WO2025226901A1 - Pairing of two-chain constructs - Google Patents

Abstract

The present invention relates to pairing of two chains of a multispecific construct and uses thereof.

Description

PAIRING OF TWO-CHAIN CONSTRUCTS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 63/638,468, filed April 25, 2024, which is hereby incorporated by reference in its entirety. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY [0002] The present application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The computer readable format copy of the Sequence Listing, which was created on April 2, 2025, is named 10050-WO01-SEC_ST26.xml and is 5,313 bytes in size. FIELD OF THE INVENTION [0003] The present invention relates to pairing of two chains of a CH3 construct, monovalent IgG, or multispecific construct, and uses thereof. Such pairing is useful for increased yield, pairing, and/or purity of the CH3 construct, monovalent IgG, or multispecific construct. BACKGROUND OF THE INVENTION [0004] Bispecific molecules are challenging to manufacture for several reasons, including chain number and chain pairing. For example, heteroIg molecules that consist of two distinct light chains and two heavy chains and can be assembled into ten different unique combinations of those polypeptide chains. Protein engineering can be done to bias chain pairing to favor the assembly of the correct heteroIg and minimize the production of the nine undesired species. Multiple technologies, including knob-in-hole (KiH) and electrostatic steering using charge pair mutations (CPM). Both KiH and CPM technologies can achieve greater than 90% correct pairing of the desired heavy chains. Correct pairing of the light chains to their cognate heavy chains is more challenging because it contains two distinct interfaces, occurs in each Fab arm, and VH/VL interface is unique to each Fab. There are some approaches to correctly pairing the light and heavy chains, including CrossMAb and DuetMab. One additional challenge beyond correct pairing is chain balance. Frequently, some chains are easier to express than others leading to imbalance in chain number, forcing the production of undesired products. [0005] One solution to improving the generation of bispecific molecules is to replace one of the Fab arms with a moiety, such an scFv or VH only binder. By using a moiety, the chain pairing problem between the light and heavy chain is solved, and the chain number is reduced from four to three. Here, we further simplify bispecific molecule production by fusing the light chain to Fc while using a moiety, thereby lowering the chain number from three to two. In addition, Fabs have conserved interactions between CH1 and Ck, and semi-conserved interactions between VH and Vk. Weakening of the CH3 interaction in CH3 domains having charge pair mutations results in more Fab-driven pairing, and was determined to improve purity and yield of constructs herein. SUMMARY OF THE INVENTION [0006] The present invention provides a CH3 construct comprising a first CH3 domain comprising a K409R mutation, and a second CH3 domain. In an embodiment, the CH3 construct comprises a V397M mutation in the first or second CH3 domain. In an embodiment, the CH3 construct comprises a V397M mutation in each of the first and second CH3 domains. [0007] The present invention provides a CH3 construct comprising a first CH3 domain and a second CH3 domain, wherein the first CH3 domain and/or the second CH3 domain comprises a V397M mutation. In an embodiment, the CH3 construct comprises a V397M mutation in the first CH3 domain and in the second CH3 domains. [0008] In an embodiment, the CH3 construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in the first CH3 domain. [0009] In an embodiment, the CH3 construct comprises a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in the second CH3 domain. [0010] In an embodiment, the CH3 construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in the first CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in the second CH3 domain. [0011] In an embodiment, the CH3 construct comprises two V397M mutations. In an embodiment, the CH3 construct comprises a K409R and a V397M mutation. In an embodiment, the CH3 construct comprises a K409R and two V397M mutations. In an embodiment, the CH3 construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation. In an embodiment, the CH3 construct comprises a V397M mutation, a K392D mutation, and a K409D mutation. In an embodiment, the CH3 construct comprises a V397M mutation, a K392D mutation, a K439D mutation, and a K409D mutation. In an embodiment, the mutations are in CH3 domain(s). In an embodiment, the CH3 construct comprises two CH3 domains, wherein the CH3 construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. [0012] In an embodiment, the CH3 construct comprises two CH3 domains, wherein the CH3 construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0013] In an embodiment, the CH3 construct comprises two CH3 domains, wherein the CH3 construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. [0014] In an embodiment, the CH3 construct comprises a CH3 domain, and a Fab, and wherein the CH3 domain comprises a K409R and/or a V397M mutation. [0015] In an embodiment, the CH3 construct comprises a CH3 disulfide. [0016] In an embodiment, the CH3 construct comprises a hinge disulfide and a Fab disulfide. [0017] In an embodiment, the CH3 construct is able to be produced at a higher yield compared to a CH3 construct that does not have the K409R and/or V397M mutations. In an embodiment, the CH3 construct is able to be produced at a higher yield compared to a CH3 construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the CH3 construct is able to be produced at a higher yield compared to a CH3 construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0018] In an embodiment, the CH3 construct is able to be produced with higher purity compared to a CH3 construct that does not have the K409R and/or V397M mutations. In an embodiment, the CH3 construct is able to be produced at a higher purity compared to a CH3 construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the CH3 construct is able to be produced at a higher purity compared to a CH3 construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. In an embodiment, purity is determined by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis. [0019] In an embodiment, the CH3 construct has improved pairing compared to a CH3 construct that does not have the K409R and/or V397M mutations. In an embodiment, the CH3 construct has improved pairing compared to a CH3 construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the CH3 construct has improved pairing compared to a CH3 construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0020] The present invention also provides a method of weakening an interaction between a first and second CH3 domains comprising charge pair mutations in a CH3 construct, comprising mutating valine at position 397 to methionine in the first and second CH3 domains, and mutating lysine at position 409 to arginine in the first CH3 domain. In an embodiment, the lysine at position 409 is mutated to arginine in a CH3 domain which further comprises a D356K mutation and a D399K mutation. [0021] The present invention also provides a method of weakening an interaction between two CH3 domains in a CH3 construct, comprising mutating valine at position 397 to methionine, and mutating lysine at position 409 to arginine. In an embodiment, the CH3 construct comprises a Fab comprising a LCVR and a HCVR pair, and a Ck and a CH1 pair. In an embodiment, the CH3 construct has improved purity compared to a CH3 construct that does not have the K409R and/or V397M mutations. In an embodiment, the CH3 construct has improved purity compared to a CH3 construct that does not have the K409R and/or V397M mutations. In an embodiment, the CH3 construct has improved pairing compared to a CH3 construct that does not have the K409R and/or V397M mutations. In an embodiment, purity is determined by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis. [0022] In an embodiment, the CH3 construct comprises a K409R and a V397M mutation. [0023] The present invention provides a method of treating a disease or disorder in a patient comprising administering to the patient a CH3 construct of the present invention. