptor subunit. In some embodiments, the antigen-binding moiety is an Fv fragment, single-chain Fv, singie-chain antibody, diabody, single-chain diabody, single-domain antibody, VHH, nanobody, oranticalin.

[0192] In addition, or alternatively, the first and second polypeptide chains of the heterodimeric protein, or polypeptide complex, may further each comprise a variable domain.

[0193] Thus, in another embodiment, the heterodimeric protein, or polypeptide complex, comprises two polypeptide chains, wherein:

[0194] (a) the first polypeptide chain comprises, in order, from the N-terminus to the C-terminus, a variable domain of a variable domain pair, and a CH3 domain of a CH3 domain pair of the invention; and

[0195] (b) the second polypeptide chain comprises, in order, from the N-terminus to the C-terminus, the second variable domain of the variable domain pair, and the second CH3 domain of the CH3 domain pair of the invention;

[0196] wherein the variable domain pair consists of a VH domain and a VL domain.

[0197] in a preferred embodiment, where a CH3 domain of the CH3 domain pair in a heterodimeric protein, or polypeptide complex, comprises substitution 398Q, the variable domain immediately N-terminal to said CH3 domain in the polypeptide chain is a VL domain.

[0198] 14

[0199] ACTIVE 718034029V 1In another preferred embodiment, where a CH3 domain of the CH3 domain pair in a heterodimeric protein, or polypeptide complex, comprises substitution 341A, the variable domain immediately N-terminal to said CH3 domain in the polypeptide chain is a VH domain.

[0200] Constant domains, as referred to herein, may be lgG1, lgG2, lgG3, or lgG4 domains, but in some embodiments, are lgG1, lgG2 or lgG4 domains, or in some embodiments are lgG1 domains.

[0201] Thus, CH3 domains, as referred to herein, may be lgG1, lgG2, lgG3, or lgG4 CH3 domains, but in some embodiments are lgG1, lgG2, or lgG4 CH3 domains, or in some embodiments are lgG1 CH3 domains.

[0202] Similarly, CH2 domains, as referred to herein, may be lgG1, lgG2, lgG3, or lgG4 CH2 domains, but in some embodiments are lgG1, lgG2, or lgG4 CH2 domains, or in some embodiments lgG1 CH2 domains.

[0203] In some embodiments, the CH2 domain has the sequence set forth in SEQ ID NO: 21 or 22.

[0204] CH3 domain pairs of the invention may be paired with lambda-germline and kappa-germline variable domains.

[0205] In some embodiments, a protein complex provided herein comprising a CH3 domain pair according to the invention, comprises at least one moiety is attached to at least one CH3 domain of the CH3 domain pair or at least one variable domain of the variable domain pair. Such at least one moiety can be attached to the N-terminus or the C-terminus of such at least one CH3 domain or variable domain. In some embodiments, at least two moieties are attached to the CH3 domain pair or variable domain. In some embodiments, when such at least two moieties are attached, the moieties are attached to the N- and / or C-terminus of the CH3 domains of the CH3 domain pair or variable domain pair.

[0206] In some embodiments, the at least one moiety is an antigen-binding moiety, enzyme, cytokine, or receptor subunit. In some embodiments, the antigen-binding moiety is an Fv fragment, single-chain Fv, single-chain antibody, diabody, single-chain diabody, single-domain antibody, VHH, nanobody,, or anticalin.

[0207] In a further aspect, the present invention provides a nucleic acid encoding a CH3 domain pair or heterodimeric protein of the invention. Also provided are vectors comprising a nucleic acid 15

[0208] ACTIVE 718034029V 1encoding a CH3 domain pair or heterodimeric protein of the invention. The present invention further provides host cells, such as mammalian host cells, comprising a nucleic acid or vector of the invention.

[0209] In another aspect, the present invention provides a method of producing the CH3 domain pair or heterodimeric protein of the invention, comprising culturing a host cell comprising a nucleic acid or vector encoding the CH3 domain pair or heterodimeric protein under condition for production of the CH3 domain pair or heterodimeric protein. The method may further comprise a step of purifying the CH3 domain pair or heterodimeric protein.

[0210] The invention includes the combination of the aspects and various features described herein except where such a combination is clearly impermissible or expressly avoided.

[0211] Brief Description of the Drawings

[0212] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0213] FIG. 1 is a graphical 3D representation of a wild-type (wt) CH3 domain pair, illustrating the CH3 domains. The left-hand side is expanded on the right-hand side to depict the interface between the CH3 domains.

[0214] FIG. 1B is a model illustrating the same region shown in FIG. 1A for a CH311- 12 domain pair. Amino acids that differ between the wt CH3 domains and the CH311 — 12 domain pair are shown in stick representation in FIG. 1A and FIG. 1B, and are labelled in FIG. 1B.

[0215] FIG. 1C is a model illustrating the same region shown in FIG. 1A for exemplary substitutions in a CH3 domain pair.

[0216] FIG. 1D is a model illustrating the same region shown in FIG. 1A for exemplary substitutions in a CH3 domain pair.

[0217] FIG. 1E is a schematic depicting a CH3 domain pair with one or more moieties attached, for example, one to four additional moieties (e.g., R-i, R2, R3, and R4) attached to the CH3 domains at the N- and / or C-terminus.

[0218] FIG. 2A is a schematic depicting relevant portions of potential products that can form in the experimental approach used to validate heterodimeric CH3 domain variants. Variants which

[0219] 16

[0220] ACTIVE 718034029V 1strongly favor heterodimerization yield only the heterodimeric product. Weaker heterodimer designs yield off-target homodimers (homodimers 1 and 2) in addition to the desired heterodimer.

[0221] FIG. 2B is a capillary electrophoresis (CE) gel image after Protein A (ProA) purification for molecules comprising wt CH3-CH3, CH3 b1— b2, CH3 t1.1 core-t2.1 core, CH3 t1.1-t2.1, or CH3 t1.1 core, K392C-t2.1 core, D399C domain pairs in the Fc region, as described in FIG. 2A.

[0222] FIG. 2C is a CE gel image after ProA purification for molecules comprising wt CH3, CH3 b1 — b2, CH311— 12, or CHS t1.1-t2.1 domain pairs in the Fc region, as described in FIG. 2A.

[0223] FIG. 3A is a graphical 3D representation of a CH3 Fab domain and an expanded view illustrating the interface between CH3 and variable domains with labelled residue positions of exemplary CH3 domain pairs.

[0224] FIG. 3B is a model highlighting specific amino acid substitutions in FIG. 3A for exemplary CH3 domain pairs.

[0225] FIG. 30 shows a model highlighting specific amino acid substitutions for exemplary CHS domain pairs.

[0226] FIG. 3D shows a model highlighting specific amino acid substitutions in FIG. 30A with those from FIG. 3C for exemplary CH3 domain pairs.

[0227] Detailed Description

[0228] The present invention relates to novel CH3 domain pairs. These domain pairs comprise two CH3 domains with specific amino acid substitutions that enable dimerization of the CH3 domains and / or modify the interface between the CH3 domain pair and an optional variable domain pair.

[0229] The novel CH3 domain pairs described herein are particularly suited for inclusion in polypeptide complexes having more than one polypeptide chain to promote high-fidelity chain heterodimerization and ensure the correct assembly of desired polypeptide complexes.

[0230] Accordingly, provided herein are polypeptide complexes having the CH3 domain pairs described herein. Such polypeptide complexes can be in a variety of different formats, wherein the CH3 domain pairs described herein are located in different positions within the polypeptide

[0231] 17

[0232] ACTIVE 718034029V 1complex, depending upon the desired number of complexes that need to be formed, such as those formed in polypeptide complexes having multiple separate polypeptides.

[0233] In addition, the CH3 domain pairs described herein are suitable for the preparation of complexes, such as polypeptide complexes, wherein one or more moieties are attached to the CH3 domain pair. Heterodimerization between the CH3 domains of the CH3 domain pair may, for example, be used to bring two desired moieties attached to the CH3 domains of the CH3 domain pair into proximity with one another. The moieties may be antigen-binding moieties, but other types of moieties, such as enzymes, cytokines and receptor subunits are also contemplated.

[0234] CH3 Domain Amino Acid Substitutions

[0235] The present invention provides a CH3 domain pair, wherein the CH3 domains of the pair comprise different amino acid substitutions which drive heterodimerization between said CH3 domains.

[0236] The present invention, in its first aspect, introduces a CH3 domain pair characterized by specific amino acid substitutions. In one CH3 domain of the pair, amino acid 368 is replaced with valine (V) or isoleucine (I), amino acid 394 with methionine (M), and amino acid 407 with either glycine (G) or alanine (A). In the corresponding CH3 domain, amino acid 366 is substituted with valine (V), amino acid 397 with leucine (L), amino acid 405 with alanine (A), and amino acid 409 with either tyrosine (Y) or phenylalanine (F). Notably, when amino acid 407 is glycine (G), amino acid 409 must be tyrosine (Y), and when amino acid 407 is alanine (A), amino acid 409 must be phenylalanine (F). The numbering of amino acids follows the Eu index.

[0237] In some embodiments, the disclosure provides a CH3 domain pair wherein one CH3 domain of the CH3 domain pair comprises amino acid substitution 368V. In some embodiments, the disclosure provides a CH3 domain pair wherein one CH3 domain of the CH3 domain pair comprises amino acid substitution 3681.

[0238] In some embodiments, the invention provides a CH3 domain pair where one domain includes amino acid substitutions 368V or 3681, 394M, and 407G, while the other domain includes 366V, 397L, 405A, and 409Y. Alternatively, the invention may feature a CH3 domain pair with amino acid substitutions 368V or 3681, 394M, and 407A in one domain, and 366V, 397L, 405A, and 409F in the other. These configurations also adhere to the Eu index for amino acid numbering.

[0239] 18

[0240] ACTIVE 718034029V 1Further embodiments describe a CH3 domain pair with one domain having amino acid substitutions L368V or L368I, T394M, and Y407G, and the other domain having T366V, V397L or M397L, F405A, and K409Y or R409Y. Alternatively, the pair may consist of one domain with L368V or L368I, T394M, and Y407A, and the other with T366V, V397L or M397L, F405A, and K409F or R409F. These amino acid substitutions are also numbered according to the Eu index.

[0241] In some embodiments, the disclosure provides a CH3 domain pair wherein one CH3 domain of the CH3 domain pair comprises amino acid substitution L368V. In some embodiments, the disclosure provides a CH3 domain pair wherein one CH3 domain of the CH3 domain pair comprises amino acid substitution L368I.

