Modified ch1 heterodimeric proteins

Abstract

Provided and exemplified herein are heterodimeric proteins having a modified CH1 domain.

Description

MODIFIED CHI HETERODIMERIC PROTEINSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 709,878, filed October 21, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Bispecific antibodies are engineered antibodies capable of binding two different antigens or epitopes simultaneously. They have offered new targeted therapeutic modalities in oncology, immunology, and infectious diseases through unique mechanisms of action arising from the binding of two different antigens or epitopes simultaneously. However, there are several challenges associated with their development. These include technical difficulties in manufacturing, potential immunogenicity, low stability, and short half-life of the proteins, as well as their complex regulatory and approval process.SUMMARY

[0003] The number of bispecific antibodies in clinical testing is growing rapidly, and consequently, so is the need for improving developability bispecific antibodies for clinical use (e.g., efficient production and purification strategies). A common challenge of producing bispecific antibodies is the purification process and potential heterogeneity that can arise from the presence of two different binding arms. Several purification strategies designed for antibodies have been developed. For example, one approach involves affinity capture with a commercially available protein A resin. Another strategy involves the use of dual-column chromatography systems, where two separate affinity chromatography columns are used in tandem. Each column is packed with a specific ligand that can selectively bind to an arm of the antibody. Additionally, the use of alternative affinity ligands, such as protein L or protein G, which have different binding specificities compared to protein A, have been explored. However, these methods can be challenging with bispecific antibodies due to the multiplicity of structures generated during the production process (e.g., mispairing of heavy and light chains). Since the variants generated during the production process often copurify with the desired bispecific antibody product. Moreover, affinity chromatography purification of bispecific antibodies is often a two-step process (e.g., Protein G purification and Protein A purification), which can present certain manufacturing challenges. For example, a two-steppurification process may be more time-consuming, result in increased production costs, lead to reduced overall yield and lower productivity, and can affect the quality and stability of the final product. Thus, there is a need for more efficient purification strategies for bispecific antibodies.

[0004] Provided and exemplified herein are bispecific antibodies having improved developability (e.g., production and isolation thereof) and methods of making thereof. Provided herein are compositions comprising a heterodimeric protein, wherein the heterodimeric protein comprises a first heavy chain (HC) constant region and a second HC constant region, wherein: (i) the first HC constant region comprises a first CHI domain comprising a first CHI amino acid sequence; and (ii) the second HC constant region comprises a second CHI domain, wherein the second CHI domain comprises a second CHI amino acid sequence that is different from the first CHI amino acid sequence. In some embodiments, the first CHI domain comprises a human CHI amino acid sequence and the second CHI domain comprises a nonhuman CHI amino acid sequence. In some embodiments, the first CHI domain comprises an IgG CHI amino acid sequence and the second CHI domain comprises a non-IgG CHI amino acid sequence. In some embodiments, the first CHI domain comprises an IgGl CHI amino acid sequence and the second CHI domain comprises a non-IgGl CHI amino acid sequence. In some embodiments, the first CHI domain comprises an IgGl CHI amino acid sequence. In some embodiments, the second CHI domain exhibits at a 2-fold or greater reduction in binding to a resin having a ligand that specifically binds the first CHI domain. In some embodiments, the second CHI domain is not retained by a column comprising a resin having a ligand that specifically binds the first CHI domain. In some embodiments, the first CHI domain comprises a first FG-loop structure and the first CHI amino acid sequence comprises a first FG-loop amino acid sequence; and the second CHI domain comprises a second FG-loop structure and the second CHI amino acid sequence comprises a second FG-loop amino acid sequence that is different from the first FG-loop sequence. In some embodiments, the first FG-loop structure and the second FG-loop structure comprises CHI domain amino acid positions 203-208 per EU numbering. In some embodiments, (i) the first FG-loop structure comprises a human FG-loop amino acid sequence and the second FG-loop structure comprises a non-human FG-loop amino acid sequence; (ii) the first FG-loop structure comprises an IgG FG-loop amino acid sequence and the second FG-loop structure comprises a non-IgG FG-loop amino acid sequence; or (iii) the first FG-loop structure comprises an IgGl FG-loop amino acid sequence and the second FG-loop structure comprises a non-IgGl FG-loop amino acid sequence. In some embodiments, the first FG-loop structure comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the second FG-loop structure comprises the amino acid sequencerepresented by the formula (N- to C-terminus): XI - His - X2 - X3 - X4 - X5 - (Formula 1) wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys. In some embodiments, the second FG-loop structure comprises the amino acid sequence of any one of SEQ ID NOs: 2-7. In some embodiments, the first CHI domain comprises: (i) the first FG-loop structure comprising the amino acid sequence of SEQ ID NO: 1; and (ii) an amino acid sequence having at least 75% or greater sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: (i) the second FG-loop structure comprising the amino acid sequence of Formula 1 or any one SEQ ID NOs: 2-7; and (ii) an amino acid sequence having at least 30% or greater sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: (i) the second FG-loop structure comprising the amino acid sequence of Formula 1 or any one SEQ ID NOs: 2-7; and (ii) an amino acid sequence having at least 75% or greater sequence identity to SEQ ID NO: 14. In some embodiments, (i) the first HC constant region further comprises a first CH2 domain and a first CH3 domain; and (ii) the second HC constant region further comprises a second CH2 domain and a second CH3 domain. In some embodiments, (i) the first CH3 domain comprises a first CH3 domain amino acid sequence comprising a knob modification; and (ii) the second CH3 domain comprises a second CH3 domain amino acid sequence comprising a hole modification. In some embodiments, (i) the first CH3 domain comprises a first CH3 domain amino acid sequence comprising a hole modification; and (ii) the second CH3 domain comprises a second CH3 domain amino acid sequence comprising a knob modification. In some embodiments, the heterodimeric protein is an antibody. In some embodiments, the heterodimeric protein is a bispecific antibody. In some embodiments, the bispecific antibody further comprises: a first light chain variable domain and a first heavy chain variable domain; and a second light chain variable domain and a second heavy chain variable domain, wherein, optionally, the first light chain variable domain and the second light chain variable domain are different. In some embodiments, the first heavy chain variable domain is covalently linked to the first CHI domain and the second heavy chain variable domain is heavy chain variable domain is linked to the first CHI domain linked to the second CHI domain. In some embodiments, the bispecific antibody further comprises first light chain constant domain covalently linked to the first light chain variable domain, and a second light chain constant domain covalently linked to the second light chain variable domain.

[0005] Provided herein are polypeptides comprising 75% to 95% sequence identity to SEQ ID NO: 14 wherein polypeptide at positions 84-89 does not comprise SEQ ID NO: 1. In some embodiments, the polypeptide exhibits at a 2-fold or greater reduction in binding to a resinhaving a ligand that binds a CHI domain comprising SEQ ID NO: 14. In some embodiments, the polypeptide is not retained by a column comprising a resin having a ligand that specifically binds a CHI domain comprising SEQ ID NO: 14. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 14 represented by the formula: - XI - His - X2 -X3 - X4 - X5 - (Formula 1) wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys.

[0006] Provided herein are polypeptides comprising the amino acid sequence at positions 84-89 of SEQ ID NO: 14 represented by the formula: - XI - His - X2 - X3 - X4 - X5 -(Formula 1) wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys; and wherein the polypeptide comprises at least 75% sequence identity to SEQ ID NO: 14. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of any one of SEQ ID NOs: 2-7. In some embodiments, the polypeptide is an immunoglobulin heavy chain constant region CHI domain.

[0007] Provided herein are one or more polynucleotides encoding the heterodimeric protein disclosed herein or the polypeptide disclosed herein. In some embodiments, the one or more polynucleotides are one or more expression vectors.

[0008] Provided herein are host cells comprising the one or more polynucleotides disclosed herein. Provided herein are one or more host cells comprising the one or more polynucleotides disclosed herein.