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [0024] The present invention provides a CH3 construct of the present invention for use in therapy. [0025] The present invention provides a CH3 construct of the present invention for use in treating a disease or disorder. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [0026] The present invention provides a CH3 construct of the present invention for the manufacture of a medicament for the treatment of a disease or disorder. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [0027] The present invention provides a pharmaceutical composition comprising a CH3 construct of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients. [0028] In an embodiment, numbering is according to Eu numbering. [0029] In an embodiment, the K409R mutation is on the same side as the HC. [0030] The present invention provides a monovalent IgG comprising a first CH3 domain comprising a K409R mutation, and a second CH3 domain. In an embodiment, the monovalent IgG comprises a V397M mutation in the first or second CH3 domain. In an embodiment, the monovalent IgG comprises a V397M mutation in each of the first and second CH3 domains. [0031] . [0032] The present invention provides a monovalent IgG comprising a first CH3 domain and a second CH3 domain, wherein the first CH3 domain and/or the second CH3 domain comprises a V397M mutation. In an embodiment, the monovalent IgG comprises a V397M mutation in the first CH3 domain and in the second CH3 domains. [0033] In an embodiment, the monovalent IgG comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in the first CH3 domain. [0034] In an embodiment, the monovalent IgG comprises a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in the second CH3 domain. [0035] In an embodiment, the monovalent IgG comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in the first CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in the second CH3 domain. [0036] In an embodiment, the monovalent IgG comprises two V397M mutations. In an embodiment, the monovalent IgG comprises a K409R and a V397M mutation. In an embodiment, the monovalent IgG comprises a K409R and two V397M mutations. In an embodiment, the monovalent IgG comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation. In an embodiment, the monovalent IgG comprises a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation. In an embodiment, the monovalent IgG comprises a V397M mutation, a K392D mutation, and a K409D mutation. In an embodiment, the mutations are in CH3 domain(s). In an embodiment, the monovalent IgG comprises two CH3 domains, wherein the monovalent IgG comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the monovalent IgG comprises two CH3 domains, wherein the monovalent IgG comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0037] In an embodiment, the monovalent IgG comprises a CH3 domain, and a Fab, and wherein the CH3 domain comprises a K409R and/or a V397M mutation. [0038] In an embodiment, the monovalent IgG comprises a CH3 disulfide. [0039] In an embodiment, the monovalent IgG comprises a hinge disulfide and a Fab disulfide. [0040] In an embodiment, the monovalent IgG is able to be produced at a higher yield compared to a monovalent IgG that does not have the K409R and/or V397M mutations. In an embodiment, the monovalent IgG is able to be produced at a higher yield compared to a monovalent IgG that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the monovalent IgG is able to be produced at a higher yield compared to a monovalent IgG that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0041] In an embodiment, the monovalent IgG is able to be produced with higher purity compared to a monovalent IgG that does not have the K409R and/or V397M mutations. In an embodiment, the monovalent IgG is able to be produced at a higher purity compared to a monovalent IgG that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the monovalent IgG is able to be produced at a higher purity compared to a monovalent IgG that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. In an embodiment, purity is determined by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis. [0042] In an embodiment, the monovalent IgG has improved pairing compared to a monovalent IgG that does not have the K409R and/or V397M mutations. In an embodiment, the monovalent IgG has improved pairing compared to a monovalent IgG that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the monovalent IgG has improved pairing compared to a monovalent IgG that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0043] The present invention also provides a method of weakening an interaction between a first and second CH3 domains comprising charge pair mutations in a monovalent IgG, comprising mutating valine at position 397 to methionine in the first and second CH3 domains, and mutating lysine at position 409 to arginine in the first CH3 domain. In an embodiment, the lysine at position 409 is mutated to arginine in a CH3 domain which further comprises a D356K mutation and a D399K mutation. [0044] The present invention also provides a method of weakening an interaction between two CH3 domains in a monovalent IgG, comprising mutating valine at position 397 to methionine, and mutating lysine at position 409 to arginine. In an embodiment, the monovalent IgG comprises a Fab comprising a LCVR and a HCVR pair, and a Ck and a CH1 pair. In an embodiment, the monovalent IgG has improved purity compared to a monovalent IgG that does not have the K409R and/or V397M mutations. In an embodiment, the monovalent IgG has improved purity compared to a monovalent IgG that does not have the K409R and/or V397M mutations. In an embodiment, purity is determined by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis. In an embodiment, the monovalent IgG has improved pairing compared to a monovalent IgG that does not have the K409R and/or V397M mutations. [0045] In an embodiment, the monovalent IgG comprises a K409R and a V397M mutation. [0046] The present invention provides a method of treating a disease or disorder in a patient comprising administering to the patient a monovalent IgG of the present invention. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [0047] The present invention provides a monovalent IgG of the present invention for use in therapy. [0048] The present invention provides a monovalent IgG of the present invention for use in treating a disease or disorder. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [0049] The present invention provides a monovalent IgG of the present invention for the manufacture of a medicament for the treatment of a disease or disorder. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [0050] The present invention provides a pharmaceutical composition comprising a monovalent IgG of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients. [0051] The present invention provides a multispecific construct comprising at least one moiety attached to a monovalent IgG. [0052] The present invention provides a multispecific construct comprising a K409R mutation. [0053] The present invention provides a multispecific construct comprising a V397M mutation. [0054] In an embodiment, the multispecific construct comprises two V397M mutations. In an embodiment, the multispecific construct comprises a K409R and a V397M mutation. In an embodiment, the multispecific construct comprises a K409R and two V397M mutations. [0055] In an embodiment, the multispecific construct comprises a monovalent IgG of the present invention. [0056] In an embodiment, the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in the CH3 domain. In an embodiment, the multispecific construct comprises a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in the CH3 domain. In an embodiment, the multispecific construct comprises a V397M mutation, a K392D mutation, and a K409D mutation in the CH3 domain. In an embodiment, the multispecific construct comprises two CH3 domains, wherein the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the multispecific construct comprises two CH3 domains, wherein the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0057] In an embodiment, the multispecific construct comprises one moiety. In an embodiment, the multispecific construct comprises two moieties. In an embodiment, the multispecific construct comprises three moieties. In an embodiment, the multispecific construct comprises four, five, six, seven, eight, or nine moieties. [0058] In an embodiment, the monovalent IgG comprises an N-terminus and a C- terminus, and wherein the moiety is attached to the N-terminus of the monovalent IgG. [0059] In an embodiment, the monovalent IgG Fab comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), and wherein the moiety is attached to the N-terminus of the LCVR. [0060] In an embodiment, the monovalent IgG Fab comprises a LCVR and a HCVR, and wherein the moiety is attached to the N-terminus of the HCVR. [0061] In an embodiment, the monovalent IgG comprises an N-terminus and a C- terminus, and wherein the moiety is attached to the C-terminus of the monovalent IgG. [0062] In an embodiment, the monovalent IgG comprises an Fc. [0063] In an embodiment, the monovalent IgG Fc comprises a light chain (LC) CH3 domain and a heavy chain (HC) CH3 domain, and wherein the moiety is attached to the C- terminus of the monovalent IgG LC CH3 domain. [0064] In an embodiment, the monovalent IgG Fc comprises a LC CH3 domain and a HC CH3 domain, and wherein the moiety is attached to the C-terminus of the monovalent IgG HC CH3 domain. [0065] In an embodiment, the multispecific construct comprises a first moiety and a second moiety each attached to the N-terminus of the monovalent IgG. [0066] In an embodiment, the multispecific construct comprises a first moiety and a second moiety each attached to the C-terminus of the monovalent IgG. [0067] In an embodiment, the multispecific construct comprises a first moiety attached to the N-terminus of the monovalent IgG, and a second moiety attached to the C-terminus of the monovalent IgG. [0068] In an embodiment, a moiety is attached to the monovalent IgG by a linker. In an embodiment, the linker is G4Q. In an embodiment, the linker is (G4Q)2. [0069] The present invention provides a monovalent IgG comprising D356K and D399K mutations on one chain, and K392D, K409D, and K439D mutations on another chain. [0070] The present invention provides a monovalent IgG comprising D356K and D399K mutations on one chain, and K392D, and K409D mutations on another chain. [0071] The present invention provides a multispecific construct comprising D356K and D399K mutations on one chain, and K392D, K409D, and K439D mutations on another chain. [0072] The present invention provides a multispecific construct comprising D356K and D399K mutations on one chain, and K392D, and K409D mutations on another chain. [0073] The present invention provides a monovalent IgG comprising K409R, D356K, V397M, and D399K mutations on one chain, and V397M, K392D, K409D, and K439D mutations on another chain. [0074] The present invention provides a monovalent IgG comprising K409R, D356K, V397M, and D399K mutations on one chain, and V397M, K392D, and K409D mutations on another chain. [0075] In an embodiment, numbering is according to Eu numbering. [0076] In an embodiment, the K409R mutation is on the same side as the HC. [0077] The present invention provides a multispecific construct comprising a first CH3 domain comprising a K409R mutation, and a second CH3 domain. In an embodiment, the multispecific construct comprises a V397M mutation in the first or second CH3 domain. In an embodiment, the multispecific construct comprises a V397M mutation in each of the first and second CH3 domains. [0078] . The present invention provides a multispecific construct comprising a first CH3 domain and a second CH3 domain, wherein the first CH3 domain and/or the second CH3 domain comprises a V397M mutation. In an embodiment, the multispecific construct comprises a V397M mutation in the first CH3 domain and in the second CH3 domains. [0079] In an embodiment, the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in the first CH3 domain. [0080] In an embodiment, the multispecific construct comprises a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in the second CH3 domain. [0081] In an embodiment, the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in the first CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in the second CH3 domain. [0082] The present invention provides a multispecific construct comprising two V397M mutations. In an embodiment, the multispecific construct comprises a K409R and a V397M mutation. In an embodiment, the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation. In an embodiment, the multispecific construct comprises a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation. In an embodiment, the multispecific construct comprises a V397M mutation, a K392D mutation, and a K409D mutation. In an embodiment, the mutations are in CH3 domain(s). In an embodiment, the multispecific construct comprises two CH3 domains, wherein the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the multispecific construct comprises two CH3 domains, wherein the multispecific construct comprises a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0083] The present invention provides a multispecific construct comprising K409R, D356K, V397M, and D399K mutations on one chain, and V397M, K392D, K409D, and K439D mutations on another chain. [0084] The present invention provides a multispecific construct comprising K409R, D356K, V397M, and D399K mutations on one chain, and V397M, K392D, and K409D mutations on another chain. [0085] In an embodiment, the moiety is a cytokine fusion, cytokine, chemokine, receptor ligand, or biologically active peptide. [0086] In an embodiment, the moiety is an scFv. In an embodiment, the scFv specifically binds CD3. [0087] In an embodiment, the moiety is a VHH. [0088] In an embodiment, the moiety is a Unidab. [0089] In an embodiment, the Fab specifically binds a tumor-associated target. [0090] In an embodiment, the multispecific construct comprises a CH3 disulfide. [0091] In an embodiment, the multispecific construct comprises a hinge disulfide and a Fab disulfide. [0092] In an embodiment, the multispecific construct is able to be produced at a higher yield compared to a multispecific construct that does not have the K409R and/or V397M mutations. In an embodiment, the multispecific construct is able to be produced at a higher yield compared to a multispecific construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the multispecific construct is able to be produced at a higher yield compared to a multispecific construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0093] In an embodiment, the multispecific construct is able to be produced with higher purity compared to a multispecific construct that does not have the K409R