[0242] In some embodiments, the CH3 domains are engineered to include amino acid substitutions that facilitate the formation of a disulfide bridge between the domains. For example, one CH3 domain may include an amino acid substitution S354C, while the other may include Y349C. As another example, one CH3 domain may include an amino acid substitution D399C, while the other may include K392C. As another example, in some embodiments, one CH3 domain of the CH3 domain pair disclosed herein further comprises amino acid substitution D399C, and the other CH3 domain of the CH3 domain pair further comprises amino acid substitution K392C or N392C, or one CH3 domain of the CH3 domain pair further disclosed herein comprises amino acid substitution K392C or N392C, and the other CH3 domain of the CH3 domain pair further comprises amino acid substitution D399C.

[0243] Thus, in certain embodiments, the invention provides a CH3 domain pair where one domain comprises amino acid substitutions L368V or L368I, T394M, D399C, and Y407G, and the other comprises T366V, K392C, V397L, F405A, and K409Y. These domains are referred to as " CH3 t1" or "t1" for the first domain, and " CH3 t2" or "t2" for the second domain. A pair comprising these domains is termed a " CH311 -t2 domain pair" or simply "t1 -t2."

[0244] In an alternative embodiment, the invention provides a CH3 domain pair where one domain comprises amino acid substitutions L368I, T394M, D399C, and Y407G, and the other comprises T366V, K392C, V397L, F405A, and K409Y.

[0245] In an alternative embodiment, the invention provides a CH3 domain pair where one domain includes amino acid substitutions L368V or L368I, T394M, D399C, and Y407A, and the other includes T366V, K392C, V397L, F405A, and K409F. Again, the amino acids are numbered according to the EU index.

[0246] 19

[0247] ACTIVE 718034029V 1In another alternative embodiment, the invention provides a CH3 domain pair comprising amino acid substitutions that are capable of modifying the interface between the CH3 domain pair and an optional variable domain pair. Such amino acid substitutions can optimize the developability of a heterodimeric protein, such as an antibody, that can accommodate a variety of variable domains. Moreover, such amino acid substitutions combine multiple interface modification strategies. Accordingly, in some embodiments, the disclosure provides a CH3 domain pair, wherein: a) the one CH3 domain of the CH3 domain pair further comprises one or more amino acid substitutions selected from the group consisting of: (i) 341A; (ii) 373F; (iii) 398Q; and (iv) 404Y; and / or b) the other CH3 domain of the CH3 domain pair further comprises one or more amino acid substitutions selected from the group consisting of: (i) 341A; (ii) 373F; and (iii) 404Y. Such amino acid substitutions can be combined with other amino acid substitutions described herein or CH3 substitutions that known in the art.

[0248] In some embodiments, the disclosure provides a CH3 domain pair, wherein: a) one CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of: (i) G341A; (ii) Y373F; (iii) L398Q; and (iv) F404Y; and / or b) the other CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of: (i) G341A; (ii) Y373F; and (iii) F404Y.

[0249] In some embodiments, the disclosure provides a CH3 domain pair, wherein:

[0250] a) one CH3 domain of the CH3 domain pair comprises:

[0251] (i) amino acid substitutions L398Q and F404Y; and optionally

[0252] (ii) amino acid substitution Y373F; and / or

[0253] b) the other CH3 domain of the CH3 domain pair comprises:

[0254] (i) amino acid substitution G341A; and / or

[0255] (ii) amino acid substitutions Y373F and F404Y.

[0256] In some embodiments, the disclosure provides a CH3 domain pair, wherein:

[0257] a) one CH3 domain of the CH3 domain pair comprises:

[0258] (i) amino acid substitutions L398Q and F404Y; and optionally

[0259] (ii) amino acid substitution Y373F; and

[0260] b) the other CH3 domain of the CH3 domain pair comprises:

[0261] (i) amino acid substitutions Y373F and F404Y; and optionally

[0262] (ii) amino acid substitution G341A.

[0263] In some embodiments, the disclosure provides a CH3 domain pair, wherein:

[0264] a) one CH3 domain of the CH3 domain pair comprises:

[0265] (i) amino acid substitutions L398Q and F404Y; and optionally

[0266] 20

[0267] ACTIVE 718034029V 1(ii) amino acid substitution Y373F; and

[0268] b) the other CH3 domain of the CH3 domain pair comprises:

[0269] (i) amino acid substitution G341A; and

[0270] (ii) amino acid substitutions Y373F and F404Y.

[0271] In some embodiments, certain amino acid substitutions, when combined together, are capable of modifying the interface between a variable domain pair and a CH3 domain pair, which result in certain shared properties. Such amino acid substitution combinations can manifest in measurable properties of a heterodimeric protein, such as an antibody, including exhibiting increases in Tm (e.g., a 2-3°C increase in Tm or a >3°C increase in Tm) as compared to the heterodimeric protein not having the amino acid substitution combination as described in Example 3.

[0272] Accordingly, in some embodiments, the disclosure provides a CH3 domain pair, when placed in an antibody results in the antibody exhibiting a 2-3°C increase in Tm, wherein:

[0273] (A)

[0274] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitution L368V; and

[0275] (b) the other CHS domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

[0276] (B)

[0277] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, L398Q and F404Y; and

[0278] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y;

[0279] (C)

[0280] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, Y373F, L398Q and F404Y; and

[0281] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;

[0282] (D)

[0283] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, Y373F, L398Q and F404Y;

[0284] (E)

[0285] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, Y373F, L398Q and F404Y; and

[0286] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitution G341A; or

[0287] 21

[0288] ACTIVE 718034029V 1(F)

[0289] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368I, Y373F, L398Q and F404Y; and

[0290] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y.

[0291] In some embodiments, the disclosure provides a CH3 domain pair, when placed in an antibody results in the antibody exhibiting a >3°C increase in Tm, wherein:

[0292] (A)

[0293] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitution L368I; and

[0294] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y;

[0295] (B)

[0296] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitution L368I; and

[0297] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;

[0298] (C)

[0299] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitution L368V, L398Q and F404Y; and

[0300] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;

[0301] (D)

[0302] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitution L368I, L398Q and F404Y; and

[0303] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y;

[0304] (E)

[0305] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitution L368I, L398Q and F404Y; and

[0306] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y; or

[0307] (F)

[0308] (a) the one CH3 domain of the CH3 domain pair comprises amino acid substitution L368I, Y373F, L398Q and F404Y; and

[0309] (b) the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

[0310] 22

[0311] ACTIVE 718034029V 1In some embodiments, the disclosure provides a CH3 domain pair, wherein: one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368I, T394M, L398Q, D399C, F404Y, and Y407G; and the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, T366V, Y373F, K392C, V397L, F404Y, F405A, and K409Y.

[0312] In some embodiments, the disclosure provides a CH3 domain pair, wherein: one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, T394M, L398Q, D399C, F404Y, and Y407G; and the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, T366V, Y373F, K392C, V397L, F404Y, F405A, and K409Y.

[0313] In some embodiments, the disclosure provides a CH3 domain pair, wherein: one CH3 domain of the CH3 domain pair comprises amino acid substitutions L368I, Y373F, T394M, L398Q, D399C, F404Y, and Y407G; and the other CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, T366V, Y373F, K392C, V397L, F404Y, F405A, and K409Y.

[0314] In some embodiments, the disclosure provides a CH3 domain pair, wherein:

[0315] c) a first CH3 domain of the CH3 domain pair comprises amino acid substitutions L398Q and F404Y; and

[0316] d) a second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

[0317] In some embodiments, the disclosure provides a CH3 domain pair, wherein:

[0318] c) a first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and

[0319] d) a second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

[0320] In some embodiments, a CH3 domain as described herein may comprise no other amino acid substitutions than those described herein. In some embodiments, a CH3 domain as described herein may comprise amino acid substitutions in addition to those described herein. For example, the CH3 domain pair may further comprise one or more substitutions that drive heterodimerization between the CH3 domains of the CH3 domain pair. As another example, in some embodiments, the CH3 domain pair further comprises knob-into-hole (KIH) amino acid substitutions. In some embodiments, one CH3 domain of the CH3 domain pair further comprises amino acid substitution Y349C and optionally amino acid substitution K370R; and the other CH3 domain of the CH3 domain pair further comprises amino acid substitutions S354C and E357W. In some embodiments, one CH3 domain of the CH3 domain pair further comprises amino acid substitution Y349C and optionally amino acid substitution K370R; and the other CH3

[0321] 23

[0322] ACTIVE 718034029V 1domain of the CH3 domain pair further comprises amino acid substitutions S354C and E357W. In some embodiments, one CH3 domain of the CH3 domain pair further comprises amino acid substitution Y349C and K370R; and other CH3 domain of the CH3 domain pair further comprises amino acid substitutions S354C and E357W.

[0323] Antibody constant region residue numbering is according to the Eu index. The Eu index is described at www. imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber. html#refs, which is hereby incorporated by reference in its entirety, and identifies the residue according to its location in an endogenous constant region sequence regardless of the residue’s physical location within a chain of a polypeptide described herein or complexes thereof.

[0324] The CH3 domains described above can be lgG1, lgG2, lgG3, or lgG4 CH3 domains, with some embodiments specifically using lgG1, lgG2, or lgG4 CH3 domains, or lgG1 CH3 domains. Exemplary amino acid sequences of lgG1, lgG2, lgG3, or lgG4 CH3 domains are provided in SEQ ID NOs: 15-18.

[0325] In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in SEQ ID NO: 1, and the other CH3 domain the sequence set forth in SEQ ID NO: 2. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in SEQ ID NO: 3, and the other CH3 domain the sequence set forth in SEQ ID NO: 4. In some embodiments of the CH3 domain pairs described herein, one CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 5, and the other CH3 domain has the sequence set forth in SEQ ID NO: 6. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in SEQ ID NO: 7, and the other CH3 domain the sequence set forth in SEQ ID NO: 8. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in SEQ ID NO: 9, and the other CH3 domain the sequence set forth in SEQ ID NO: 10. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in SEQ ID NO: 11, and the other CH3 domain the sequence set forth in SEQ ID NO: 12.