[0009] Provided herein are methods of isolating a bispecific antibody comprising the heterodimeric protein disclosed herein or the polypeptide disclosed herein, the method comprising: (a) contacting the bispecific antibody with a support that binds first CHI domain; (b) washing the support; and (c) detaching the bispecific antibody from the support.

[0010] Provided herein are methods of separating a bispecific antibody comprising the heterodimeric protein disclosed herein or the polypeptide disclosed herein from a monomeric antibody component thereof or mono-specific antibody, the method comprising: (a) contacting the bispecific antibody with a support that binds first CHI domain; (b) washing the support; and (c) detaching the bispecific antibody from the support. In some embodiments, the support exhibits at least a 2-fold reduction in binding to the second CHI domain compared to binding to the first CHI. In some embodiments, the support comprises a resin having a ligand that specifically binds the first CHI domain. In some embodiments, the ligand specifically binds a human IgG CHI domain.

[0011] Provided herein are methods of isolating a bispecific antibody comprising a first CHI domain and a second CHI domain, wherein the second CHI domain comprises a different sequence from the first CHI domain, the method comprising: (a) contacting the bispecific antibody with a support that binds the first CHI domain, wherein the support does not bind the second CHI domain; and (b) eluting the bispecific antibody comprising the first CHI domain from the support.

[0012] Provided herein are methods of separating a bispecific antibody comprising a first CHI domain and a second CHI domain from a monomeric antibody component thereof or monovalent antibody, wherein the second CHI domain comprises a different sequence from the first CHI domain, the method comprising: (a) contacting the bispecific antibody with a support that binds the first CHI domain, wherein the support does not bind the second CHI domain; and (b) eluting the bispecific antibody comprising the first CHI domain from the support. In some embodiments, the first CHI domain comprises SEQ ID NO: 1. In some embodiments, the different sequence has less than 90%, less than 80%, less than 60%, or less than 50% sequence identity to SEQ ID NO:1. In some embodiments, the second CHI does not comprise SEQ ID NO: 1. In some embodiments, the second CHI domain at amino acid positions corresponding to 84-89 of SEQ ID NO: 14 comprises the amino acid sequence represented by the formula: - XI - His - X2 - X3 - X4 - X5 - (Formula 1) wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys. In some embodiments, the second CHI domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-7 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14. In some embodiments, the support comprises a resin having a ligand that specifically binds the first CHI domain. In some embodiments, the ligand specifically binds a human IgG CHI domain. In some embodiments, after (a) and before (b), the method comprises washing the support. In some embodiments, the contacting is performed on a column comprising the support that binds the first CHI domain.

[0013] Provided herein are methods of purifying an unpurified composition comprising one or more bispecific antibodies, wherein each of the one or more bispecific antibodies comprises a heterodimer formed by a first heavy chain (HC) and a second HC, wherein the method comprises: (a) contacting the one or more bispecific antibodies with a support that binds the first HC; (b) washing the support; and (c) detaching the one or more bispecific antibodies from the support, wherein the purifying results in a purified composition comprising one or more bispecific antibodies at a purity of greater than about 95%.

[0014] Provided herein are methods of purifying an unpurified composition comprising one or more bispecific antibodies, wherein each of the one or more bispecific antibodies comprises a heterodimer formed by a first heavy chain (HC) and a second HC, wherein the method comprises: (a) contacting the one or more bispecific antibodies with a support that binds the first HC; (b) washing the support; and (c) detaching the one or more bispecific antibodies from the support, wherein the purifying results in a purified composition comprising greater than 95% of the one or more bispecific antibodies with less than 10% protein impurity.

[0015] Provided herein are methods of purifying an unpurified composition comprising one or more bispecific antibodies, wherein each of the one or more bispecific antibodies comprises a heterodimer formed by a first heavy chain (HC) and a second HC, wherein the method comprises: (a) contacting the one or more bispecific antibodies with a support that binds the first HC; (b) washing the support; and (c) detaching the one or more bispecific antibodies from the support, wherein purifying the one or more bispecific antibodies takes less than about 2 hours. In some embodiments, (i) the first HC comprises a first domain, or portion thereof, comprising a first amino acid sequence; and (ii) the second HC comprises a second domain, or portion thereof, wherein the second domain comprises a second amino acid sequence that is different from the first amino acid sequence. In some embodiments, the first domain comprises a first CHI domain and the second domain comprises a second CHI domain. In some embodiments, the first domain comprises a first CH2 domain and the second domain comprises a second CH2 domain. In some embodiments, the first domain comprises a first CH3 domain and the second domain comprises a second CH3 domain. In some embodiments, the one or more bispecific antibodies comprises a knob and hole bispecific IgG antibody. In some embodiments, the first domain comprises a first VH domain and the second domain comprises a second VH domain. In some embodiments, each of the one or more bispecific antibodies comprises the heterodimeric protein disclosed herein or the polypeptide disclosed herein. In some embodiments, purifying the one or more bispecific antibodies is performed in the absence of a protein tag. In some embodiments, each of the one or more bispecific antibodies does not comprise a protein tag. In some embodiments, purifying the one or more bispecific antibodies is performed in the absence of a pH gradient. In some embodiments, purifying the one or more bispecific antibodies is performed in the absence of a buffer gradient. In some embodiments, purity is determined by SDS-PAGE or size-exclusion chromatography. In some embodiments, the purified composition comprises monovalent antibodies at a concentration of less than 5%. In some embodiments, the purified composition comprises from about 0.05 mgs to about 50 kgs of the one or more bi specific antibodies. In some embodiments, purifying the one or morebispecific antibodies takes less than about 1 hour. In some embodiments, 1.35 equivalents of the one or more bispecific antibodies are produced from 1 equivalent of the first HC and 1.25 equivalents of the second HC. In some embodiments, the purified composition comprises greater than 80% of the one or more bispecific antibodies from the unpurified composition.

[0016] Provided herein are methods of producing a composition comprising about 50 kgs or more of a heterodimeric protein, wherein the composition comprises less than 5% protein impurity, and wherein the heterodimeric protein comprises a first heavy chain (HC) constant region and a second HC constant region, wherein the first HC constant region comprises: (i) a first CHI domain comprising a first CHI amino acid sequence, (ii) a first CH2 domain comprising a first CH2 amino acid sequence, (iii) a first CH3 domain comprising a first CH3 amino acid sequence, or (iv) a first VH domain comprising a first VH amino acid sequence; and the second HC constant region comprises: (i) a second CHI domain, wherein the second CHI domain comprises a second CHI amino acid sequence that is different from the first CHI amino acid sequence, (ii) a second CH2 domain, wherein the second CH2 domain comprises a second CH2 amino acid sequence that is different from the first CH2 amino acid sequence, (iii) a second CH3 domain, wherein the second CH3 domain comprises a second CH3 amino acid sequence that is different from the first CH3 amino acid sequence, or (iv) a second VH domain, wherein the second VH domain comprises a second VH amino acid sequence that is different from the first VH amino acid sequence.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0018] FIG. 1 depicts an exemplary SEC chromatogram of the purified heterodimeric protein (RSVF Protein / CD163 bispecific antibody).

[0019] FIG. 2 shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD163 bispecific antibody) and a corresponding antigen (RSV F Protein).

[0020] FIG. 3 depicts an exemplary SEC chromatogram of the purified heterodimeric protein (EGFR / RNF43 bispecific antibody).

[0021] FIG. 4 shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (EGFR / RNF43 bispecific antibody) and a corresponding antigen (EGFR).

[0022] FIG. 5 depicts an exemplary SEC chromatogram of the purified heterodimeric protein (EGFR / RSV F Protein bispecific antibody).

[0023] FIG. 6 shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (EGFR / RSV F Protein bispecific antibody) and a corresponding antigen (EGFR).

[0024] FIG. 7 depicts an exemplary SEC chromatogram of the purified heterodimeric protein (EGFR / RSV F Protein bispecific antibody).