and/or V397M mutations. In an embodiment, the multispecific construct is able to be produced at a higher purity compared to a multispecific construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the multispecific construct is able to be produced at a higher purity compared to a multispecific construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. In an embodiment, purity is determined by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis. [0094] In an embodiment, the multispecific construct has improved pairing compared to a multispecific construct that does not have the K409R and/or V397M mutations. In an embodiment, the multispecific construct has improved pairing compared to a multispecific construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the multispecific construct has improved pairing compared to a multispecific construct that does not have the K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. [0095] The present invention also provides a method of weakening an interaction between a first and second CH3 domains comprising charge pair mutations in a multispecific construct, comprising mutating valine at position 397 to methionine in the first and second CH3 domains, and mutating lysine at position 409 to arginine in the first CH3 domain. In an embodiment, the lysine at position 409 is mutated to arginine in a CH3 domain which further comprises a D356K mutation and a D399K mutation. [0096] The present invention also provides a method of weakening an interaction between two CH3 domains in a multispecific construct, comprising mutating valine at position 397 to methionine, and mutating lysine at position 409 to arginine. In an embodiment, the multispecific construct comprises a Fab comprising a LCVR and a HCVR pair, and a Ck and a CH1 pair. In an embodiment, the multispecific construct has improved purity compared to a multispecific construct that does not have the K409R and/or V397M mutations. In an embodiment, the multispecific construct has improved purity compared to a multispecific construct that does not have the K409R and/or V397M mutations. In an embodiment, the multispecific construct has improved pairing compared to a multispecific construct that does not have the K409R and/or V397M mutations. In an embodiment, purity is determined by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis. [0097] The present invention provides a method of treating a disease or disorder in a patient comprising administering to the patient a multispecific construct of the present invention. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [0098] The present invention provides a multispecific construct of the present invention for use in therapy. [0099] The present invention provides a multispecific construct of the present invention for use in treating a disease or disorder. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [00100] The present invention provides a multispecific construct of the present invention for the manufacture of a medicament for the treatment of a disease or disorder. In an embodiment, the disease or disorder is cancer. In an embodiment, the disease or disorder is an immune disease. [00101] The present invention provides a pharmaceutical composition comprising a multispecific construct of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients. [00102] In an embodiment, numbering is according to Eu numbering. [00103] In an embodiment, the K409R mutation is on the same side as the HC. BRIEF DESCRIPTION OF THE DRAWINGS [00104] Figure 1 displays molecules with different moiety locations. scFvs are shown as exemplary moieties, but it is understood that any moiety can be used. [00105] Figure 2 depicts molecule pairing. [00106] Figure 3 depicts the addition of charge pair mutations (CPM) into CH3 to guide pairing. [00107] Figure 4 depicts weakening CH3 to guide pairing. [00108] Figure 5 depicts exemplary orientations and disulfide positions of multispecific constructs. [00109] Figure 6a and Figure 6b depict the results of the matrix study supporting lower CH3 affinity and positions for scFv. [00110] Figure 7 depicts a comparison of multispecific constructs to other formats. [00111] Figure 8 depicts the strategy for determining if CH3 weakening improves pairing with knob-in-hole technology. [00112] Figure 9 depicts the results of the matrix study supporting lower CH3 affinity for knob-in-hole technology. [00113] Figure 10 depicts the strategy for determining if CH3 weakening improves pairing in the absence of an additional binding moiety. [00114] Figure 11 depicts the results supporting lower CH3 affinity improves pairing without an additional binding moiety. [00115] Figure 12 depicts a comparison of multispecific constructs using a VH binding domain as the moiety. DETAILED DESCRIPTION [00116] The use of only two chains offers a major advantage by simplifying construct development and selection in process development. The monovalent IgG chassis will also provide an opportunity to improve Fc pairing and reduce product related impurities that are often problematic for other 1 + 1 type formats. The use of a moiety as the second targeting domain removes the need to solve the HC/LC pairing problem, which can be a challenge for formats such as the hetero-IgG and IgG-Fab. Additionally, the ability to move the moiety to different termini will allow us to select for molecules with optimal geometry for activity. Adding additional moiety modules will create higher valency molecules. [00117] In addition, the charge distribution due to the native differences between CH1 and CL and the introduced charge from the CPMs simply purification. The CPMs KK and DDD (D356K and D399K (“KK”) and K392D, K409D, and K439D (“DDD”), have been shown to increase the percent of monovalent IgG after protein A purification (Gunasekaran et. al, Immunology, vol.285,issue 25 (June 2010)). However, to optimize the CH3 affinity to improve pairing, purity, and yield (for example) of a construct, a lower affinity CH3-CH3 heterodimer was engineered. Relying on the natural Fab pairing (VH/Vk and CH1/Ck) results in better pairing specificity than the non-natural heterodimer comprising the CPMs D356K and D399K, and K392D, K409D, and K439D. Therefore, constructs comprising the KK and DDD CPMs and the CH3 weakening mutations of the present invention demonstrate improved purity and yield compared to constructs comprising only the KK and DDD CPMs. [00118] CH3-CH3 interactions are described in Rispens et al., J Biol Chem, 2014 Feb 28; 289(9): 6098-6109. [00119] Constructs of the present invention demonstrate better pairing, improved purity, improved stability, higher production yields, less side products, and shorter development time than alternative formats. Determination of pairing, purity, stability, yield, and side products can be determined as described herein and according to methods known in the art. For example, purity, which is a higher percentage of heterodimer formation compared to homodimer formation, can be measured by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis. If a construct has higher purity during expression, and similar expression levels, it will have higher yield (amount of pure construct that is produced). [00120] V397M is preferred in KK and DDD CH3 chains, whereas K409R can only be used in the KK chain. V397M + K409R in KK and V397M in DDD chain can be referred to as “weak” CH3 pairing. The KK and DDD mutations are able to result in chain pairing, however the addition of V397M and/or K409R mutations substantially improve yield and pairing. It is understood that V397M and K409R mutations can also be used with KK and DD charge pair mutations ((D356K and D399K (“KK”) and K392D and K409D (“DD”; see e.g. Gunasekaran et. al, Immunology, vol.285,issue 