[0326] In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 28, 31, 34, 37, and 40, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 43. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 28, 31, 34, 37, and 40, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 46. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 28, 31, 34, 37, and 24

[0327] ACTIVE 718034029V 140, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 49. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 44. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 47. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 50. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 45. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 48. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 51. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 67. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 70. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 73. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 68. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 71. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 74. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 69. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the other CH3 domain may have the sequence set 25

[0328] ACTIVE 718034029V 1forth in SEQ ID NO: 72. In some embodiments of the CH3 domain pairs described herein, one CH3 domain may have the sequence set forth in any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the other CH3 domain may have the sequence set forth in SEQ ID NO: 75.

[0329] In some embodiments of the CH3 domain pairs described herein, the CH3 domains in the CH3 domain pair can comprise specific combinations of CH3 domains. The combination of CH3 domains may be any one of the following: t1 (e.g., SEQ ID NO: 1) / t2 (e.g., SEQ ID NO: 2); 12 (e.g., SEQ ID NO: 2) / t1 (e.g., SEQ ID NO: 1); t1.1 (e.g., SEQ ID NO: 3) / t2.1 (e.g., SEQ ID NO: 4); t2.1 (e.g., SEQ ID NO: 4) / t1.1 (e.g., SEQ ID NO: 3); t1 core (e.g., SEQ ID NO: 5) / t2 core (e.g., SEQ ID NO: 6); t2 core (e.g., SEQ ID NO: 6) / t1 core (e.g., SEQ ID NO: 5); t1.1 core (e.g., SEQ ID NO: 7) / t2.1 core (e.g., SEQ ID NO: 8); 12.1 core (e.g., SEQ ID NO: 8) / l1.1 core (e.g., SEQ ID NO: 7); t1 core, K392C (e.g., SEQ ID NO: 9) / t2 core, D399C (e.g., SEQ ID NO: 10); t2 core, D399C (e.g., SEQ ID NO: 10) / t1 core, K392C (e.g., SEQ ID NO: 9); H.1 core, K392C (e.g., SEQ ID NO: 11) / t2.1 core, D399C (e.g., SEQ ID NO: 12); 12.1 core, D399C (e.g., SEQ ID NO: 12) / 11.1, K392C core (e.g., SEQ ID NO: 11); ot1a (e.g., SEQ ID NO: 28) / ot2x (e.g., SEQ ID NO: 43); ot1b (e.g., SEQ ID NO: 31) / ot2x (e.g., SEQ ID NO: 43); otic (e.g., SEQ ID NO: 34) / ot2x (e.g., SEQ ID NO: 43); ot1d (e.g., SEQ ID NO: 37) / ot2x (e.g., SEQ ID NO: 43); ot1e (e.g., SEQ ID NO: 40) / ot2x (e.g., SEQ ID NO: 43); ot1a (e.g., SEQ ID NO: 28) / ot2y (e.g., SEQ ID NO: 46); ot1b (e.g., SEQ ID NO: 31) / ot2y (e.g., SEQ ID NO: 46); otic (e.g., SEQ ID NO: 34) / ot2y (e.g., SEQ ID NO: 46); ot1d (e.g., SEQ ID NO: 37) / ot2y (e.g., SEQ ID NO: 46); ot1e (e.g., SEQ ID NO: 40) / ot2y (e.g., SEQ ID NO: 46); ot1a (e.g., SEQ ID NO: 28) / ot2z (e.g., SEQ ID NO: 49); ot1b (e.g., SEQ ID NO: 31) / ot2z (e.g., SEQ ID NO: 49); otic (e.g., SEQ ID NO: 34) / ot2z (e.g., SEQ ID NO: 49); ot1d (e.g., SEQ ID NO: 37) / ot2z (e.g., SEQ ID NO: 49); ot1e (e.g., SEQ ID NO: 40) / ot2z (e.g., SEQ ID NO: 49); ot1a core (e.g., SEQ ID NO: 29) / ot2x core (e.g., SEQ ID NO: 44); ot1b core (e.g., SEQ ID NO: 32) / ot2x core (e.g., SEQ ID NO: 44); otic core (e.g., SEQ ID NO: 35) / ot2x core (e.g., SEQ ID NO: 44); ot1d core (e.g., SEQ ID NO:

[0330] 38) / ol2x core (e.g., SEQ ID NO: 44); ot1e core (e.g., SEQ ID NO: 41) / ot2x core (e.g., SEQ ID NO: 44); ot1a core (e.g., SEQ ID NO: 29) / ot2y core (e.g., SEQ ID NO: 47); ot1b core (e.g., SEQ ID NO: 32) / ot2y core (e.g., SEQ ID NO: 47); otic core (e.g., SEQ ID NO: 35) / ot2y core (e.g., SEQ ID NO: 47); ot1d core (e.g., SEQ ID NO: 38) / ot2y core (e.g., SEQ ID NO: 47); ot1e core (e.g., SEQ ID NO: 41) / ol2y core (e.g., SEQ ID NO: 47); ol1a core (e.g., SEQ ID NO: 29) / ot2z core (e.g., SEQ ID NO: 50); ot1b core (e.g., SEQ ID NO: 32) / ot2z core (e.g., SEQ ID NO: 50); otic core (e.g., SEQ ID NO: 35) / ot2z core (e.g., SEQ ID NO: 50); ot1d core (e.g., SEQ ID NO: 38) / ot2z core (e.g., SEQ ID NO: 50); ol1e core (e.g., SEQ ID NO: 41) / ot2z core (e.g., SEQ ID NO: 50); ot1a core, K392C (e.g., SEQ ID NO: 30) / ot2x core, D399C (e.g., SEQ ID NO: 45); ot1b core, K392C (e.g., SEQ ID NO: 33) / ot2x core, D399C (e.g., SEQ ID NO: 45); otic core, K392C (e.g., SEQ ID NO: 36) / ot2x core, D399C (e.g., SEQ ID NO: 45); ot1d core, K392C (e.g., SEQ ID NO: 39) / ot2x core, D399C (e.g., SEQ ID NO: 45); ot1e core, K392C (e.g., SEQ ID NO: 42) / ot2x 26

[0331] ACTIVE 718034029V 1core, D399C (e.g., SEQ ID NO: 45); ot1a core, K392C (e.g., SEQ ID NO: 30) / ot2y core, D399C (e.g., SEQ ID NO: 48); ot1b core, K392C (e.g., SEQ ID NO: 33) / ot2y core, D399C (e.g., SEQ ID NO: 48); otic core, K392C (e.g., SEQ ID NO: 36) / ot2y core, D399C (e.g., SEQ ID NO: 48); ot1d core, K392C (e.g., SEQ ID NO: 39) / ot2y core, D399C (e.g., SEQ ID NO: 48); ot1e core, K392C (e.g., SEQ ID NO: 42) / ot2y core, D399C (e.g., SEQ ID NO: 48); ot1a core, K392C (e.g., SEQ ID NO: 30) / ot2z core, D399C (e.g., SEQ ID NO: 51); ot1b core, K392C (e.g., SEQ ID NO: 33) / ot2z core, D399C (e.g., SEQ ID NO: 51); otic core, K392C (e.g., SEQ ID NO: 36) / ot2z core, D399C (e.g., SEQ ID NO: 51); ot1d core, K392C (e.g., SEQ ID NO: 39) / ot2z core, D399C (e.g., SEQ ID NO: 51); ot1e core, K392C (e.g., SEQ ID NO: 42) / ot2z core, D399C (e.g., SEQ ID NO: 51); ot1.1a (e.g., SEQ ID NO: 52) / ot2.1x (e.g., SEQ ID NO: 67); ot1.1b (e.g., SEQ ID NO: 55) / ot2.1x (e.g., SEQ ID NO: 67); ot1.1c (e.g., SEQ ID NO: 58) / ot2.1x (e.g., SEQ ID NO: 67); ot1.1d (e.g., SEQ ID NO: 61) / ot2.1x (e.g., SEQ ID NO: 67); ot1.1e (e.g., SEQ ID NO: 64) / ot2.1x (e.g., SEQ ID NO: 67); ot1.1a (e.g., SEQ ID NO: 52) / ot2.1y (e.g., SEQ ID NO: 70); ot1.1b (e.g., SEQ ID NO: 55) / ot2.1y (e.g., SEQ ID NO: 70); ot1.1c (e.g., SEQ ID NO: 58) / ot2.1y (e.g., SEQ ID NO: 70); ot1.1d (e.g., SEQ ID NO: 61) / ot2.1y (e.g., SEQ ID NO: 70); ot1.1e (e.g., SEQ ID NO:

[0332] 64) / ot2.1y (e.g., SEQ ID NO: 70); ot1.1a (e.g., SEQ ID NO: 52) / ot2.1z SEQ ID NO: 73); ot1.1b (e.g., SEQ ID NO: 55) / ot2.1z SEQ ID NO: 73); ot1.1c (e.g., SEQ ID NO: 58) / ot2.1z SEQ ID NO: 73); ot1.1d (e.g., SEQ ID NO: 61) / ot2.1z SEQ ID NO: 73); ot1.1e (e.g., SEQ ID NO: 64) / ot2.1z SEQ ID NO: 73); ot1.1a core (e.g., SEQ ID NO: 53) / ot2.1x core (e.g., SEQ ID NO: 68); ot1.1b core (e.g., SEQ ID NO: 56) / ot2.1x core (e.g., SEQ ID NO: 68); ot1.1c core (e.g., SEQ ID NO: 59) / ot2.1x core (e.g., SEQ ID NO: 68); ot1.1d core (e.g., SEQ ID NO: 62) / ot2.1x core (e.g., SEQ ID NO: 68); ot1.1e core (e.g., SEQ ID NO: 65) / ot2.1x core (e.g., SEQ ID NO: 68); ot1.1a core (e.g., SEQ ID NO: 53) / ot2.1y core (e.g., SEQ ID NO: 71); ot1.1b core (e.g., SEQ ID NO:

[0333] 56) / ot2.1y core (e.g., SEQ ID NO: 71); ot1.1c core (e.g., SEQ ID NO: 59) / ot2.1y core (e.g., SEQ ID NO: 71); ot1.1 d core (e.g., SEQ ID NO: 62) / ot2.1y core (e.g., SEQ ID NO: 71); ot1.1e core (e.g., SEQ ID NO: 65) / ot2.1y core (e.g., SEQ ID NO: 71); ot1.1a core (e.g., SEQ ID NO:

[0334] 53) / ot2.1z core (e.g., SEQ ID NO: 74); ot1.1b core (e.g., SEQ ID NO: 56) / ot2.1z core (e.g., SEQ ID NO: 74); ot1.1c core (e.g., SEQ ID NO: 59) / ot2.1z core (e.g., SEQ ID NO: 74); ot1.1d core (e.g., SEQ ID NO: 62) / ot2.1z core (e.g., SEQ ID NO: 74); ot1.1e core (e.g., SEQ ID NO:

[0335] 65) / ot2.1z core (e.g., SEQ ID NO: 74); ot1.1a core, K392C (e.g., SEQ ID NO: 54) / ot2.1x core, D399C (e.g., SEQ ID NO: 69); ot1.1b core, K392C (e.g., SEQ ID NO: 57) / ot2.1x core, D399C (e.g., SEQ ID NO: 69); ot1.1b core, K392C (e.g., SEQ ID NO: 57) / ot2.1x core, D399C (e.g., SEQ ID NO: 69); ot1.1ccore, K392C (e.g., SEQ ID NO: 60) / ot2.1x core, D399C (e.g., SEQ ID NO: 69); ot1.1d core, K392C (e.g., SEQ ID NO: 63) / ot2.1x core, D399C (e.g., SEQ ID NO: 69); ot1.1e core, K392C (e.g., SEQ ID NO: 66) / ot2.1x core, D399C (e.g., SEQ ID NO: 69); ot1.1a core, K392C (e.g., SEQ ID NO: 54) / ot2.1y core, D399C (e.g., SEQ ID NO: 72); ot1.1b core, K392C (e.g., SEQ ID NO: 57) / ot2.1y core, D399C (e.g., SEQ ID NO: 72); ot1.1 b core, K392C (e.g., SEQ ID NO: 57) / ot2.1y core, D399C (e.g., SEQ ID NO: 72); ot1.1ccore, K392C (e.g.,

[0336] 27

[0337] ACTIVE 718034029V 1SEQ ID NO: 60) / ot2.1y core, D399C (e.g., SEQ ID NO: 72); ot1.1d core, K392C (e.g., SEQ ID NO: 63) / ot2.1y core, D399C (e.g., SEQ ID NO: 72); ot1.1e core, K392C (e.g., SEQ ID NO: 66) / ot2.1y core, D399C (e.g., SEQ ID NO: 72); ot1.1a core, K392C (e.g., SEQ ID NO: 54) / ot2.1z core, D399C (e.g., SEQ ID NO: 75); ot1.1b core, K392C (e.g., SEQ ID NO: 57) / ot2.1z core, D399C (e.g., SEQ ID NO: 75); ot1.1b core, K392C (e.g., SEQ ID NO: 57) / ot2.1z core, D399C (e.g., SEQ ID NO: 75); ot1.1c core, K392C (e.g., SEQ ID NO: 60) / ot2.1z core, D399C (e.g., SEQ ID NO: 75); ot1.1d core, K392C (e.g., SEQ ID NO: 63) / ot2.1z core, D399C (e.g., SEQ ID NO: 75); orotl.le core, K392C (e.g., SEQ ID NO: 66) / ot2.1z core, D399C (e.g., SEQ ID NO: 75). In a preferred embodiment, the combination of CH3 domains is t1 (SEQ ID NO: 1) and t2 (SEQ ID NO: 2). In a preferred embodiment, the combination of CH3 domains is ot1a (SEQ ID NO: 28) and ot2x (e.g., SEQ ID NO: 43). In a preferred embodiment, the combination of CH3 domains is (SEQ ID NO: 31) and ot2x (e.g., SEQ ID NO: 43). In a preferred embodiment, the combination of CH3 domains is otic (e.g., SEQ ID NO: 34) and ot2x (e.g., SEQ ID NO: 43).

[0338] In some embodiments of the molecules that may be connected to a CH3 domain described herein, CH2 domains are used. An exemplary CH2 domain comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments of the molecules described herein, CH2 domains are used that are variants of a WT CH2 domain. Such a variant may result in a silent Fc (sFc). sFc CH2 variant domains are part of an engineered immunoglobulin Fc region designed to reduce or eliminate effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (GDC), by altering specific amino acids within the CH2 domain. Among the well-known CH2 variants in the literature are “LALAPK,” “LALAPA,” and “LALAPG,” which refer to specific amino acid substitutions at positions 234, 235, and 329 according to the Eu numbering system. In the LALAPK variant, the substitutions are L234A, L235A, and P329K. In the LALAPA variant, the substitutions are L234A, L235A, and P329A, which may also be combined with a D265A substitution. In the LALAPG variant, the substitutions are L234A, L235A, and P329G. These modifications are strategically designed to minimize interactions with Fc receptors and complement proteins, thereby reducing the immune effector functions typically mediated by the Fc region of antibodies. An exemplary CH2 domain comprises the amino acid sequence of SEQ ID NO: 22.

[0339] Nucleic acids, vectors, host cells and methods of production

[0340] Provided is an isolated nucleic acid encoding a CH3 domain pair or heterodimeric protein (polypeptide complex) of the invention. Nucleic acids may comprise DNA and / or RNA and may be partially or wholly synthetic. The terms nucleic acid and nucleic acid molecule are used interchangeably herein.

[0341] 28

[0342] ACTIVE 718034029V 1An isolated nucleic acid may be used to express a CH3 domain pair or heterodimeric protein of the invention. The nucleic acid will generally be provided in the form of an expression vector. Another aspect of the invention thus provides an expression vector comprising a nucleic acid as described above. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

[0343] Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate in the context.

[0344] A nucleic acid or expression vector as described herein may be introduced into a host cell. A further aspect of the invention thus provides a host cell comprising a vector or nucleic acid of the invention. Techniques for the introduction of nucleic acids or vectors into host cells are well established in the art and any suitable technique may be employed. A range of host cells suitable for the production of recombinant CH3 domain pairs or heterodimeric proteins are known in the art, and include bacterial, yeast, insect or mammalian host cells. A preferred host cell is a mammalian cell, such as a CHO, NSO, or HEK cell, for example a HEK293 cell.

[0345] Another aspect of the invention provides a method of producing a CH3 domain pair or heterodimeric protein of the invention comprising expressing a nucleic acid encoding the CH3 domain pair or heterodimeric protein in a host cell. Methods for culturing host cells are well- known in the art. The method may further comprise isolating and / or purifying the CH3 domain pair or heterodimeric protein. Techniques for the purification of recombinant proteins are well-known in the art and include, for example HPLC, FPLC, or affinity chromatography, e.g. using Protein A or Protein L. In some embodiments, purification may be performed using an affinity tag on the CH3 domain pair or heterodimeric protein.

[0346] The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims. It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included,

[0347] 29

[0348] ACTIVE 718034029V 1unless otherwise Indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0349] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the embodiments, is / are referred to as comprising particular elements, features, steps, etc., certain embodiments of the disclosure or aspects of the embodiments consist, or consist essentially of, such elements, features, steps, etc. Thus, for each embodiment of the disclosure that comprises one or more elements, features, steps, etc., the disclosure also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

[0350] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0351] In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

[0352] Although the disclosure has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the scope of the disclosure. Accordingly, the above examples are intended to illustrate but not limit the present disclosure.

[0353] 30

[0354] ACTIVE 718034029V 1Examples

[0355] General methods

[0356] Transient transfection of HEK mammalian cells

[0357] Proteins were produced through transient transfection of human embryonic kidney (HEK) ceils using the Expi293 Expression System (ThermoFisher), following the manufacturer’s instructions. Unless otherwise noted, transfections were carried out in 15 mL volumes of HEK cells with 15 pg of total DNA, using an equal mass ratio of the respective plasmids. For example, to produce heteromeric complexes consisting of three polypeptide chains, a 1:1:1 chain ratio was used, corresponding to 5.0 pg of each DNA construct. Transfected cells were incubated at 37°C in an 8% CO2environment while shaking at 125 rpm for 5 days.

[0358] Supernatants were harvested by centrifugation at 800 x g for 10 minutes.

[0359] Affinity purification using Protein A resin

[0360] Clarified transfection supernatants were affinity purified using MabSelect SuRe™ L bulk protein A resin (Cytiva). The resin was equilibrated with Dulbecco’s PBS (DPBS), pH 7.4, to remove storage buffer. Supernatant was incubated with the resin for a minimum of 1 hour at room temperature on a tube rotator. The resin was then captured on a gravity filtration column and washed with phosphate-buffered saline (PBS). Bound proteins were eluted with 0.1 M sodium acetate (pH 3.5) into neutralization buffer (2 M Tris-HCI, pH 9.0). Eluted samples were buffer-exchanged into DPBS using 30 kDa molecuiar weight cutoff (MWCO) centrifugai filters (Amicon Ultra-15; EMD-Millipore).

[0361] Capillary Electrophoresis

[0362] Samples, both before (affinity-purified only) and after polishing, were characterized under reducing and non-reducing conditions using capillary electrophoresis (CE). CE was performed with the ProteinEXact HT LabChip and Reagent Kit (Revvity) on the LabChip GXII Touch Protein Characterization System (Revvity). Proteins in 96-well sample plates were mixed with sample buffer and Bond Breaker TCEP reducing agent (Thermo Scientific) for reducing runs and denatured at 70°C for 10 minutes. The chip was prepared according to the manufacturer’s protocol, and the plate, chip, and ladder were loaded onto the instrument. The ‘HT ProteinEXact’ method was initiated, with a run time of 65 seconds per sample.

[0363] Electropherogram peaks were integrated to determine % homogeneity and migration time. Data analysis was performed using LabChip GX Reviewer software.

[0364] 31

[0365] ACTIVE 718034029V 1Example 1

[0366] A novel heterodimeric CH3 domain pair (CH3 t1-t2) was designed, engineered, and systematically evaluated for improved heterodimeric interface stabilization and prevention of homodimer formation. Multiple interface designs were generated and assessed for their impact on expression, stability, and proper assembly. Surprisingly, the CH3 t1-t2 domain pair exhibited superior performance relative to alternative designs evaluated, as well as existing CH3 domain pairs, reliably directing correct pairing and affording high expression and assembly efficiency.

[0367] The CH311 -t2 domains include multiple strategic amino acid substitutions that combine multiple interface modification strategies. Evaluation of the engineered design revealed good assembly of the heterodimeric CH3 domain pair (disregarding the disulfide) and weak off-target CH3 domain homodimer production. FIGS. 1A and 1B illustrate the structural differences between the wild-type CH3 domain pair (FIG 1A) and the engineered CH3 t1-t2 interface (FIG 1B), highlighting the positions of the amino acid substitutions.