[0025] FIG. 8 shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (EGFR / RSV F Protein bispecific antibody) and a corresponding antigen (EGFR).

[0026] FIG. 9 depicts an exemplary SEC chromatogram of the purified heterodimeric protein (PD1 / RSV F Protein bispecific antibody).

[0027] FIG. 10 shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (PD1 / RSV F Protein bispecific antibody) and a corresponding antigen (PD1).

[0028] FIG. 11 depicts an exemplary SEC chromatogram of the purified heterodimeric protein (BAFF / RSV F Protein bispecific antibody).

[0029] FIG. 12 shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (BAFF / RSV F Protein bispecific antibody) and a corresponding antigen (BAFF).

[0030] FIG. 13 depicts an exemplary SEC chromatogram of the purified heterodimeric protein (CD19 / RSV F Protein bispecific antibody).

[0031] FIG. 14 shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (CD19 / RSV F Protein bispecific antibody) and a corresponding antigen (CD 19).

[0032] FIG. 15 depicts an exemplary binding curve from a binding affinity assay for a half antibody containing wild-type IgGl, mouse-human chimeric, mouse, IgA, IgD, IgE, or IgM CHI domain.

[0033] FIG. 16 shows an exemplary SDS-PAGE gel of heterodimeric protein purified by either Capture Select™ CH1-XL (see lanes 1-6). Lane 1 shows abispecific antibody containing a mouse-human chimeric CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lane 2shows a bispecific antibody containing a mouse CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lane 3 shows a bispecific antibody containing an IgA CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lane 4 shows abispecific antibody containing an IgD CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lane 5 shows a bispecific antibody containing an IgE CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lane 6 shows a bispecific antibody containing an IgM CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Quantification of the percent purity for each purified heterodimeric protein is reported at the bottom of each lane.

[0034] FIG. 17A shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mousehuman chimeric CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0035] FIG. 17B depicts an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mouse CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0036] FIG. 17C shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgA CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0037] FIG. 17D depicts an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgD CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0038] FIG. 17E shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgE CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0039] FIG. 17F depicts an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgM CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0040] FIG. 18A shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mouse-human chimeric CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0041] FIG. 18B depicts an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mouse CHI (RSVF arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0042] FIG. 18C shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgA CHI (RSVF arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0043] FIG. 18D depicts an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgD CHI (RSVF arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0044] FIG. 18E shows an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgE CHI (RSVF arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0045] FIG. 18F depicts an exemplary binding curve from a binding affinity assay for the purified heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgM CHI (RSVF arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0046] FIG. 19 shows an exemplary SDS-PAGE gel of heterodimeric protein (RSV F Protein / CD71 bispecific antibody) purified by either MabSelect PrismA™ (see lanes 1-5) or CaptureSelect™ CH1-XL (see lanes 6-10). Lanes 1 and 6 show an RSV F Protein / CD71 bispecific antibody containing a mouse-human chimeric CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lanes 2 and 7 show an RSV F Protein / CD71 bispecific antibody containing a mouse CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lanes 3 and 8 show an RSV F Protein / CD71 bispecific antibody containing an IgD CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lanes 4 and 9 show an RSV F Protein / CD71 bispecific antibody containing an IgE CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Lanes 5 and 10 show an RSV F Protein / CD71 bispecific antibody containing an IgM CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Quantification of the percent purity for each purified heterodimeric protein is reported at the bottom of each lane.

[0047] FIG. 20 depicts an exemplary SDS-PAGE gel of heterodimeric protein (RSV F Protein / CD71 bispecific antibody) purified by either MabSelect PrismA™ (see lane 1) or CaptureSelect™ CH1-XL (see lane 2). The RSVF Protein / CD71 bispecific antibodies contain an IgA CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm). Quantification of the percent purity for each purified heterodimeric protein is reported at the bottom of each lane.

[0048] FIG. 21A shows an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mouse-human chimeric CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSVF Protein).

[0049] FIG. 21B depicts an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mouse CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0050] FIG. 21C depicts an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgD CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0051] FIG. 21D shows an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgE CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0052] FIG. 21E depicts an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgM CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (RSV F Protein).

[0053] FIG. 22A shows an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mouse-human chimeric CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0054] FIG. 22B depicts an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing a mouse CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0055] FIG. 22C depicts an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgD CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0056] FIG. 22D shows an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgE CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).

[0057] FIG. 22E depicts an exemplary binding curve from a binding affinity assay for the CaptureSelect™ CH1-XL purified (see FIG. 19) heterodimeric protein (RSV F Protein / CD71 bispecific antibody) containing an IgM CHI (RSV F arm) and a wild-type IgGl CHI (CD71 arm), and a corresponding antigen (CD71 Protein).DETAILED DESCRIPTIONHeterodimeric proteins

[0058] Provided and exemplified herein are compositions comprising heterodimeric proteins and methods of making and uses thereof. A heterodimeric protein can refer to a protein composed of two polypeptide chains differing in composition in the order, number, and / or kind of their amino acid residues. In some embodiments, the heterodimeric proteins described herein provide an efficient purification strategy for the purification of heterodimeric proteins (such as bispecific antibodies). The heterodimeric proteins provided herein can take the form of multispecific antibodies, bispecific antibodies or antibody derivatives.Antibodies

[0059] Provided and exemplified herein are heterodimeric antibodies. In some embodiments, the heterodimeric protein provided herein is an antibody. The term “antibody” is used in the broadest sense and generally refers to and / or includes monoclonal antibodies, multi-valent antibodies, multi-specific, and antigen-binding fragments of antibodies. Antigenbinding fragments of antibodies generally refer to and / or include antibody-derived moieties or proteins that comprise a functional set of CDRs (e.g., a CDR-H1-3 and CDR-L1-3) that bind an antibody and have a molecular weight less than a full-length antibody (e.g., a molecular weight less than -150,000 Daltons). In certain embodiments, an antigen-binding antibody fragment includes: fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab' fragments, Fv fragments, IgG (rlgG) fragments, and single chain antibody fragments, including single chain variable fragments (sFv or scFv). Antibodies and antigen-binding fragments ofantibodies generally encompass genetically engineered, and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multi-specific antibodies, multi-valent antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. A full-length antibody, intact antibody, and / or whole antibody are interchangeable, and generally include and / or refer to an antibody having a structure substantially similar to a native antibody structure having heavy chains that contain an Fc region and / or include antibodies of any class or sub-class, including IgG and sub-classes thereof (e.g., IgGl and IgG4), IgM, IgE, IgA, and IgD.

[0060] In some embodiments, heterodimeric proteins provided herein comprise an Fc region. An Fc region generally encompasses and / or refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The Fc region of an antibody can interact with a number of receptors or ligands including Fc Receptors (e.g., FcyRI, FcyRIIA, FcyRIIIA), the complement protein Clq, and other molecules such as proteins A and G. These interactions can influence a variety of effector functions and downstream signaling events including: antibody dependent cell-mediated cytotoxicity (ADCC), Antibody-dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC). Generally, the Fc domain includes an immunoglobulin CH2 and CH3 domain. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the Fc region is a wildtype Fc region, e.g., a wildtype human Fc region. In some embodiments, the Fc region comprises a variant, e.g., an Fc region comprising an addition, substitution, or deletion of at least one amino acid residue in the Fc region which results in, e.g., reduced or ablated affinity for at least one Fc receptor. In some embodiments, the heterodimeric protein comprising a variant Fc region has one or more of the following properties: (1) reduced effector function (e.g., reduced ADCC, ADCP and / or CDC); (2) reduced binding to one or more Fc receptors; and / or (3) reduced binding to Clq complement. In some embodiments, the reduction in any one, or all of properties ( 1 )-(3) is compared to an otherwise similar antibody with a wildtype Fc region. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In certain embodiments, the Fc region includes IgG and sub-classes thereof (e.g., IgGl and IgG4), IgM, IgE, IgA, and / or IgD heavy chain constant regions and / or heavy chain constant regions derived from IgG and sub-classes thereof (e.g., IgGl and IgG4),IgM, IgE, IgA, and IgD. In some embodiments, the heterodimeric protein comprises an IgG antibody.