25 (June 2010)). D356K and D399K (“KK”) and K392D, K409D, and K439D (“DDD”) mutations are described in Estes et al., iScience, 2021, Dec.17; 24(12). [00121] Unless otherwise indicated, all numbering described herein are according to Eu numbering, which is known in the art (see e.g. Edelman et al., PNAS 1969 May;63(1):78-85). It is envisaged that the mutations described herein might be made at the indicated position wherein the indicated position has a different natural amino acid (e.g. different allotype). For example, a CH3 domain that has E356 instead of D356 can also be mutated to lysine. [00122] Sequences for the Fc region are as follows: Fc with KK mutations (SEQ ID NO: 1), Fc with DDD mutations (SEQ ID NO: 2), Fc with KK and V397M and K409R mutations (SEQ ID NO: 3), Fc with DDD and V397M mutations (SEQ ID NO: 4). [00123] The K409R mutation can be on the same side as the HC, as shown in Figure 4. It is also envisaged that the K409R mutation may be on the same side as the LC. However, the DDD side and the LC are both more acidic, and it is easier to separate mispairs if acidic domains and basic domains are stacked to the same side. [00124] As used herein, “monovalent IgG” refers to a fusion of an Fc to the C-terminus of a light chain (LC), and the resulting LC-Fc fusion pairs with the natural heavy chain forming a heterodimeric structure. During expression, the correctly paired monovalent IgG and the LC- Fc homodimer are secreted; however, the heavy chain dimer is not secreted, which simplifies purification. The resulting monovalent IgG is an Fc-Fab core to which moieties can be attached. Attaching moieties to the monovalent IgG results in a “multispecific construct” that is able to specifically bind more than one target. For example, a multispecific construct may comprise a Fab that specifically binds one target, and an attached scFv that specifically binds another target. A monovalent IgG has been previously described (see e.g. Gunasekaran et. al, Immunology; vol.285, issue 25, June 2010; PCT Publication Number WO2015048272; and PCT Publication Number WO2014151910A1). [00125] It is envisaged that the mutations described herein will be useful for dimerization of asymmetric constructs. An “asymmetric construct” is a protein that comprises at least two non-identical chains, each having a CH3 domain. For example, an asymmetric construct includes a multispecific construct of the present invention. [00126] As used herein, a K409R or V397M mutation refers to changing the lysine to arginine at position 409 (for K409R) or the valine to a methionine at position 397 (for V397M). Mutating residues can be performed according to procedures that are well-understood (see e.g. Kenneth Walker and Jeremy King, Encyclopedia of Cell Biology; vol.1, 2023, pgs.161-169). [00127] A “CH3 construct” refers to a multichain construct that contains paired CH3 domains. In an embodiment, the CH3 domains comprise V397M and/or K409R mutations. In an embodiment, the CH3 domains comprise D356K and D399K mutations on one chain, and K392D, K409D, and K439D mutations on another chain. In an embodiment, the CH3 domains comprise D356K and D399K mutations on one chain, and K392D and K409D mutations on another chain. In an embodiment, the CH3 domains comprise a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation in a second CH3 domain. In an embodiment, the CH3 domains comprise a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation in one CH3 domain; and a V397M mutation, a K392D mutation, and a K409D mutation in a second CH3 domain. In an embodiment, the CH3 construct is an IgG. In an embodiment, the CH3 construct is a monovalent IgG. In an embodiment, the CH3 construct is a multispecific construct. [00128] As used herein, a “moiety” refers to a polypeptide, protein, or other construct (biological or synthetic). Said moiety may be attached to a CH3 construct, monovalent IgG, or an asymmetric construct. A moiety can be attached according to methods known in the art (e.g. a linker). Examples of a moiety include cytokine fusions, cytokines, chemokines, receptor ligands, and biologically active peptides. A moiety also includes a light chain single domain antibody, multimers and mixtures thereof, Fab, VHH, or scFv. A moiety is able to specifically bind to a target. [00129] “Specifically binds” refers to binding to an antigen (target) with a dissociation constant (KD) is ≤10-7 M as measured via a surface plasma resonance technique (e.g., BIACore, GE-Healthcare Uppsala, Sweden) or Kinetic Exclusion Assay (KinExA, Sapidyne, Boise, Idaho). [00130] As used herein, an “antibody” is an immunoglobulin molecule comprising 2 heavy chains (HCs) and 2 light chains (LCs) interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the CDRs contained therein. The CDRs are separated with regions that are more conserved, termed framework regions (“FR”). Each LCVR and HCVR is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the LC are referred to as “LCDR1, LCDR2, and LCDR3,” and the 3 CDRs of the HC are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of the residues which form specific interactions with the antigen. The functional ability of an antibody to bind a particular antigen is, thus, largely influenced by the amino acid residues within the six CDRs. [00131] An Fc comprises a CH2 domain and a CH3 domain. A CH2 domain is the second constant domain of the heavy chain, and a CH3 domain is the third constant domain of the heavy chain. As described herein, a CH2 and CH3 domain can be attached to a LC, thus forming an LC-CH2-CH3 polypeptide. A “LC CH3” domain is the CH3 domain that is on the same polypeptide (chain) as the LC. [00132] A CH3 construct or multispecific construct of the present invention can have one or more disulfide bonds (“disulfide” or “disulfides”) between the hinge (hinge disulfide), Fab (Fab disulfide), and/or CH3 (CH3 disulfide). Exemplary disulfides are shown in Figure 5. [00133] The CH3 constructs, monovalent IgG, and multispecific constructs of the present invention may be used to treat a disease or disorder in a patient. It is envisioned that said CH3 constructs, monovalent IgG, and multispecific constructs can be useful in the treatment of a disease or disorder in which binding at least two targets and eliciting a biological effect is beneficial. [00134] Also provided herein are one or more nucleic acid sequences encoding a CH3 construct or multispecific construct of the present invention. [00135] A DNA molecule of the present invention is a DNA molecule that comprises a non-naturally occurring polynucleotide sequence encoding a polypeptide chain having the amino acid sequence of at least one of the polypeptide chains in a CH3 construct or multispecific construct of the present invention. [00136] Polynucleotides of the present invention can be expressed in a host cell after the sequences have been operably linked to an expression control sequence. The expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences. [00137] Transformed cells can be cultured under conditions that promote expression of the polypeptide chain, and the polypeptide chain recovered by conventional protein purification procedures. Polypeptide chains contemplated for use herein include substantially homogeneous recombinant mammalian polypeptides substantially free of contaminating endogenous materials. Cells containing the nucleic acid encoding a CH3 construct or multispecific construct polypeptide chain of the present invention also include hybridomas. [00138] A polynucleotide encoding an amino acid sequence of a polypeptide chain of a CH3 construct or multispecific construct of the present invention can comprise one or more additional sequences, for example, regulatory sequences, and/or can be part of a larger nucleic