[0368] Initial validation of the heterodimeric properties was conducted using asymmetrical polypeptides in which the engineered CH3 t1-t2 domains replaced the wild-type CH3 domains in an Fc region. This approach enabled rapid differentiation between heterodimer and homodimer formation by CE analysis of ProA-purified protein products. A -100 kDa species on the GE trace indicates successful heterodimer formation, while molecules comprising homodimer 1 and molecules comprising homodimer 2 represent as -60 kDa and -150 kDa species, respectively. Schematic representations of the relevant portion of the heterodimer, homodimer 1 and of the relevant portion of homodimer 2 are shown in FIG. 2A.

[0369] These studies revealed that the wild-type CH3 domains yielded approximately 50% of the heterodimer, whereas a previously known reference engineered pair (CH3 b1-b2) achieved 57- 69% heterodimer formation, regardless of domain orientation. The CH311.1-t2.1 domain pair exhibited 92-93% heterodimer formation (FIG. 2B). Because of the symmetry of the CH3 dimer interface, the t1.142.1 domain pair was also tested with both symmetric forms of the D399C-K392C disulfide, or with no disulfide. The t1.1-t2.1 domain pair by itself, without a disulfide, resulted in formation of 87-90% heterodimer (FIG. 2B, lanes F11 and C4). Adding in the D399C-K392C disulfide resulted in formation of heterodimer up to -93%, irrespective of which symmetric form of the disulfide was used (FIG. 2B, lanes H3 and C9 compared to lanes F8 and E8). In a study, the CH3 t1-t2 pair exhibited 85-96% heterodimer formation compared to 79-84% heterodimer formation for the CH311.142.1 domain pair (FIG. 2C). Additionally, even in the presence of competing CH3 chains, the CH311 -t2 domain pair maintained preferential mutual pairing, thereby minimizing off-target chain assemblies.

[0370] 32

[0371] ACTIVE 718034029V 1Example 2 (Additional CH3 domain pairs and substitutions)

[0372] Additional novel CH3 domain pairs were designed, engineered, and systematically evaluated to optimize the developability of antibody constructs comprising a variety of variable domains that are in combined with CH3 domains, including but not limited to the novel CH3 domains described above. Multiple VH / VL-CH3 / CH3 interface modification strategies were developed using both rational design and computational modeling approaches.

[0373] The additional CH3 domain pairs include multiple strategic amino acid substitutions that combine multiple interface modification strategies. Candidate amino acid substitutions were selected based on multiple criteria, including their proximity to the variable domains in a polypeptide complex comprising two variable domains and a CH3 dimer (see e.g. FIG. 3A). This included residues near the interface between the variable domains and a variety of CH3 domains, including the CH3 t1-t2 domains described in Example 1. Each candidate amino acid substitution was computationally screened for predicted T cell epitopes using NetMHCIIpan-4.0 to assess and minimize potential immunogenicity risk.

[0374] FIGS. 3A and 3B illustrate the structural differences between a wild-type CH3 domain pair (FIG.

[0375] 3A) at the VH / VL interface and the engineered interface, highlighting the positions of candidate amino acid substitutions of the wild-type CH3 domain (FIG. 3B). FIGS. 3C and 3D illustrate the structural differences between the t1 -t2 CH3 domain pair (FIG. 30) at the VH / VL interface and the engineered interface highlighting the positions of candidate amino acid substitutions of the t1-t2 domain pair. The models of the structures illustrated in FIGS. 3A and 30 superimpose closely, as do those in FIGS. 3B and 3D. This structural similarity underscores that the candidate amino acid substitutions are compatible with a variety of CH3 domains, including the wildtype CH3 domains and the t1-t2 domains.

[0376] Exemplary CH3 domain pairs modified with selected amino acid substitutions were further evaluated, including in various antibody formats, as described in the following Examples.

[0377] Example 3

[0378] A number of the candidate amino acid substitutions as described in Example 2 and illustrated in FIGS. 3A-3D were experimentally evaluated in the context of both lambda-germline and kappa¬ germline variable domains. These substitutions were incorporated into both one-armed antibody (OAA) constructs and bispecific antibody (bsAb) constructs that were expressed in triplicate in 96-well plates using 1 mL transfection volumes.

[0379] 33

[0380] ACTIVE 718034029V 1Expression yields and melting temperatures of a multiplicity of exemplary CH3 domain pairs modified with selected amino acid substitutions are shown in TABLE 1 (OAA) and TABLE 2 (bsAb). The results showed robust expression and increased melting temperatures for the multiple modified CH3 domain pairs when paired with both exemplary lambda-germline and kappa-germline variable domains.

[0381] 34

[0382] ACTIVE 718034029V 1ABLE 1: Experimental results for OAAs

[0383] Evolocumab (lambda) B7H3 (kappa)

[0384] OAAs OAAs

[0385] HEK Expr. Yield i HEK Expr. Yield i Avg OAA Tm id VL-tl substitutions VH-t2 substitutions (ug / mL) Tm (°C) (ug / mL) Tm (°C) (°C) ti-t2 L- L- 63 ± 19 58.2 ± 0.1 79 ± 13 61.8 ± 1.2 60.0 t1-ot2y L... IG341A 44 ± 10 58.3 ± 0.0 130 ± 32 61.9 ± 0.4 60.1 t1-ot2z L- IY373F-F404Y 71 ± 30 59.6 ± 0.2 67 ± 14 63.1 ± 1.8 61.4 t1-ot2x L. *3341 A-Y373F-F404Y 109 ± 10 60.7 ± 0.1 83 ± 13 64.7 ± 0.1 62.7 ot1 d-t2 IL368I i— 89 ± 26 58.6 ± 0.2 114 ± 3 64.0 ± 0.1 61.3 ot: d-ot2y 13681 G341A 63 ± 13 59.4 ± 0.2 137 ± 23 64.1 ± 0.1 61.8 ot1d-ot2z 1368I IY373F-F404Y 96 ± 10 60.3 ± 0.2 133 ± 3 65.5 ± 0.1 62.9 ot1d-ot2x IL368I IG341 A-Y373F-F404Y 103 + 12 61.3 ± 0.1 137 ± 10 65.9 ± 0.0 63.6 ot1 b-t2 1398Q-F404Y 81 ± 14 59.2 ± 0.2 137 ± 33 63.0 ± 0.9 61.1 ot1 b-ot2y 1398Q-F404Y IG341A 68 ± 18 59.3 ± 0.1 150 ± 49 63.9 ± 0.2 61.6 ot1 b-ot2z 1398Q-F404Y Y373F-F404Y 89 ± 23 60.4 ± 0.2 141 ± 23 64.9 ± 0.5 62.7 ot1b-ot2x IL398Q-F404Y IG341 A-Y373F-F404Y 130 ± 8 60.9 ± 0.3 137 ± 38 65.5 ± 0.3 63.2 ot1e-t2 IY373F-L398Q-F404Y i— 53 ± 9 58.0 ± 0.0 129 ± 41 62.8 ± 0.0 60.4 ot1e-ot2y IY373F-L398Q-F404Y IG341A 40 ± 23 57.8 ± 0.1 116 ± 40 63.1 ± 0.2 60.5 ot1e-ot2z IY373F-L398Q-F404Y IY373F-F404Y 70 ± 5 59.2 ± 0.1 130 ± 34 63.7 ± 0.8 61.5 ot1e-ot2x IY373F-L398Q-F404Y IG341 A-Y373F-F404Y 111 ± 7 60 2 + 0.1 152.+ 23 64.6 + 0.1 62.4 ot1a-t2 ii_368l-L398Q-F404Y i— 104 ± 49 59.0 ± 0.5 134 ± 13 65.0 ± 0.1 62.0 ot1a-ot2y 1368I-L398Q-F404Y IG341A 68 ± 22 60.1 ± 0.4 135 ± 38 65.3 ± 0.1 62.7 ot1a-ot2z 1368I-L398Q-F404Y Y373F-F404Y 86 ± 7 61.2 ± 0.5 106 ± 18 65.7 ± 1.4 63.5 ot1a-ot2x 1368I-L398Q-F404Y *3341 A-Y373F-F404Y 116 ± 5 62.3 ± 0.1 160 ± 14 66.6 ± 0.2 64.5 ot1c-t2 iL368l-Y373F-L398Q-F404Y i— 83 ± 38 58.5 ± 0.3 114 ± 37 63.3 ± 1.1 60.9 ot1c-ot2y iL368l-Y373F-L398Q-F404Y IG341A 65 ± 21 59.0 ± 0.1 126 ± 33 64.3 ± 0.1 61.7 ot1c-ot2z 1368I-Y373F-L398Q-F404Y Y373F-F404Y 80 ± 6 60.0 ± 0.3 139 ± 47 64.2 ± 1.4 62.1

[0386]

[0387] ot1c-ot2x 1368I-Y373F-L398Q-F404Y *3341 A-Y373F-F404Y 123 ± 10 61.0 ± 0.5 170 ± 12 65.7 ± 0.1 63.4

[0388] 35

[0389] CTIVE 718034029v1ABLE 2: Experimental results for bispecifics

[0390] Evolocumab (lambda) B7H3 (kappa)