[0061] Complementarity determining regions (CDRs) generally include amino acids within antibody variable regions (e.g., contiguous or non-contiguous) that confer antigen specificity and / or binding affinity (e.g., to an IgE constant domain). In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). Framework regions (FRs) generally refer to and / or include non-CDR regions of the heavy and light chain variable regions. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-Ll, FR-L2, FR-L3, and FR-L4).

[0062] Variable regions (also referred to as variable domains) generally refer to and / or include the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen (e.g., a single variable domain comprises a CDR 1, CDR 2, and CDR 3). The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. In certain instances, a single VH or VL domain can be sufficient to confer antigen-binding specificity.

[0063] In some embodiments, the heterodimeric protein provided herein comprises a multispecific protein. As used herein, a “multifunctional” or a “multispecific” protein can refer to a polypeptide that has two or more functionalities, e.g., two or more binding specificities. In some embodiments, the functionalities can include one or more immune cell engagers, one or more tumor binding molecules, and other moi eties described herein. In some embodiments, the multispecific protein is a multispecific antibody. An antibody, or antibody fragment referred to as “multispecific” refers to a molecule that possesses the ability to specifically bind to at least two or more structurally distinct targets. A “multispecific antibody” including grammatical equivalents refers to a multispecific molecule that preserves at least one fragment of an antibody able to specifically bind with a target, for example, a variable region, heavy or light chain, or complementarity determining region from an antibody molecule. In some embodiments, a multispecific (or multifunctional) antibody comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., thesame protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a multispecific antibody comprises a third, fourth or fifth immunoglobulin variable domain. In some embodiments, a multispecific antibody is a bispecific antibody binding agent, a trispecific antibody, or a tetraspecific antibody.Bispecific Antibodies

[0064] Provided and exemplified herein are bispecific antibodies. In some embodiments, the heterodimeric protein provided herein is a bispecific antibody. A bispecific antibody generally refers to and includes antibody formats that possess the ability to specifically bind to at least two structurally distinct targets. The specific binding can be the result of two distinct binding moieties that are structurally distinct at the molecular level, including but not limited to distinct non-identical amino acid sequences; or a single binding moiety that is able to specifically bind to two structurally distinct targets with high affinity (e.g., with a KD less than about lxlOA-6). A “bispecific antibody” including grammatical equivalents refers to a bispecific molecule that preserves at least one fragment of an antibody able to specifically bind a target, for example, a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule.

[0065] A bispecific antibody can be characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In some embodiments, a bispecific antibody comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody comprises a half antibody, or fragment thereof, havingbinding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.

[0066] Bispecific antibodies can be conceived and designed to alter functionality or binding properties of the composite binding molecules or bispecific antibodies (see e.g., “Bispecific antibodies: a mechanistic review of the pipeline.” Nat Rev Drug Discovery. 2019 Aug;18(8):585-608) (see e.g., “The making of bispecific antibodies” MAbs. 2017 Feb-Mar; 9(2): 182-212). For example, bispecific antibodies can be selected from any of the following non-limiting formats: a common light chain bispecific IgG, a Fab-Fc-Fab:Fc bispecific IgG, a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG, a Fab-Fc-Fab:Fab-Fc bispecific IgG, an scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG, a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG, and a Fab-Fc-scFv:Fab-Fc bispecific IgG.

[0067] In some embodiments, the heterodimeric protein comprises a bispecific IgG antibody. In some embodiments, the heterodimeric protein comprises a knob and hole bispecific IgG antibody. Embodiments of the disclosure include “knob-into-hole” bispecific antibodies, wherein the otherwise symmetric dimerization region of a bispecific binding agent is altered so that it is asymmetric. For example, a knob-into-hole bispecific IgG that is specific for antigens A and B can be altered so that the Fc portion of the A-binding chain has one or more protrusions (“knobs”), and the Fc portion of the B-binding chain has one or more hollows (“holes”), where the knobs and holes are arranged to interact. This reduces the homodimerization (A-A and B-B antibodies) and promotes the heterodimerization desired for a bispecific binding agent. See, e.g., Y. Xu et al., mAbs (2015) 7(l):231-42. In some embodiments, the bispecific binding agent has a knob-into-hole design. In some embodiments, the “knob” comprises a T336W alteration of the CH3 domain, i.e., the threonine at position 336 is replaced by a tryptophan. In some embodiments, the “hole” comprises one or a combination of T366S, L368A, and Y407V. In some embodiments, the “hole” comprises T366S, L368A, and Y407V.

[0068] Knob-in-Hole as described in US 5,731,116, US 7,476,724 and Ridgway, J. et al. (1996) Prot. Engineering 9(7): 617-621, broadly involves: (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. “Knobs” or “protuberances” are typically created by replacing a small amino acid in a parental antibody with a larger amino acid (e.g., T366Y or T366W); “Holes” or “cavities” are created by replacing a larger residue in a parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and / or Y407V).

[0069] For bispecific antibodies including an Fc domain, introduction of specific mutations into the constant region of the heavy chains to promote the correct heterodimerization of the Fc portion can be utilized. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the “knobs-into-holes” (KiH) approach which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary “hole” in the other CH3 domain of the paired heavy chain so as to promote correct pairing of heavy chains (see e.g., US7642228).

[0070] Exemplary Fc mutations are provided by Igawa and Tsunoda who identified 3 negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are: E356-K439, E357-K370, D399-K409 and vice versa. By introducing at least two of the following three mutations in chain A: E356K, E357K and D399K, as well as K370E, K409D, K439E in chain B, alone or in combination with newly identified disulfide bridges, they were able to favor very efficient heterodimerization while suppressing homodimerization at the same time (Martens T et al. A novel one-armed antic- Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 2006; 12:6144-52; PMID: 17062691). Xencor defined 41 variant pairs based on combining structural calculations and sequence information that were subsequently screened for maximal heterodimerization, defining the combination of S364H, F405A (HA) on chain A and Y349T, T394F on chain B (TF) (Moore GL et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011; 3:546-57; PMID: 22123055).

[0071] Other exemplary Fc mutations to promote heterodimerization of multispecific antibodies include those described in the following references, the contents of each of which is incorporated by reference herein, WO2016071377A1, US20140079689A1, US20160194389A1, US20160257763, WO2016071376A2, W02015107026A1, W02015107025A1, W02015107015A1, US20150353636A1, US20140199294A1, US7750128B2, US20160229915 Al, US20150344570A1, US8003774A1, US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1, US20120149876A1, US20140200331A1, US9309311B2, US8586713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1 and US20110054151A1.

[0072] Stabilizing cysteine mutations have also been used in combination with KiH and other Fc heterodimerization promoting variants, see e.g., US7183076. Other exemplarycysteine modifications include, e.g., those disclosed in US20140348839A1, US7855275B2, and US9000130B2.Modified CHI domains

[0073] Provided and exemplified herein are polypeptides. In some embodiments, the polypeptide comprises an amino sequence having at least 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 14. In some embodiments, the polypeptide does not comprise an amino sequence of SEQ ID NO: 1 at positions 84-89. In some embodiments, the polypeptide exhibits at a 2-fold or greater (3 -fold) reduction in binding to a resin having a ligand that binds a CHI domain comprising SEQ ID NO: 14. In some embodiments, the polypeptide exhibits at a 2-fold or greater (e.g., 3 -fold) reduction in binding to the resin compared to a polypeptide without CHI modifications. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 14 represented by the formula: - XI - His - X2 - X3 - X4 - X5 - (Formula 1), wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 14 represented by the formula: -XI - His - X2 - X3 - X4 - X5 - (Formula 1), wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys; and wherein the polypeptide comprises at least 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 14 (e.g., at positions 1-83 and 90-96).