acid, for example, a vector. [00139] Vectors containing the polynucleotide sequences of the present invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. Examples of vectors include, but are not limited to, plasmids, viral vectors, non- episomal mammalian vectors and expression vectors, for example, recombinant expression vectors. [00140] As used interchangeably herein, "treatment" and/or "treating" and/or "treat" are intended to refer to all processes wherein there may be a slowing, interrupting, arresting, controlling, stopping, or reversing of the progression of the disorders described herein, but does not necessarily indicate a total elimination of all disorder symptoms. Treatment includes administration of a CH3 construct, monovalent IgG, or multispecific construct of the present invention for treatment of a disease or condition in a human that would benefit from activity of a CH3 construct, monovalent IgG, or multispecific construct of the present invention, and includes: (a) inhibiting further progression of the disease; and (b) relieving the disease, i.e., causing regression of the disease or disorder or alleviating symptoms or complications thereof. [00141] Therapeutically effective amounts (or dose) of a CH3 construct, monovalent IgG, or multispecific construct of the present invention can be administered. The amount of a CH3 construct, monovalent IgG, or multispecific construct that constitutes a therapeutically dose may vary with the indication treated, the weight of the patient, the calculated skin surface area of the patient. Dosing of a CH3 construct, monovalent IgG, or multispecific construct can be adjusted to achieve the desired effects. In many cases, repeated dosing may be required. Dosages and the frequency of administration may vary according to such factors as the route of administration, the particular CH3 construct, monovalent IgG, or multispecific construct employed, the nature and severity of the disease to be treated, whether the condition is acute or chronic, and the size and general condition of the subject. [00142] A CH3 construct, monovalent IgG, or multispecific construct, or a pharmaceutical composition containing such a molecule, can be administered by any feasible method. Protein therapeutics will ordinarily be administered by a parenteral route, for example by injection, since oral administration, in the absence of some special formulation or circumstance, would lead to hydrolysis of the protein in the acid environment of the stomach. Subcutaneous, intramuscular, intravenous, intraarterial, intralesional, or peritoneal bolus injection are possible routes of administration. A CH3 construct, monovalent IgG, or multispecific construct also be administered via infusion, for example intravenous or subcutaneous infusion. [00143] CH3 constructs, monovalent IgGs, or multispecific constructs can be administered in the form of a composition comprising one or more additional components such as a physiologically acceptable carrier, excipient or diluent. Optionally, the composition additionally comprises one or more physiologically active agents. In various particular embodiments, the composition comprises one, two, three, four, five, or six physiologically active agents in addition to one or more CH3 constructs, monovalent IgGs, or multispecific constructs. [00144] As used herein, an “effective amount” means the amount of a CH3 construct, monovalent IgG, or multispecific construct of the present invention or pharmaceutical composition comprising such a CH3 construct, monovalent IgG, or multispecific construct that will elicit the biological or medical response of or desired therapeutic effect on a tissue, system, animal, mammal, or human that is being sought by the researcher, medical doctor, or other clinician. An effective amount of the CH3 construct, monovalent IgG, or multispecific construct may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the CH3 construct, monovalent IgG, or multispecific construct to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effect of the CH3 construct, monovalent IgG, or multispecific construct is outweighed by the therapeutically beneficial effects. Such benefit includes improving signs or symptoms of inflammatory disease(s). An effective amount can be readily determined by one skilled in the art, by the use of known techniques, and by observing results obtained under analogous circumstances. An effective amount of a CH3 construct, monovalent IgG, or multispecific construct of the present invention may be administered in a single dose or in multiple doses. In determining the effective amount for a patient, a number of factors are considered by the attending medical practitioner, including, but not limited to: the patient's size (e.g., weight or mass), body surface area, age, and general health; the specific disease or disorder involved; the degree of, or involvement, or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances known to medical practitioners. EXAMPLES EXAMPLE 1: MULTISPECIFIC CONSTRUCT ENGINEERING [00145] The formats disclosed herein are a heterodimer between a light chain-Fc fusion and a heavy chain with an scFv that binds CD3 attached to one of the four termini (Figure 1). To maximize yield and purity, it is critical to produce as much heterodimer material as possible while minimizing the production of light chain-Fc fusion homodimers. Protein engineering provides the best opportunity to maximize heterodimerization. The format contains three interfaces for heterodimerization: 1) CH3 on heavy chain Fc with CH3 on light chain Fc, 2) CH1 with Ck, and 3) VH with VL (Figure 2). Since every antibody contains a unique VH/Vk interface, CH3/CH3 and CH1/Ck interfaces were examined. To improve pairing, engineering was performed to introduce heterodimerization into the CH3 interface, optimize the CH3 interface affinity, and alternative disulfide positions between light chain-Fc fusion and the heavy chain were tested. [00146] CH3 heterodimerization was accomplished by charge-pair mutations (CPMs) (Figure 3). In Eu numbering, the CH3(++) CPMs consist of D356K and D399K, and the CH3(- --) CPMs consist of K392D, K409D, and K439D. The light chain was fused to the Fc with the CH3(---) CPM Fc. [00147] For optimizing the CH3 affinity, a lower affinity CH3-CH3 heterodimer was compared to a higher affinity CH3-CH3 heterodimer. Relying on the natural Fab pairing (VH/Vk and CH1/Ck) was thought to have better specificity than the non-natural CH3(++)/CH3(---) heterodimer. To weaken the CH3 interface, K409R and V397M mutations were introduced. K409R is a naturally occurring variation between IgG1 and IgG4 that results in greatly weakening the CH3 interface. K409R was only introduced into the CH3(++) chain. As mentioned above, the CH3(---) uses K409D as part of the engineered charge pairs. V397M also weakens the CH3 interface and occurs in some IgG2 and IgG3 allotypes. V397M was introduced into both CH3 domains. EXAMPLE 2: MUTLISPECIFIC CONSTRUCT PURITY AND YIELD [00148] For determining the effect of interface disulfides on heterodimerization, multiple different disulfide patterns between the light chain-Fc fusion and the heavy chain were compared. The following disulfide pairs were tested: 1.) the hinge disulfides and Ck/CH1 disulfide, 2.) only the hinge disulfides, or 3.) only the Ck/CH1 disulfide, 4.) only an asymmetric CH3 disulfide. Different disulfides may provide higher selectivity for chain pairing. [00149] To determine which strategy for heterodimerization is best, a matrix comparing each disulfide pattern with both high affinity CH3 (DDD + KK mutations) and low affinity CH3 (K409R, D356K, V397M, and D399K mutations on one chain, and V397M, K392D, K409D, and K439D mutations on another chain) was performed (Figure 5). To control for differences in VH and Vk on protein production, four different Fabs were tested. scFv attached to all four termini was performed to determine if there were positional effects. To estimate final purity, the protein A yield was corrected with analytical CEX MP%. [00150] The molecules were expressed at 4 mL scaling using an internal CHO cell line. The molecules were purified using magnetic proA beads (Genscript, catalog # L00695) using a KingFisher™ Flex Purification System (ThermoFisher). ProA beads were added to the media without cell removal for overnight protein capture. The beads were removed from the cultures and were washed 3x with PBS and 2x with molecular grade water. The samples were eluted from the beads using 100 mM acetic acid, pH 3.6. The eluted material was neutralized with 3M Tris (1.5% v/v). The approximate pH of the material is 5.0-5.3. [00151] The eluted material was analyzed using microcapillary electrophoresis (MCE), analytical size-exclusion chromatography (SEC), and analytical cation exchange chromatography (CEX). MCE was performed on LabChip GXII Touch HT Protein Characterization System (PerkinElmer). Reduced MCE was used to characterize chain number, chain ratio, and clipping. Non-reduced MCE was used to characterized disulfide formation and chain pairing. Analytical CEX was performed using a YMC BioPro SP-F 3 µm 4.6x30. The gradient consisted of 0 mM sodium chloride to 250 mM sodium chloride in 20 mM MES, pH 6.2. Analytical SEC was performed using a ACQUITY Protein BEH SEC 200Å 1.7 µm 4.6x300. The running buffer was 100mM Sodium Phosphate, 250mM NaCl, pH 6.8 at a flow rate of 0.4 ml/min. [00152] These data are shown in Figure 6. The low affinity CH3 outperformed the high affinity version for yield and purity for each binder tested. The disulfide position had higher levels of variation. In general, the constructs with the CH3 disulfide only or the hinge with the Fab disulfides had better overall yield and purity compared to the Fab only disulfides or the hinge only disulfides. Similar results were observed between the CH3 disulfide or the hinge disulfides with Fab disulfide combination. [00153] The lower affinity CH3 and Hinge+CH1/Ck disulfide was selected for comparison to BiTE^® molecules and Fab-scFv-Fc bispecific antibodies using the same targeting arms (Figure 7). Pairing and product quality were measured using analytical CEX and analytical SEC. Disulfide formation and pairing were monitored using microcapillary electrophoresis. We found that the molecule format of the present invention had greater yield (roughly ~2x) and comparable analytics with the BiTE molecules and Fab-scFv-Fc. Table 1: Comparison of molecule of the present invention with Fab-scFv-Fc and HLE-BiTE molecules. EXAMPLE 3: MULTISPECIFIC CONSTRUCT STABILITY [00154] The weak CH3 affinity with standard disulfide pattern and the CH3 only disulfide were selected for stability testing. These variants were compared with Fab-scFv-Fc and HLE-BiTE format. The selected variants were scaled up for stability testing 70 mg / ml. The scaled up material was purified using MabSelect™ SuRe™ (Cytiva). The resin was washed with 1X TBS, and the protein was eluted with 100 mM sodium acetate. As the material was eluted, it was immediately neutralized with 20 mM MES, pH 6.2 at ratio of 1 part elution buffer with 4 part neutralization buffer by in-line dilution. The material was further purified using CEX. The buffer system consisted of 20 mM MES, pH 6.2 with 0 mM sodium chloride in buffer A and 1M sodium chloride in buffer B. The gradient was 0-40% B over 40 CV. The material was concentrated to 70 mg / ml using Pierce™ Protein Concentrator PES (ThermoFisher). [00155] Accelerated stability testing was performed by incubating the material at 40°C for 2 weeks at 70 mg/ml concentration. Stability was tested by comparing the material before temperature stress (T0) to the material after 2 weeks (2wk40) by SEC. MP is main-peak. It indicates how much of the material is still a monomer. Pre-MP is pre-main peak. It indicates how much aggregation has happened. Post-MP is post main-peak. It indicates how much clipping has happened. [00156] Regardless of the location of the scFv, these new formats showed high stability and resistance to aggregation with time, further validating their utility as potential therapeutics. The amount of aggregation in all positions was also similar to the Fab-scFv-Fc format, indicating no loss in stability from V397M and K409R substitutions. The hinge and Ck/CH1 disulfides version has similar stability as the CH3 only disulfide at 70 mg/ml after 2 weeks at 40°C treatment. Table 2: Accelerated stability results. Ck/CH1 and hinge disulfide CH3 disulfide EXAMPLE 4: WEAKENED CH3 MUTATIONS APPLIED TO KNOB IN HOLE TECHNOLOGY [00157] To determine if the strategy of weakening the CH3 interaction could also improve pairing for knob-in-hole heterodimerization technology, four different Fabs were tested, and the scFv locations were varied to control for positional effects. A comparator benchmark included disulfides between Ck and CH1 and the hinge, the weakened CH3, and charge pair mutations (Figure 8). [00158] As above, the molecules were expressed at 4 mL scaling using an internal CHO cell line. The molecules were purified using magnetic proA beads (Genscript, catalog # L00695) using a KingFisher™ Flex Purification System (ThermoFisher). ProA beads were added to the media without cell removal for overnight protein capture. The beads were removed from the cultures and were washed 3x with PBS and 2x with molecular grade water. The samples were eluted from the beads using 100 mM acetic acid, pH 3.6. The eluted material was neutralized with 3M Tris (1.5% v/v). The approximate pH of the material is 5.0-5.3. [00159] The eluted material was analyzed using microcapillary electrophoresis (MCE) and analytical size-exclusion chromatography (SEC). MCE was performed on LabChip GXII Touch HT Protein Characterization System (PerkinElmer). Reduced MCE was used to characterize chain number, chain ratio, and clipping. Non-reduced MCE was used to characterized disulfide formation and chain pairing. Analytical SEC was performed using a ACQUITY Protein BEH SEC 200Å 1.7 µm 4.6x300. The running buffer was 100mM Sodium Phosphate, 250mM NaCl, pH 6.8 at a flow rate of 0.4 ml/min. [00160] To predict final yield, protein A yield was calculated and corrected with MCE main peak %. The MCE main peak % was able to identify the percentage of correctly paired species. For all Fabs tested and for all locations, there was improved calculated yield for the weakened CH3 interaction for knob-in-hole technology (Figure 9). EXAMPLE 5: WEAKENED CH3 MUTATIONS WITHOUT ADDITIONAL BINDER [00161] To determine if the improved yield observed for the weakened CH3 binding could be applied to constructs lacking a secondary binding moiety, four different Fab constructs were tested (Figure 10). Material was expressed at 4 mL scale and purified using protein A beads on KingFisher as above. The pairing efficiency was determined using SEC and MCE. Final yield was predicted by correcting the protein A yield by the SEC purity. The weakened CH3 mutations were determined to improve final predicted yield. EXAMPLE 6: WEAKENED CH3 MUTATIONS WITH OTHER SECONDARY BINDER MOIETY [00162] To determine if the weakened CH3 strategy could be extended to molecules with moieties other than an scFv, three different Fabs with 9 different VH binders were tested (Figure 12). These molecules were expressed at 4 mL scale and purified with KingFisher as above. Purity was assessed with microcapillary electrophoresis and analytical size-exclusion chromatography. The average calculated yield was higher than the Fab-VH-heteroFc molecules overall (Figure 12).