[0391] bispecifics bispecifics

[0392] Avg HEK Expr. Yield i; HEK Expr. Yield; bispecific Id IVL-11 substitutions IVH-t2 substitutions (ug / mL) Tm (°C) (ug / mL) Tm (°C) Tm (°C) t1-t2........ 11 ± 6 57.9 ± 0.3 86 ± 16 62.3 ± 0.3 60.1 t1 -ot2y G341. A 6 ± 3 58.8 ± 0.4 119 + 24 62.6 + 0.5 60.7 t1-ot2z L- Y373F-F404Y 13 ± 7 59.6 ± 0.3 68 ± 7 64.5 ± 0.1 62.1 t1-ot2x L„ iG341 A-Y373F-F404Y 23 ± 5 60.7 + 0.2 104 ± 25 64.7 ± 0.1 62.7 ot1 d-t2 L368I 19 ± 5 58.9 ± 0.2 99 ± 32 64.5 ± 0.4 61.7 ot1d-ot2y i|_368l iG341A 11 ± 3 59.7 ± 0.2 125 + 18 64.5 ± 0.3 62.1 ot1d-ot2z iL368l iY373F-F404Y 20 ± 4 60.8 ± 0.1 118 ± 7 65.9 ± 0.1 63.4 ot1d-ot2x il_368l iG341 A-Y373F-F404Y 35 + 6 62.0 + 0.1 132 ± 17 66.2 + 0.3 64.1 o 11 b-t2 I..398G -F404Y ■■■■■ 14 ± 6 59.3 ± 0.2 96 + 60 64.2 ± 0.3 61.8 ot1 b-ot2y L398G-F404Y G341A 12 ± 4 59.5 ± 0.3 153 ± 17 64.3 ± 0.3 61.9 ot1 b-ot2z il_398Q-F404Y iY373F-F404Y 15 ± 1 60.8 + 0.1 104 ± 64 65.7 ± 0.1 63.3 ot1b-ot2x i|_398Q-F404Y iG341 A-Y373F-F404Y 34 ± 6 61.4 ± 0.1 160 ± 10 65.6 ± 0.1 63.5 ot1e-t2 iY373F-L398Q-F404Y i— 10 ± 6 58.5 ± 0.2 85 + 30 63.5 ± 0.1 61.0 ot1e-ot2y iY373F-L398Q-F404Y iG341A 10 ± 5 58.1 ± 0.3 102 ± 18 63.5 ± 0.2 60.8 ot1e-ot2z iY373F-L398Q-F404Y iY373F-F404Y 12 ± 5 59.4 ± 0.3 106 ± 24 64.7 ± 0.1 62.1 ot1e-ot2x iY373F-L398Q-F404Y iG341 A-Y373F-F404Y 31 ± 16 60.4 ± 0.1 114 + 31 65.0 ± 0.2 62.7 ot1a-t2 iL368l-L398Q-F404Y i— 30 ± 9 59.9 + 0.2 103 ± 57 65.5 ± 0.2 62.7 ot1a-ot2y il368l-L398Q-F404Y IG341A 16 ± 2 60.4 ± 0.1 164 ± 27 65.3 ± 0.1 62.9 ot1a-ot2z il_368l-L398Q-F404Y iY373F-F404Y 21 ± 3 61.7 + 0.1 124 ± 41 66.8 ± 0.1 64.3 ot1a-ot2x iL368l-L398Q-F404Y iG341 A-Y373F-F404Y 37 ± 8 62.8 ± 0.1 144 ± 9 66.9 ± 0.1 64.9 ot1c-t2 iL368l-Y373F-L398Q-F404Y i— 18 + 8 58.8.+ 0.2 101 + 34 64.6 ± 0.3 61.7 ot1c-ot2y il_368l-Y373F-L398Q-F404Y iG341A 11 ± 1 59.3 + 0.2 128 ± 32 64.5 ± 0.1 61.9 ot1c-ot2z iL368l-Y373F-L398Q-F404Y iY373F-F404Y 26 ± 10 60.4 ± 0.1 106 ± 35 65.6 ± 0.1 63.0

[0393]

[0394] ot1c-ot2x iL368l-Y373F-L398Q-F404Y iG341 A-Y373F-F404Y 36 ± 8 61.7 ± 0.1 142 ± 23 65.8 ± 0.1 63.8

[0395] 36

[0396] CTIVE 718034029v1Example 4

[0397] Among the exemplary CH3 domain pairs shown in TABLE 1 and TABLE 2, many of the CH3 domain pairs modified with selected amino acid substitutions showed marked increases in Tm in OAA and bispecific antibody formats comprising those CH3 domain pairs when compared with an OAA and bispecific antibodies (bsAb) comprising a t1 -t2 CH3 domain pair, with many exhibiting marked increases in Tm. For example, OAAs and bispecifics comprising CH3 domain pairs designated t1-ot2x, ot1b-ot2z, ot1e-ot2x, ot1a-t2, ot1a-ot2y, and ot1c-ot2z as well as ot1d-ot2z, ot1d-ot2x, ot1b-ot2x, ot1a-ot2z, ot1a-ot2x, and ot1 c-ot2x showed increased Tm over the t1 -t2 domain pair by 2-5°C on average. Interestingly, these CH3 domain pairs modified with selected amino acid substitutions also had a positive impact on expression yield of OAAs and bsAbs comprising the designated domain pairs. The yields obtained after CH1 purification were increased by substantial amounts in a number of the OAAs and bsAbs compared with those of an OAA and bsAb comprising a t1 -t2 CH3 domain pair. Importantly, no marked decrease in the efficiency of assembly was noted for any of these exemplary CHS domain pairs modified with the selected amino acid substitutions.

[0398] The exemplary OAAs and bsAb described in this Example were further evaluated for a propensity for self-association and / or non-specific binding (e.g., poly-reactivity). Self-association propensity was measured using AC-SINS and the DNA and insulin poly-reactivity was measured with standard ELISA methods. The exemplary OAAs and bsAb had little or no reactivity in AC-SINS and poly-reactivity assays, with levels comparable to a reference antibody and far below that of the assay positive control.

[0399] 37

[0400] ACTIVE 718034029V1Sequences

[0401] SEQ ID NO: 1 (tl ) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENNYKTMPPVLCSDGSFFLGSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0402] SEQ ID NO: 2 (t2 ) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYCTTPPLLDSDGSFALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0403] SEQ ID NO:

[0404]

[0405] (tl. l ) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENNYKTMPPVLCSDGSFFLASKI TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0406] SEQ ID NO: 4 ( t2.1 ) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYCTTPPLLDSDGSFALYSFL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0407] SEQ ID NO: 5 (tl core) GQPREPQVYTLPPSRDELTKNQVSLTCWKGFYPSDIAVEWESNGQPENNYKTMPPVLDSDGSFFLGSKL TVDKSRWQQGNVFSCSWHEALHNHYTQKSLSLSPGK

[0408] SEQ ID NO: 6 (t.2 core) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLDSDGSFALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0409] SEQ ID NO: 7 (tl. l core) GQPREPQVYTLPPSRDELTKNQVSLTCWKGFYPSDIAVEWESNGQPENNYKTMPPVLDSDGS FELAS KL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0410] SEQ ID NO: 8 (t2.1 core ) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLDSDGSFALYSFL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0411] SEQ ID NO: 9 (tl core, K392C) GQPREPQVYTLPPSRDELTKNQVSLTCWKGFYPSDIAVEWESNGQPENNYCTMPPVLDSDGSFFLGSKL TVDBOSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0412] SEQ ID NO: 10 ( t2 core, D399C)

[0413] 38

[0414] ACTIVE 718034029V1GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLCSDGSFALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0415] SEQ ID NO: 11 (tl. l core, K392C) GQPREPQVYTLPPSRDELTKNQVSLTCWKGFYPSDIAVEWESNGQPENNYCTMPPVLDSDGSFFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0416] SEQ ID NO: 12 (t2. 1 core, D399C) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLCSDGSFALYSFL TVDIISRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0417] SEQ ID NO: 13 (bl ) GQPREPQVCTLPPSRDELTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKI TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0418] SEQ ID NO: 14 (b2 ) AQPREPQVYTLPPCRDWLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKI TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0419] SEQ ID NO: 13 (WT CH3 ( IgGl ) ) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKI TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0420] SEQ ID NO: 16 (WT CH3 ( IgG2 ) ) GQPREPQVYTLPPSREEMTKNQVSDTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKI TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPEI

[0421] SEQ ID NO: 17 (WT CH3 ( IgG3 ) ) GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKL TVDKSR. WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0422] SEQ ID NO: 18 (WT CH3 ( IgG4 ) ) GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSV14HEALHNHYTQKSLSDSLGK

[0423] SEQ ID NO: 19 (CH3 Knob - T366W) GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKI, TVDKSRWQQGNVFSCSV14HEALHNHYTQKSLSDSPGK

[0424] 39

[0425] ACTIVE 718034029V1SEQ ID NO: 20 (CH3 Hole - D356E, L358M, T366S, E368A, Y407V) GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL

[0426] 40 TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0427] SEQ ID NO: 21 (CH2 ) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVS VLT VL HQ DWLNGKE Y KCKVSNKAL PAP I E KT I S KAK

[0428] SEQ ID NO: 22 (CH2 ( sFc - LALAPK) ) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIEKTISKAK

[0429] SEQ ID NO: 28 (otla) GQPREPQVYTLPPSRDELTKNQVSDTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVQCSDGSYFLGSKL TVDKSR. WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0430] SEQ ID NO: 29 (otla core ) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVQDSDGSYFLGSKL TVDKSR. WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0431] SEQ ID NO: 30 (otla core, K392C) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLGSKI TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSDSPGK

[0432] SEQ ID NO: 31 ( otlb) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENNYKTMPPVQCSDGSYFLGSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSDSPGK

[0433] SEQ ID NO: 32 (otlb core ) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENNYKTMPPVQDSDGSYFLGSKL TVDKSR. WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0434] SEQ ID NO: 33 (otlb core, K392C) GQPREPQVYTLPPSRDELTKNQVSIJTCVVKGFYPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLGSKI TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSDSPGK

[0435] SEQ ID NO: 34 (otic) GQPREPQVYTLPPSRDELTKNQVSDTCIVKGFFPSDIAVEWESNGQPENNYKTMPPVQCSDGSYFLGSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0436] 40

[0437] ACTIVE 718034029V1SEQ ID NO: 35 GQPREPQVYTLPPSRDELT: KNQVSLTCIVKGFFPSDIAVEWESNGQPENNY: KTMPPY7QDSDGSYFLGSKL TVDKSRWQQGNVFSCSIWIEALHNHYTQKSLSLSPGK

[0438] SEQ ID NO: 36 (otic core, K392C) GQPREPQVYTLPPSRDELTZKNQVSLTCIVKGFFPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLGSKL TVDKSRWQQGNVFSCSIWIEALHNHYTQKSLSLSPGK

[0439] SEQ ID NO: 37 (otld) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVLCSDGSFFLGSKL TVDB6SRWQQGNVFSCSVMHEALHNHYTQKSLSESPGK

[0440] SEQ ID NO: 38 (otld core ) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVLDSDGSFFLGSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0441] SEQ ID NO: 39 (otld core, K392C) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYCTMPPVLDSDGSFFLGSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0442] SEQ ID NO: 40 (otle) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFFPSDIAVEWESNGQPENNYKTMPPVQCSDGSYFLGSKL TVDBOSRWQQGNVFSCSVMHEALHNHYTQKSLSESPGK

[0443] SEQ ID NO: 41 (otle core ) GQPREPQVYTLPPSRDELTKNQVSETCVVKGFFPSDIAVEWESNGQPENNYKTMPPVQDSDGSYFLGSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0444] SEQ ID NO: 42 (otle core, K392C) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFFPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLGSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0445] SEQ ID NO: 43 (ot2x) AQPREPQVYTLPPSRDELTKNQVSEVCLVKGFFPSDIAVEWESNGQPENNYCTTPPELDSDGSYALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0446] SEQ ID NO: 44 ( ot2x core )