[0074] In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of any one of SEQ ID NOs: 2-7. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the polypeptide is an immunoglobulin heavy chain constant region CHI domain.

[0075] The CHI antibody domain (constant heavy chain 1 domain) is part of the immunoglobulin heavy chain and can influence the stability and flexibility of the antibodymolecule. It consists of a compact, globular structure that is composed of about 110-120 amino acid residues. The domain is connected to the variable heavy chain (VH) region by a flexible hinge region. CHI domains can be modified to reduce mispairing. For example, CrossMab technology exchanges CHI and CL domains in the Fab of one half of a bispecific antibody to avoid non-specific L chain mispairing. Such crossover variants retain binding specificity and affinity but make the two arms so different that L chain mispairing is prevented. The CrossMab technology (as reviewed in Klein et al. Supra) involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings (see e.g., Cain, C. SciBX 4(28); doi: 10.1038 / scibx.2011.783, the contents of which are incorporated by reference herein).

[0076] One downstream approach utilized to remove mis-paired products is the use of affinity chromatography. This approach often also requires that the bispecific antibody be generated with modifications made on one of the HCs of the bispecific antibody to alter binding affinity. For example, for Protein G purification, a combination of mutations in the Fc and CHI regions can be introduced to disrupt Protein G binding and achieve differential elution. However, even this approach requires a two-step affinity chromatography purification process (e.g., Protein G purification and Protein A purification). A two-step affinity chromatography purification process can present manufacturing challenges during bispecific antibody production. For example, a two-step purification process may be more time-consuming, result in increased production costs, lead to reduced overall yield and lower productivity, and can affect the quality and stability of the final product. In some embodiments, the heterodimeric proteins provided herein comprise differing CHI domains (e.g., a first CHI domain and a second CHI domain) to facilitate a one-step purification protocol (e.g., with anti-CHl resins). Bispecific antibodies comprising modified CHI domain

[0077] Provided and exemplified herein are heterodimeric proteins. In some embodiments, the heterodimeric protein, as described herein, is a bispecific antibody comprising a modified CHI domain. In some embodiments, the heterodimeric proteins comprise a heterodimer formed by a first heavy chain (HC) constant region and a second HC constant region. In some embodiments, the first HC constant region comprises a first CHI domain comprising a first CHI amino acid sequence. In some embodiments, the second HC constant region comprises a second CHI domain comprising a second CHI amino acid sequence. In some embodiments, the second CHI amino acid sequence is different from the first CHI amino acid sequence. In certain embodiments, a different amino acid sequence, as described throughout, is a nonidentical amino acid sequence (e.g., a contiguous sequence of amino acids that are not identicalto a reference or second sequence of amino acids). For example, a different or non-identical amino acid sequence can comprise a single amino acid sequence difference. A different amino acid sequence can comprise one or more (e.g., 1, 2, 3, 4, 5, 10, 20, or greater) amino acid sequence differences. In some embodiments, the heterodimeric protein, as described herein, comprises one or more amino acid sequences having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1-21.

[0078] In some embodiments, a second CHI amino acid sequence that is different from the first CHI amino acid sequence can refer to a second CHI amino acid sequence that is not identical to the first CHI amino acid sequence. For example, the second CHI amino acid sequence may be from a different species compared to the first CHI amino acid sequence. In some embodiments, the first CHI domain comprises a human CHI amino acid sequence. In some embodiments, the second CHI domain comprises a non-human (e.g., mouse, rat, etc.) CHI amino acid sequence. In some embodiments, the second CHI domain comprises a mouse CHI amino acid sequence. In some embodiments, the second CHI domain comprises a rat CHI amino acid sequence.

[0079] In some case, a second CHI amino acid sequence that is non-identical (e.g., different) to the first CHI amino acid sequence may refer to the second CHI amino acid sequence and the first CHI amino acid sequence comprising different immunoglobulin sequences. For example, the first CHI domain can comprise an IgG CHI amino acid sequence, and the second CHI domain can comprise a non-IgG (e.g., IgM, IgE, IgA, IgD) CHI amino acid sequence. In some embodiments, the second CHI domain comprises an IgM CHI amino acid sequence. In some embodiments, the second CHI domain comprises an IgE CHI amino acid sequence. In some embodiments, the second CHI domain comprises an IgA CHI amino acid sequence. In some embodiments, the second CHI domain comprises an IgD CHI amino acid sequence. In some embodiments, the second CHI domain comprises a non-IgGl (e.g., IgG4, IgM, IgE, IgA, IgD) CHI amino acid sequence. In some embodiments, the second CHI domain comprises an IgG4 CHI amino acid sequence. In some embodiments, the first CHI domain comprises an IgGl CHI amino acid sequence.

[0080] In some case, a second CHI amino acid sequence that is non-identical to the first CHI amino acid sequence may refer to the second CHI amino acid sequence and the first CHI amino acid sequence comprising different binding affinities. For example, the second CHI domain exhibits at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or greater reduction in binding to a resin having a ligand that specificallybinds the first CHI domain (e.g., a first CHI domain comprising SEQ ID NO: 14). In some embodiments, the second CHI domain is not retained by a column comprising a resin having a ligand that specifically binds the first CHI domain (e.g., a first CHI domain comprising SEQ ID NO: 14)

[0081] In some cases, a second CHI amino acid sequence that is non-identical to the first CHI amino acid sequence may refer to the second CHI amino acid sequence comprising one or more amino acid substitutions or mutations relative to the first CHI amino acid sequence. For example, the second CHI amino acid sequence may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more amino acid substitutions or mutations relative to the first CHI amino acid sequence. The one or more amino acid substitutions or mutations can be located within specific amino acid positions of a CHI domain, such as 203-208 per EU numbering. The one or more amino acid substitutions or mutations can be contiguous or non-contiguous. For example, the one or more amino acid substitutions or mutations that are contiguous can refer to the second CHI amino acid sequence comprising one or more amino acid substitutions or mutations at positions 203-208, per EU numbering, relative to the first CHI amino acid sequence. In some cases, the one or more amino acid substitutions or mutations that are non-contiguous can refer to the second CHI amino acid sequence comprising one or more amino acid substitutions or mutations at positions 203, 205, and 207, per EU numbering, relative to the first CHI amino acid sequence. In some cases, the second CHI amino acid sequence that is non-identical to the first CHI amino acid sequence retains the same immunogenicity relative to the first CHI amino acid sequence.

[0082] In some embodiments, the first CHI domain comprises a first FG-loop structure and the first CHI amino acid sequence comprises a first FG-loop amino acid sequence. In some embodiments, the second CHI domain comprises a second FG-loop structure and the second CHI amino acid sequence comprises a second FG-loop amino acid sequence that is different from the first FG-loop sequence. In some embodiments, the first FG-loop structure and the second FG-loop structure comprises CHI domain amino acid positions 203-208 per EU numbering (or analogous positions in IgA, IgD, IgE, or IgM), such as the amino acids listed in Table 1. In some embodiments, the first FG-loop structure and the second FG-loop structure comprises CHI domain amino acid positions 211-216 per Kabat numbering (or analogous positions in IgA, IgD, IgE, or IgM), such as the amino acids listed in Table 1. In some embodiments, the first FG-loop structure and the second FG-loop structure comprises CHI domain amino acid positions 107-114 per IMGT unique numbering (or analogous positions inIgA, IgD, IgE, or IgM), such as the amino acids listed in Table 1. In some embodiments, the first FG-loop structure comprises a human FG-loop amino acid sequence and the second FG-loop structure comprises a non-human FG-loop amino acid sequence. In some embodiments, the first FG-loop structure comprises an IgG FG-loop amino acid sequence and the second FG-loop structure comprises a non-IgG FG-loop amino acid sequence. In some embodiments, the first FG-loop structure comprises an IgGl FG-loop amino acid sequence and the second FG-loop structure comprises a non-IgGl FG-loop amino acid sequence.Table 1. Exemplary FG-loop for Human IGHG CHI DomainIMGT unique IGHG1 amino IMGT exon Eu numbering Kabat numbering numbering for acid translation numbering (98 aa)CHI domain 1-98 118-215105 N 84 201 209106 V 85 202 210107 N 86 203 211108 H 87 204 212109 K 88 205 213110 P 89 206 214111 - - - - 112 - - - - 113 s 90 207 215114 N 91 208 216115 T 92 209 217116 K 93 210 218117 V 94 211 219

[0083] In some embodiments, the first FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the first CHI domain comprises a first FG-loop structure comprising an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1; and an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14.