10050-US01-PRI SEQUENCES

Claims

CLAIMS What is claimed: 1. A multispecific construct comprising at least one moiety attached to a monovalent IgG, wherein the monovalent IgG comprises a first CH3 domain comprising a K409R mutation according to Eu numbering, and a second CH3 domain.

2. The multispecific construct of Claim 1, further comprising a V397M mutation according to Eu numbering in the first or second CH3 domain.

3. The multispecific construct of Claim 1, comprising a V397M mutation according to Eu numbering in each of the first and second CH3 domains.

4. A multispecific construct comprising at least one moiety attached to a monovalent IgG, wherein the monovalent IgG comprises a first CH3 domain and a second CH3 domain, wherein the first CH3 domain and/or the second CH3 domain comprises a V397M mutation according to Eu numbering.

5. The multispecific construct of Claim 4, comprising a V397M mutation according to Eu numbering in the first CH3 domain and in the second CH3 domains.

6. The multispecific construct of any one of Claims 1-5, comprising a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation according to Eu numbering in the first CH3 domain.

7. The multispecific construct of any one of Claims 1-6, comprising a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation according to Eu numbering in the second CH3 domain.

8. The multispecific construct of any one of Claims 1-7, comprising a K409R mutation, a D356K mutation, a V397M mutation, and a D399K mutation according to Eu numbering in the first CH3 domain; and a V397M mutation, a K392D mutation, a K409D mutation, and a K439D mutation according to Eu numbering in the second CH3 domain.

9. The multispecific construct of any one of claims 1-8, comprising one moiety.

10. The multispecific construct of any one of claims 1-8, comprising two moieties.

11. The multispecific construct of any one of claims 1-8, comprising three moieties.

12. The multispecific construct of any one of claims 1-8, comprising four, five, six, seven, eight, or nine moieties.

13. The multispecific construct of any one of Claims 1-12, wherein the monovalent IgG comprises an N-terminus and a C-terminus, and the moiety is attached to the N-terminus of the monovalent IgG.

14. The multispecific construct of Claim 13, wherein the monovalent IgG comprises a Fab comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), and the moiety is attached to the N-terminus of the LCVR.

15. The multispecific construct of Claim 13, wherein the monovalent IgG Fab comprises a LCVR and a HCVR, and the moiety is attached to the N-terminus of the HCVR.

16. The multispecific construct of any one of Claims 1-12, wherein the monovalent IgG comprises an Fc, N-terminus and a C-terminus, and the moiety is attached to the C- terminus of the monovalent IgG.

17. The multispecific construct of Claim 16, wherein the monovalent IgG Fc comprises a light chain (LC) CH3 domain and a heavy chain (HC) CH3 domain, and the moiety is attached to the C-terminus of the monovalent IgG LC CH3 domain.

18. The multispecific construct of Claim 16, wherein the monovalent IgG Fc comprises a LC CH3 domain and a HC CH3 domain, and the moiety is attached to the C-terminus of the monovalent IgG HC CH3 domain.

19. The multispecific construct of any one of Claims 13-15, wherein the multispecific construct comprises a first moiety and a second moiety each attached to the N-terminus of the monovalent IgG.

20. The multispecific construct of any one of Claims 16-18, wherein the multispecific construct comprises a first moiety and a second moiety each attached to the C-terminus of the monovalent IgG.

21. The multispecific construct of any one of Claims 13-20, wherein the multispecific construct comprises a first moiety attached to the N-terminus of the monovalent IgG, and a second moiety attached to the C-terminus of the monovalent IgG.

22. The multispecific construct of any one of Claims 1-21, wherein the moiety is a cytokine fusion, cytokine, chemokine, receptor ligand, or biologically active peptide.

23. The multispecific construct of any one of Claims 1-21, wherein the moiety is an scFv.

24. The multispecific construct of any one of Claims 1-21, wherein the moiety is a VHH.

25. The multispecific construct of any one of Claims 1-24, comprising a Fab, wherein the Fab specifically binds a tumor-associated target.

26. The multispecific construct of any one of Claims 1-25, wherein the multispecific construct is produced at a higher yield compared to a construct that does not have the K409R and/or V397M mutations according to Eu numbering.

27. A method of weakening an interaction between a first CH3 and second CH3 domains comprising charge pair mutations in a multispecific construct, comprising mutating valine at position 397 to methionine according to Eu numbering in the first and second CH3 domains, and mutating lysine at position 409 to arginine according to Eu numbering in the first CH3 domain.

28. The method of Claim 27, wherein the lysine at position 409 according to Eu numbering is mutated to arginine in the CH3 domain which further comprises a D356K mutation and a D399K mutation according to Eu numbering.

29. The method of Claim 27 or 28, wherein the multispecific construct has improved purity compared to a construct that does not have K409R and/or V397M mutations according to Eu numbering.

30. The method of Claim 29, wherein the purity is determined by analytical cation exchange, analytical size exclusion, or microcapillary electrophoresis.

31. The multispecific construct of any one of Claims 1-30 for use in treating a disease or disorder.

32. The multispecific construct of any one of Claims 1-30 for the manufacture of a medicament for the treatment of a disease or disorder.

33. A pharmaceutical composition comprising the multispecific construct of any one of Claims 1-29 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

34. The multispecific construct of any one of Claims 1-30, comprising a CH3 disulfide.

35. The multispecific construct of any one of Claims 1-30, comprising a hinge disulfide and a Fab disulfide.

36. A monovalent IgG comprising a CH3 domain comprising a K409R and/or a V397M mutation according to Eu numbering.