[0447] 41

[0448] ACTIVE 718034029V1AQPREPQVYTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYKTTPPLLDSDGSYALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0449] SEQ ID NO: 45 (ot2x core, D399C) AQPREPQVYTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYKTTPPLLCSDGSYALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0450] SEQ ID NO: 46 (ot2y) QPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYCTTPPLLDSDGSFALYSYL TVDBOSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0451] SEQ ID NO: 47 (ot2y core ) AQPREPQVYTLPPSRDELTKNQVSL'FCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLDSDGSFALYSYD TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0452] SEQ ID NO: 48 (ot2y core, D399C) AQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLCSDGSFALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0453] SEQ ID NO: 49 (ot2z ) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYCTTPPLLDSDGSYALYSYL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0454] SEQ ID NO: 50 ( ot2z core ) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYKTTPPLLDSDGSYALYSYI TVDKSRWQQGNVFSCSV14HEALHNHYTQKSLSDSPGK

[0455] SEQ ID NO: 51 (ot2z core, D399C) GQPREPQVYTLPPSRDELTKNQVSLY7CLVKGFFPSDIAVEWESNGQPENNYKTTPPLLCSDGSYALYSYL TVDKSRWQQGNVFSCSV14HEALHNHYTQKSLSLSPGK

[0456] SEQ ID NO: 52 (otl. la) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVQCSDGSYFLASKL TVDKSRWQQGNVFSCSV14HEALHNHYTQKSLSLSPGK

[0457]

[0458] GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0459] 42

[0460] ACTIVE 718034029V1SEQ ID NO: 54 ( otl. la core, K392C ) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0461] SEQ ID NO: 55 ( otl. lb ) GQPREPQVYTLPPSRDELTKNQ\'TSLTCVVKGFYPSDIA\'TEWESNGQPENNYKTMPPV^CSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0462] SEQ ID NO: 56 ( otl. lb core ) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENNYKTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0463] SEQ ID NO: 57 ( otl. lb core, K392C ) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFYPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0464] SEQ ID NO: 58 ( otl. lc ) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFFPSDIAVEWESNGQPENNYKTMPPVQCSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0465] SEQ ID NO: 59 ( otl. lc core ) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFFPSDIAVEWESNGQPENNYKTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0466] SEQ ID NO: 60 ( otl. lc core, K392C ) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFFPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0467] SEQ ID NO: 61 ( otl. ld) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVLCSDGS FELAS KL TVDKSRWQQGNVFSCSWHEALHNHYTQKSLSLSPGK

[0468] SEQ ID NO: 62 ( otl. ld core ) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTMPPVLDSDGS FELAS KL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0469] SEQ I D NO: 63 ( otl. ld core, K392C) GQPREPQVYTLPPSRDELTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYCTMPPVLDSDGSFFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0470] 43

[0471] ACTIVE 718034029V1SE'. Q ID NO: 64 (otl. le) GQPREPQVYTLPPSRDELTKNQVSLTCIZVKGFFPSDIAVEWESNGQPENNYKTMPPVQCSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0472] SEQ ID NO: 65 (otl. le core ) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFFPSDIAVEWESNGQPENNYKTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0473] SEQ ID NO: 66 ( otl. le core, K392C) GQPREPQVYTLPPSRDELTKNQVSLTCVVKGFFPSDIAVEWESNGQPENNYCTMPPVQDSDGSYFLASKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0474] SEQ ID NO: 67 ( ot2.1x) AQPREPQl'YTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYCTTPPLLDSDGSYALYSFL TVDKSRWQQGNVFSCSV14HEALHNHYTQKSLSLSPGK

[0475] SEQ ID NO: 68 (ot2.1x core ) AQPREPQl'YTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYKTTPPLLDSDGSYALYSFL TVDKSRWQQGNVFSCSV14HEALHNHYTQKSLSLSPGK

[0476] SEQ ID NO: 69 (ot2.1x core, D399C) AQPREPQVYTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYKTTPPLLCSDGSYALYSFL TVDKSRWQQGNVFSCSV14HEALHNHYTQKSLSLSPGK

[0477] SEQ ID NO: 70 (ot2.1y) AQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYCTTPPLLDSDGSFALYSFL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0478] SEQ ID NO: 71 (ot2.1y core ) AQPREPQVYTLPPSRDELTK14QVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLDSDGSFALYSFL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0479] SEQ ID NO: 72 (ot2.1y core, D399C) AQPREPQVYTLPPSRDELTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPLLCSDGSEALYSFL TVDP1SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0480] SEQ ID NO: 73 (ot2.1z )

[0481] 44

[0482] ACTIVE 718034029v1GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYCTTPPLLDSDGSYALYSFL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0483] SEQ ID NO: 74 (ot2.1z core ) GQPREPQVYTLPPSRDELTKNQVSLVCLTKGFFPSDIAVEWESNGQPENNYKTTPPLLDSDGSY. ALYSFL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0484] SEQ ID NO: 75 (ot2.1z core, D399C) GQPREPQVYTLPPSRDELTKNQVSLVCLVKGFFPSDIAVEWESNGQPENNYKTTPPLLCSDGSYALYSFL TVDB5SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0485] 45

[0486] ACTIVE 718034029V1

Claims

Claims1. A CH3 domain pair, wherein:a) the first CH3 domain of the CH3 domain pair comprises substitutions 368V or 368I, 394M, and 407G; andb) the second CH3 domain of the CH3 domain pair comprises substitutions 366V, 397L, 405A, and 409Y;wherein the amino acids are numbered according to the Eu index.

2. The CH3 domain pair according to claim 1, wherein:a) the first CH3 domain of the CH3 domain pair comprises substitutions L368V or 368I, T394M, and Y407G; andb) the second CH3 domain of the CH3 domain pair comprises substitutions T366V, V397L or M397L, F405A, and K409Y or R409Y;wherein the amino acids are numbered according to the Eu index.

3. The CH3 domain pair according to any one of the preceding claims, wherein the first CH3 domain of the CHS domain pair further comprises substitution D399C, and the second CH3 domain of the CH3 domain pair further comprises substitution K392C or N392C, or wherein the first CH3 domain of the CH3 domain pair further comprises substitution K392C or N392C, and the second CH3 domain of the CH3 domain pair further comprises substitution D399C.

4. The CH3 domain pair according to any one of the preceding claims, wherein:a) the first CH3 domain of the CH3 domain pair comprises substitutions L368V or L368I, T394M, D399C, and Y407G; andb) the second CHS domain of the CH3 domain pair comprises substitutions T366V, K392C, V397L, F405A, and K409Y.

5. The CH3 domain pair according to any one of the preceding claims, wherein the first CH3 domain of the CH3 domain pair comprises substitution L368V.

6. The CH3 domain pair according to any one of the preceding claims, wherein the first CH3 domain of the CH3 domain pair comprises substitution L368I.

7. The CH3 domain pair according to any one of the preceding claims, wherein:a) the first CH3 domain of the CH3 domain pair further comprises one or more amino acid substitutions selected from the group consisting of:(I) 341A;46ACTIVE 718034029V1(ii) 373F;(iii) 398Q; and(iv) 404Y; and / orb) the second CH3 domain of the CH3 domain pair further comprises one or more amino acid substitutions selected from the group consisting of:(i) 341A;(ii) 373F; and(iii) 404Y.

8. The CH3 domain pair according to claim 7, wherein:a) the first CH3 domain of the CH3 domain pair further comprises one or more amino acid substitutions selected from the group consisting of:(i) G341A;(ii) Y373F;(iii) L398Q; and(iv) F404Y; and / orb) the second CH3 domain of the CH3 domain pair further comprises one or more amino acid substitutions selected from the group consisting of:(i) G341A(ii) Y373F; and(iii) F404Y.

9. The CH3 domain pair according to ciaim 7 or 8, wherein:a) the first CH3 domain of the CH3 domain pair further comprises:(i) amino acid substitutions L398Q and F404Y; and optionally(ii) amino acid substitution Y373F; and / orb) the second CH3 domain of the CH3 domain pair further comprises:(i) amino acid substitution G341A; and / or(ii) amino acid substitutions Y373F and F404Y.

10. The CH3 domain pair according to any one of claims 7 to 9, wherein:a) the first CH3 domain of the CH3 domain pair further comprises:(i) amino acid substitutions L398Q and F404Y; and optionally(ii) amino acid substitution Y373F; andb) the second CH3 domain of the CH3 domain pair further comprises:(i) amino acid substitutions Y373F and F404Y; and optionally(ii) amino acid substitution G341A.47ACTIVE 718034029V111. The CH3 domain pair according to claim 10, wherein:a) the first CH3 domain of the CHS domain pair further comprises:(i) amino acid substitutions L398Q and F404Y; and optionally(ii) amino acid substitution Y373F; andb) the second CH3 domain of the CH3 domain pair further comprises:(i) amino acid substitution G341A; and(ii) amino acid substitutions Y373F and F404Y.

12. The CH3 domain pair according to any one of claims 7 to 9, wherein:(A)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L368V; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;(B)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y;(C)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;(D)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, Y373F, L398Q and F404Y;(E)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitution G341A; or(F)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368I, Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y.48ACTIVE 718034029V113. The CH3 domain pair according to any one of claims 7 to 9, wherein:(A)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L368I; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y;(B)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L368I; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;(C)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L368V, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;(D)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L368I, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y;(E)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L368I, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y; or(F)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L368I, Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

14. The CH3 domain pair according to claim 13, wherein:a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368I, T394M, L398Q, D399C, F404Y, and Y407G; andb) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, T366V, Y373F, K392C, V397L, F404Y, F405A, and K409Y.49ACTIVE 718034029V115. The CH3 domain pair according to claim 13, wherein:a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368V, T394M, L398Q, D399C, F404Y, and Y407G; andb) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, T366V, Y373F, K392C, V397L, F404Y, F405A, and K409Y.

16. The CH3 domain pair according to claim 13, wherein:a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L368I, Y373F, T394M, L398Q, D399C, F404Y, and Y407G; andb) the second CHS domain of the CH3 domain pair comprises amino acid substitutions G341A, T366V, Y373F, K392C, V397L, F404Y, F405A, and K409Y.

17. The CH3 domain pair according to any one of claims 1 to 16, wherein the first and second CH3 domains of the CH3 domain pair are I gG 1, I gG2, IgGS, or lgG4 CH3 domains.