[0084] In some embodiments, the second FG-loop structure comprises the amino acid sequence represented by the formula (N- to C-terminus): - XI - His - X2 - X3 - X4 - X5 -(Formula 1), wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys. In some embodiments, the second FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 2-7. In some embodiments, the second FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, thesecond FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the second FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the second FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the second FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the second FG-loop structure comprises an amino sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7.

[0085] In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising the amino acid sequence of Formula 1 or at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 2-7; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising the amino acid sequence of Formula 1; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%,or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the second CHI domain comprises: the second FG-loop structure comprising at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7; and an amino acid sequence having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14.

[0086] In some embodiments, each of the one or more bispecific antibodies comprises the heterodimeric protein disclosed herein or the polypeptide disclosed herein. In some embodiments, the heterodimeric protein, as described herein, comprise a first HC constant region and a second HC constant region. In some embodiments, the first HC constant region further comprises a first CH2 domain and a first CH3 domain. In some embodiments, the second HC constant region further comprises a second CH2 domain and a second CH3 domain. In some embodiments, the heterodimeric protein, as described herein, is a bispecific antibody comprising a modified CHI domain. In some embodiments, the first CH3 domain comprises a first CH3 domain amino acid sequence comprising a knob modification (e.g., W366 or Y366 per EU numbering). In some embodiments, the second CH3 domain comprises a second CH3 domain amino acid sequence comprising a hole modification (e.g., S366, A368, and / or V407 per EU numbering). In some embodiments, the first CH3 domain comprises a first CH3 domain amino acid sequence comprising a hole modification. In some embodiments, the second CH3 domain comprises a second CH3 domain amino acid sequence comprising a knob modification. In some embodiments, the heterodimeric protein, as described herein, is an antibody. In some embodiments, the heterodimeric protein, as described herein, is a bispecific antibody. In some embodiments, the heterodimeric protein, as described herein, is a bispecific antibody comprising a modified CHI domain.

[0087] In some embodiments, the bispecific antibody further comprises: a first light chain variable domain and a first heavy chain variable domain; and a second light chain variable domain and a second heavy chain variable domain, wherein, optionally, the first light chain variable domain and the second light chain variable domain are different. In some embodiments, the first heavy chain variable domain is covalently linked to the first CHI domain and the second heavy chain variable domain is heavy chain variable domain is linked to the first CHI domain linked to the second CHI domain. In some embodiments, the bispecificantibody further comprises first light chain constant domain covalently linked to the first light chain variable domain, and a second light chain constant domain covalently linked to the second light chain variable domain.Methods

[0088] Provided herein are methods of making the heterodimeric proteins as described herein. Provided herein are one or more polynucleotides encoding the heterodimeric protein disclosed herein. In some embodiments, encoding can refer to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. For example, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. In some cases, the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. In some embodiments, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain one or more introns.

[0089] In some embodiments, the polynucleotide is an isolated recombinant polynucleotide. In some embodiments, “nucleic acid” or “polynucleotide” can refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Sequences encoding the heterodimeric proteins, or fragments thereof, can be inserted by standard methods, and the resulting nucleic acids encoding the heterodimeric proteins, or fragments thereof, are transduced into suitable host cells and expressed. Alternatively, the nucleic acids can be expressed in a cell-free expression system, which can provide more control over oxidation and reduction conditions, pH, folding, glycosylation, and the like.

[0090] Described herein, in certain embodiments, is a vector comprising the nucleotide sequences described herein. In some embodiments, the vectors comprise one or more of the polynucleotides as described herein. Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC). Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY : Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY : Wiley, (including supplements through 2014); and Makrides,S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0091] Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors may be transfected or introduced into an appropriate host cell. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in media and screened for the appropriate activity.

[0092] Methods and conditions for culturing the resulting transfected cells and for recovering the antibody binding agent produced are known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed, based upon the present description.

[0093] Also provided herein are host cells comprising the one or more polynucleotides disclosed herein. The polynucleotides may be present in a single vector or separate vectors present in the same host cell or separate host cell. The host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.

[0094] In some embodiments, described herein are host cells comprising one or more polynucleotides encoding the heterodimeric protein as described herein. In some embodiments, the host cells are genetically engineered by using an expression cassette. The phrase “expression cassette,” refers to nucleotide sequences, which are capable of affecting expression of a gene in hosts compatible with such sequences. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression may also be used, such as, for example, an inducible promoter.

[0095] In some embodiments, described herein are host cells comprising the vectors described herein. The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0096] In some embodiments, the methods described can be performed in a host cell, or in vitro, in cell-free synthetic systems. Host cells may be any that can be robustly recoded. These can be bacterial cells that have well developed genetic systems, of which E. coli is exemplary. Other bacterial species can also be used. In some embodiments, cell-free systems for producing the proteins may be coupled transcription / translation systems or only translation systems. Notably, in some embodiments, biological syntheses are utilized rather than chemical synthesis.

[0097] In one aspect the present disclosure provides a cell comprising the one or more polynucleotides disclosed herein. Culturing of recoded cells with the constructed nucleic acid sequences may be by any means known in the art. In some embodiments, the culturing may be batch or continuous, in shaker flasks or in fermenters or immobilized on solid surfaces, such as small particles contained in larger vessels.

[0098] In some embodiments, the cell can be a plurality of cells. In some cases, the plurality of cells can be from about 1 cell to about 1 billion cells. In some cases, the plurality of cells can be about 0 cells, about 10 cells, about 100 cells, about 1,000 cells, about 10,000 cells, about 100,000 cells, about 1,000,000 cells, about 10,000,000 cells, about 100,000,000 cells, about 1,000,000,000 cells. In some cases, the plurality of cells can be at least about 0 cells, about at least 10 cells, about at least 100 cells, about at least 1,000 cells, about at least 10,000 cells, about at least 100,000 cells, about at least 1,000,000 cells, about at least 10,000,000 cells, about at least 100,000,000 cells, about at least 1,000,000,000 cells, or more.

[0099] In some embodiments, the cell can be a bacterial cell. In some cases, the bacterial cell can be a bio-contained strain. In some cases, the bacterial cell can be a multi-virus resistance bacterial cell. In some embodiments, the bacterial cell can be from a bacterial genus. Non-limiting examples of bacterial geneses include Staphylococcus, Streptococcus, Enterococcus, Moraxella, Neisseria, Corynebacterium, Bacillus, Lactobacillus, Listeria, Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Serratia, Hafnia, Morganella, Providencia, Salmonella, Shigella, Yersinia, Acinetobacter, Pseudomonas, Strenotrophomonas, Burkholderia, Haemophilus, Legionella, Achromobacter, Aeromonas, Alcaligenes, Campylobacter, Flavobacterium, Helicobacter, Pasteurella, Bacteroides, Clostridium, Propionibacterium, Prevotella, Mycobacterium, Mycoplasma, Actinomyces, Acetobacter, Bordetella, Vibrio, and Nocardia. In some embodiments, the bacterial cell can be abacterial species. Non-limiting examples of bacterial species include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Moraxella catharralis, Neisseria meningitidis, Listeria monocytogenes, Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus mycoides, Bacillus subtilis, Bacteroides fragilis, Bacteroides gingivalis, Bordetella bronchiseptica, Bordetella pertussis, Burkholderia mallei, Burkholderiahosphora, Campylobacter jejuni, Campylobacter pylori, Clostridium botulinum, Clostridium difficule, Cory neb acterium diphtheria Corynebacterium fusiforme, Enterococcus avium, Enterococcus durans, Enterococcus gallinarum, Enterococcus maloratus, Haemophilus influenzae, Haemophilus pertussis, Haemophilus parainfluenzae, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus lactis, Legionella pneumophila, Mycobacterium avium, Mycobacterium bovis, Mycoplasma hominis, Mycoplasma fermentans, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tulanensis, Prevotella melaninogenica, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Vibrio comma, Vibrio vulnificus, and Yersinia enterocolitica.

[0100] In some embodiments, the cell can be a mammalian cell. In some embodiments, the cell can be a mammalian cell line. Non-limiting examples of mammalian cell lines include HeLa cells, HEK293 cells, CHO cells, NIH / 3T3 cells, Jurkat cells, RAW 264.7 cells, SH-SY5Y cells, MCF-7 cells, A549 cells, and U87 cells.

[0101] Provided herein are methods of isolating the heterodimeric protein described herein (such as a bispecific antibody). In some embodiments, the method comprises: (a) contacting the bispecific antibody with a support that binds first CHI domain; (b) washing the support; and (c) detaching (e.g., eluting) the bispecific antibody from the support. Also provided herein are methods of isolating a bispecific antibody (such as a heterodimeric protein described herein) comprising a first CHI domain and a second CHI domain, wherein the second CHI domain comprises a different sequence from the first CHI domain. In some embodiments, the method comprises: (a) contacting the bispecific antibody with a support that binds the first CHI domain, wherein the support does not bind the second CHI domain; and (b) eluting the bispecific antibody comprising the first CHI domain from the support.

[0102] Provided herein are methods of purifying an unpurified composition comprising one or more bispecific antibodies, wherein each of the one or more bispecific antibodies comprises a heterodimer formed by a first heavy chain (HC) and a second HC, wherein the method comprises: (a) contacting the one or more bispecific antibodies with a support that binds the first HC; (b) washing the support; and (c) detaching the one or more bispecific antibodies from the support.

[0103] In some embodiments, the purifying results in a purified composition comprising one or more bi specific antibodies at a purity of greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or about 100%. In some embodiments, the purifying results in a purified composition comprising greater than about 95% of the one or more bispecific antibodies with less than about 10% protein impurity. In some embodiments, the purifying results in a purified composition comprising greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or about 100% of the one or more bispecific antibodies. In some embodiments, the purified composition of the one or more bispecific antibodies comprises less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or about 0% protein impurity. In some embodiments, the purified composition comprises monovalent antibodies at a concentration of less than about 5%. In some embodiments, the purified composition comprises monovalent antibodies at a concentration of less than about 10%, less than about 9%, less than about 9%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or about 0%. In some embodiments, the purified composition comprises greater than about 80% of the one or more bispecific antibodies from the unpurified composition. In some embodiments, the purified composition comprises greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or about 100% of the one or more bispecific antibodies from the unpurified composition.

[0104] In some embodiments, purifying the one or more bispecific antibodies takes less than about 4 hours, less than about 3 hours, less than about 2 hours, less than about 1.9 hours, less than about 1.8 hours, less than about 1.7 hours, less than about 1.6 hours, less than about 1.5 hours, less than about 1.4 hours, less than about 1.3 hours, less than about 1.2 hours, less than about 1.1 hours, or less than about 1 hour.

[0105] In some embodiments, the purified composition comprises about 0.05 mgs, about 0.1 mgs, about 0.2 mgs, about 0.3 mgs, about 0.4 mgs, about 0.5 mgs, about 1 mgs, about 5 mgs, about 10 mgs, about 20 mgs, about 50 mgs, about 100 mgs, about 200 mgs, about 500 mgs, or more of the one or more bispecific antibodies. In some embodiments, the purified composition comprises kgs of the one or more bispecific antibodies. In some embodiments, there is no upper limit for the amount (e.g., kgs) of one or more bispecific antibodies that can be purified when using the methods described herein. For example, the purified composition can comprise about 1 kg, about 2 kgs, about 5 kgs, about 10 kgs, about 20 kgs, about 50 kgs, or more of the one or more bispecific antibodies. In some embodiments, about 1.35 equivalents of the one or more bispecific antibodies are produced from about 1 equivalent of the first HC and about 1.25 equivalents of the second HC. In some embodiments, about 1 equivalents, about 1.35 equivalents, about 1.5 equivalents, about 1.75 equivalents, or about 2 equivalents of the one or more bispecific antibodies are produced from about 0.5 equivalents, about 1 equivalent, about 1.5 equivalents, or about 2 equivalents of the first HC and about 0.5 equivalents, about 1 equivalents, about 1.25 equivalents, about 1.5 equivalents, about 2 equivalents, or about 3 equivalents of the second HC.

[0106] Provided herein are methods of producing a composition comprising kgs of a heterodimeric protein, wherein the composition comprises less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or about 0% protein impurity, and wherein the heterodimeric protein comprises a first heavy chain (HC) constant region and a second HC constant region, wherein the first HC constant region comprises: (i) a first CHI domain comprising a first CHI amino acid sequence, (ii) a first CH2 domain comprising a first CH2 amino acid sequence, (iii) a first CH3 domain comprising a first CH3 amino acid sequence, and / or (iv) a first VH domain comprising a first VH amino acid sequence; and the second HC constant region comprises: (i) a second CHI domain, wherein the second CHI domain comprises a second CHI amino acid sequence that is different from the first CHI amino acid sequence, (ii) a second CH2 domain, wherein the second CH2 domain comprises a second CH2 amino acid sequence that is different from the first CH2 amino acid sequence, (iii) a second CH3 domain, wherein the second CH3 domain comprises a second CH3 amino acid sequence that is different from the first CH3 amino acid sequence, and / or (iv) a second VH domain, wherein the second VH domain comprises a second VH amino acid sequence that is different from the first VH amino acid sequence. In some embodiments, the composition comprises from about 0.05 mgs to about 50 kgs of the heterodimeric protein. In someembodiments, the composition comprises about 0.05 mgs, about 0.1 mgs, about 0.2 mgs, about 0.3 mgs, about 0.4 mgs, about 0.5 mgs, about 1 mgs, about 5 mgs, about 10 mgs, about 20 mgs, about 50 mgs, about 100 mgs, about 200 mgs, about 500 mgs, or more of the heterodimeric protein. In some embodiments, the composition comprises kgs of the heterodimeric protein (e.g., there is no upper limit for the amount of heterodimeric protein that can be purified when using the methods described herein). For example, the composition can comprise about 1 kg, about 2 kgs, about 5 kgs, about 10 kgs, about 20 kgs, about 50 kgs, or more of the heterodimeric protein.

[0107] In some embodiments, each of the one or more bispecific antibodies comprises the heterodimeric protein disclosed herein. In some embodiments, the first HC comprises a first domain, or portion thereof, comprising a first amino acid sequence; and the second HC comprises a second domain, or portion thereof, wherein the second domain comprises a second amino acid sequence that is different from the first amino acid sequence. In some embodiments, the first domain comprises a first CHI domain and the second domain comprises a second CHI domain. In some embodiments, the first domain comprises a first CH2 domain and the second domain comprises a second CH2 domain. In some embodiments, the first domain comprises a first CH3 domain and the second domain comprises a second CH3 domain. In some embodiments, the one or more bispecific antibodies comprises a knob and hole bispecific IgG antibody. In some embodiments, the first domain comprises a first VH domain and the second domain comprises a second VH domain.