18. The CH3 domain pair according to any one of claims 1 to 16, wherein the first and second CH3 domains of the CH3 domain pair are I gG 1 CH3 domains.

19. The CH3 domain pair according to claim 1, wherein:(i) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 1, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 2;(ii) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 3, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 4;(iii) the first CHS domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 5, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 6;(iv) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 7, and the second CHS domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 8;(v) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 9, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 10;(vi) the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 11, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 12;(vii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one 50ACTIVE 718034029V1of SEQ ID NOS: 28, 31, 34, 37, and 40, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43;(viii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 31, 34, 37, and 40, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 46;(ix) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 28, 31, 34, 37, and 40, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 49;(x) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 44;(xi) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 47;(xii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 29, 32, 35, 38, and 41, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 50;(xiii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 45;(xiv) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 48;(xv) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 30, 33, 36, 39, and 42, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 51;(xvi) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 67;(xvii) the first CHS domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 70;(xviii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 52, 55, 58, 61, and 64, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 73;(xix) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 68;51ACTIVE 718034029V1(xx) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 71;(xxi) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 53, 56, 59, 62, and 65, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 74;(xxii) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 69;(xxiii) the first CH3 domain of the CHS domain pair has a sequence selected from any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 72; or(xxiv) the first CH3 domain of the CH3 domain pair has a sequence selected from any one of SEQ ID NOS: 54, 57, 60, 63, and 66, and the second CH3 domain of the CH3 domain pair has a sequence set forth in SEQ ID NO: 75.

20. The CH3 domain pair according to claim 19, wherein:the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 28, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43.

21. The CH3 domain pair according to claim 19, wherein:the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 31, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43.

22. The CH3 domain pair according to claim 19, wherein:the first CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 34, and the second CH3 domain of the CH3 domain pair has the sequence set forth in SEQ ID NO: 43.

23. A polypeptide complex comprising two polypeptide chains, wherein:(a) the first polypeptide chain comprises, in order, from the N-terminus to the C-terminus, a variable domain of a variable domain pair, and a CH3 domain of a CH3 domain pair; and (b) the second polypeptide chain comprises, in order, from the N-terminus to the C-terminus, the second variable domain of the variable domain pair, and the second CH3 domain of the CH3 domain pair;wherein the CH3 domain pair is a CH3 domain pair according to any one of claims 1 to 52ACTIVE 718034029V122; andwherein the variable domain pair consists of a VH domain and a VL domain.

24. The polypeptide complex according to claim 23, wherein where a CH3 domain of the CH3 domain pair comprises substitution 398Q, the variable domain immediately N-terminal to said CH3 domain in the polypeptide chain is a VL domain.

25. The polypeptide complex according to claim 23 or 24, wherein where a CH3 domain of the CH3 domain pair comprises substitution 341A, the variable domain immediately N-terminal to said CH3 domain in the polypeptide chain is a VH domain.

26. A CH3 domain pair, wherein:a) the first CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of:(i) 341A;(ii) 373F;(iii) 398Q; and(iv) 404Y; and / orb) the second CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of:(I) 341A;(ii) 373F; and(iii) 404Y;wherein the positions are numbered according to the Eu index.

27. The CH3 domain pair according to claim 26, wherein:a) the first CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of:(i) G341A;(ii) Y373F;(iii) L398Q; and(iv) F404Y; and / orb) the second CH3 domain of the CH3 domain pair comprises one or more amino acid substitutions selected from the group consisting of:(i) G341A(ii) Y373F; and(iii) F404Y.53ACTIVE 718034029V128. The CH3 domain pair according to claim 26 or 27, wherein:a) the first CH3 domain of the CHS domain pair comprises:(i) amino acid substitutions L398Q and F404Y; and optionally(ii) amino acid substitution Y373F; and / orb) the second CH3 domain of the CH3 domain pair comprises:(i) amino acid substitution G341A; and / or(ii) amino acid substitutions Y373F and F404Y.

29. The CH3 domain pair according to any one of claims 26 to 28, wherein:a) the first CH3 domain of the CHS domain pair comprises:(i) amino acid substitutions L398Q and F404Y; and optionally(ii) amino acid substitution Y373F; andb) the second CH3 domain of the CH3 domain pair comprises:(i) amino acid substitutions Y373F and F404Y; and optionally(ii) amino acid substitution G341A.

30. The CH3 domain pair according to claim 29, wherein:a) the first CH3 domain of the CHS domain pair comprises:(i) amino acid substitutions L398Q and F404Y; and optionally(ii) amino acid substitution Y373F; andb) the second CH3 domain of the CH3 domain pair comprises:(i) amino acid substitution G341A; and(ii) amino acid substitutions Y373F and F404Y.

31. The CH3 domain pair according to any one of claims 26 to 28, wherein:(A)(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;(B)(a) the first CHS domain of the CH3 domain pair comprises amino acid substitutions L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F and F404Y;(C)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;54ACTIVE 718034029V1(D)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y;(E)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitution G341A; or(F)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, and F404Y.

32. The CH3 domain pair according to any one of claims 26 to 28, wherein:(A)(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F and F404Y;(B)(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;(C)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y;(D)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F and F404Y; OR(F)(a) the first CH3 domain of the CH3 domain pair comprises amino acid substitution Y373F, L398Q and F404Y; and(b) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

33. The CH3 domain pair according to claim 32, wherein:55ACTIVE 718034029V1a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions L398Q and F404Y; andb) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

34. The CH3 domain pair according to claim 32, wherein:a) the first CH3 domain of the CH3 domain pair comprises amino acid substitutions Y373F, L398Q and F404Y; andb) the second CH3 domain of the CH3 domain pair comprises amino acid substitutions G341A, Y373F, and F404Y.

35. The CH3 domain pair according to any one of claims 26 to 34, wherein the first CH3 domain or the second CH3 domain of the CH3 domain pair comprises amino acid substitution L368V or L368I.

36. The CH3 domain pair according to any one of claims 26 to 35, wherein the first and / or second CH3 domain comprises one or more further amino acid substitutions, for example one or more further amino acid substitutions.

37. The CH3 domain pair according to claim 36, wherein the CH3 domain pair further comprises knob-into-hole (KIH) amino acid substitutions.

38. The CH3 domain pair according to claim 36, wherein in the CH3 domain pair:a) one CH3 domain of the CH3 domain pair further comprises amino acid substitution Y349C and optionally amino acid substitution K370R; andb) the other CH3 domain of the CH3 domain pair further comprises amino acid substitutions S354C and E357W.

39. The CH3 domain pair according to claim 38, wherein:a) the first CH3 domain of the CH3 domain pair further comprises amino acid substitution Y349C and optionally amino acid substitution K370R; andb) the second CH3 domain of the CH3 domain pair further comprises amino acid substitutions S354C and E357W.

40. The CH3 domain pair according to claim 39, wherein:a) the first CH3 domain of the CH3 domain pair further comprises amino acid substitution Y349C and K370R; and56ACTIVE 718034029V1b) the second CH3 domain of the CH3 domain pair further comprises amino acid substitutions S354C and E357W.

41. The CH3 domain pair according to any one of ciaims 26 to 40, wherein the first and second CH3 domains of the CH3 domain pair are IgG 1, I gG2, I gG3, or lgG4 CH3 domains.

42. The CH3 domain pair according to any one of ciaims 26 to 40, wherein the first and second CH3 domains of the CH3 domain pair are lgG1 CH3 domains.

43. A complex comprising a CH3 domain pair according to any one of claims 1 to 22 and 26 to 42, wherein at least one moiety is attached to at least one CH3 domain of the CH3 domain pair.

44. The complex according to claim 44, wherein the at least one moiety is attached to the N-terminus or the C-terminus of the CH3 domain.

45. The complex according to claim 43, wherein at least two moieties are attached to the CH3 domain pair.

46. The complex according to claim 45, wherein the moieties are attached to the N- and / or C-terminus of the CH3 domains of the CH3 domain pair.

47. The complex according to any one of claims 43 to 46, wherein the at least one moiety is an antigen-binding moiety, enzyme, cytokine, or receptor subunit.

48. The complex according to claim 47, wherein the antigen-binding moiety is an Fv fragment, single-chain Fv, single-chain antibody, diabody, single-chain diabody, single-domain antibody, VHH, nanobody, oranticalin.

49. A polypeptide complex comprising two polypeptide chains, wherein:(a) the first polypeptide chain comprises, in order, from the N-terminus to the C-terminus, a variable domain of a variable domain pair, and a CH3 domain of a CH3 domain pair; and (b) the second polypeptide chain comprises, in order, from the N-terminus to the C-terminus, the second variable domain of the variable domain pair, and the second CH3 domain of the CH3 domain pair;wherein the CH3 domain pair is a CH3 domain pair according to any one of claims 26 to 42; andwherein the variable domain pair consists of a VH domain and a VL domain.57ACTIVE 718034029V150. The polypeptide complex according to claim 49, wherein where a CH3 domain of the CH3 domain pair comprises substitution 398Q, the variable domain immediately N-terminal to said CH3 domain in the polypeptide chain is a VL domain.

51. The polypeptide complex according to claim 49 or 50, wherein where a CH3 domain of the CH3 domain pair comprises substitution 341A, the variable domain immediately N-terminal to said CH3 domain in the polypeptide chain is a VH domain.

52. The polypeptide complex according to any one of claims 49 to 51, wherein at least one moiety is attached to at least one CH3 domain of the CH3 domain pair or at least one variable domain of the variable domain pair.

53. The polypeptide complex according to claim 52, wherein the at least one moiety is attached to the N-terminus or the C-terminus of the CH3 domain or variable domain.

54. The polypeptide complex according to claim 52, wherein at least two moieties are attached to the CH3 domain pair or variable domain.

55. The polypeptide complex according to claim 54, wherein the moieties are attached to the N- and / or C-terminus of the CH3 domains of the CH3 domain pair or variable domain pair.

56. The polypeptide complex according to any one of claims 52 to 55, wherein the at ieast one moiety is an antigen-binding moiety, enzyme, cytokine, or receptor subunit.

57. The polypeptide complex according to claim 56, wherein the antigen-binding moiety is an Fv fragment, single-chain Fv, singie-chain antibody, diabody, single-chain diabody, singledomain antibody, VHH, nanobody, or anticalin.

58. A nucleic acid encoding the CH3 domain pair according to any one of claims 1 to 22 and 26 to 42, the complex according to any one of ciaims 43-48 or the polypeptide complex according to any one of claims 23 to 25 and 49 to 57.58ACTIVE 718034029V1

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