[0108] In some embodiments, purifying the one or more bispecific antibodies is performed in the absence of a protein tag. In some embodiments, each of the one or more bispecific antibodies does not comprise a protein tag. In some embodiments, each of the one or more bispecific antibodies does not comprise an amino acid residue from a cleaved protein tag. For example, other purifying techniques (such as those commonly used) may use a Tobacco Etch Virus (TEV) protein tag or a 3C Protease to purify one or more bispecific antibodies. In the case of the TEV protein tag, when it is cleaved after purification it leaves behind an amino acid residue or “fingerprint” from the cleaved protein tag of ENLYFQ at the C-terminus of a protein, and / or G or S at the N-terminus of a protein. For the 3C Protease, when it is cleaved after purification it leaves behind an amino acid residue or “fingerprint” from the cleaved protein tag of LEVLFQ at the C-terminus of a protein, and / or GP at the N-terminus of a protein.

[0109] In some embodiments, purifying the one or more bispecific antibodies is performed in the absence of a pH gradient. In some embodiments, purifying the one or more bispecific antibodies is performed in the absence of acetic acid. In some embodiments, purifying the oneor more bispecific antibodies is performed in the absence of a buffer gradient. In some embodiments, purifying the one or more bispecific antibodies is performed in the absence of a buffer exchange. In some embodiments, purity is determined by SDS-PAGE or size-exclusion chromatography.

[0110] Provided herein are methods of separating the heterodimeric protein described herein, such as a bispecific antibody, from a monomeric antibody component thereof or mono-specific antibody. In some embodiments, the method comprises of isolating or separating a bispecific antibody comprising the heterodimeric protein described herein from a monomeric antibody component thereof or mono-specific antibody. In some embodiments, the method comprises: (a) contacting the bispecific antibody with a support that binds first CHI domain; (b) washing the support; and (c) detaching (e.g., eluting) the bispecific antibody from the support. Also provided herein are methods of separating a bispecific antibody (such as a heterodimeric protein described herein) comprising a first CHI domain and a second CHI domain from a monomeric antibody component thereof or monovalent antibody, wherein the second CHI domain comprises a different sequence from the first CHI domain. In some embodiments, the method comprises: (a) contacting the bispecific antibody with a support that binds the first CHI domain, wherein the support does not bind the second CHI domain; and (b) eluting the bispecific antibody comprising the first CHI domain from the support.[oni] In some embodiments, the support exhibits at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or greater reduction in binding to the second CHI domain (e.g., compared to a first CHI domain comprising SEQ ID NO: 14). In some embodiments, the support comprises a resin having a ligand that specifically binds the first CHI domain. In some embodiments, the ligand specifically binds a human IgG CHI domain. In some embodiments, the ligand binds a human IgGl CHI domain. In some embodiments, the ligand binds a human IgG2 CHI domain. In some embodiments, the ligand binds a human IgG3 CHI domain. In some embodiments, the ligand binds a human IgG4 CHI domain. In some embodiments, after (a) and before (b), the method comprises washing the support. In some embodiments, the contacting is performed on a column comprising the support that binds the first CHI domain.

[0112] In some embodiments, the first CHI domain comprises SEQ ID NO: 1. In some embodiments, the different sequence has less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, or less than 50% sequence identity to SEQ ID NO:1. In some embodiments, the second CHI does not comprise SEQ IDNO: 1. In some embodiments, the second CHI domain at amino acid positions corresponding to 84-89 of SEQ ID NO: 14 comprises the amino acid sequence represented by the formula:-XI - His - X2 - X3 - X4 - X5 -(Formula 1), wherein: XI is Ala, Lys, or Gin; X2 is Pro, Tyr, or Thr; X3 is Ala, Thr, Asn, or Pro; X4 is Asn or Ser; and X5 is Asn, Ser, Pro, or Lys. In some embodiments, the second CHI domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-7 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14. In some embodiments, the second CHI domain comprises the amino acid sequence of SEQ ID NO: 2 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14. In some embodiments, the second CHI domain comprises the amino acid sequence of SEQ ID NO: 3 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14. In some embodiments, the second CHI domain comprises the amino acid sequence of SEQ ID NO: 4 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14. In some embodiments, the second CHI domain comprises the amino acid sequence of SEQ ID NO: 5 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14. In some embodiments, the second CHI domain comprises the amino acid sequence of SEQ ID NO: 6 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14. In some embodiments, the second CHI domain comprises the amino acid sequence of SEQ ID NO: 7 at amino acid positions corresponding to 84-89 of SEQ ID NO: 14.Definitions

[0113] As described herein, the term “percent (%) sequence identity,” and terms related thereto, in the context of amino acid sequences or nucleic acid sequences, is the percentage of amino acid residues or nucleic acid residues in a candidate sequence that are identical with the amino acid residues or nucleic acid residues, respectively, in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity or percent nucleic acid identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as Clustal Omega, BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software, with BLAST being the alignment algorithm of preference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, although for simplicity it maybe preferred to use default parameters.

[0114] As used herein, the term “individual” is synonymous with patient and / or subject and includes and / or refers to a human and may be a human that has been diagnosed as needing to treat a disease or condition as disclosed herein. However, examples are not limited to humans and include, chimpanzees, marmosets, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. The term “individual” includes vertebrates. The individual is typically a human and may be a human that has been diagnosed as needing to treat a disease or condition as disclosed herein.

[0115] As used herein, treating or treatment of includes and / or refers to ameliorating the disease or disorder or symptoms thereof (e.g., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In certain embodiments, treating or treatment also includes and / or refers to alleviating or ameliorating at least one physical and / or biological parameters including those which may not be discernible by the patient. In certain embodiments, treating or treatment includes and / or refers to modulating a disease, disorder, or biological process either physically, (e.g, stabilization of a discernible symptom), physiologically, (e.g, stabilization of a physical and / or biological parameter), or both. In certain embodiments, treating or treatment includes and / or refers to preventing or delaying the onset or development or progression of the disease or disorder. In certain embodiments, treating or treatment includes and / or refers to preventing or delaying or inhibiting the deterioration of (i) a healthy physiological state or (ii) a baseline physiological state (e.g., the progression of a disease or disorder).

[0116] As used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of’ and / or “consisting of,” unless context clearly connotes otherwise. Similarly, as used herein, in any instance or embodiment described herein, “comprises” may be replaced with “consists essentially of’ and / or “consists of,” unless context clearly connotes otherwise.

[0117] As used herein, the term “about,” in the context of a given value or range, includes and / or refers to a value or range that is within 10% of the given value or range.

[0118] As used herein, the term “and / or” is to be taken as specific disclosure of each of the specified features or components alone or with any combination of other features or components. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each were set out individually herein.EXAMPLES

[0119] The application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concepts.Example 1: Expression and Purification of Knob and Hole Half IgG Proteins

[0120] Knob and Hole half IgG proteins are expressed using standard Mammalian expression systems and can be purified with high yield and purity.Cell Culture Assay

[0121] Cells were grown in complete growth medium and maintained at 37°C with shaking at 150 RPM, 8% CO2, and 80% humidity.Knob and Hole protein expression

[0122] Knob and Hole protein fragments (half IgG proteins) were expressed and purified from mammalian cells (e.g., Expi293F™ cells, ExpiCHO-S™ cells) using transient transfection following the manufacturer's protocol. At designated time point (e.g., 4-14 days), media was harvested by centrifugation at 4,000xg for 10 minutes.Knob and Hole protein purification

[0123] Knob and Hole protein fragments were purified by affinity chromatography (e.g., Protein A affinity chromatography) and buffer exchanged into PBS containing 10% glycerol. The Knob and Hole protein fragments were then concentrated (e.g., 150 uM Knob concentration, 200 uM Hole concentration), and flash frozen for storage at -80°C. Purity and integrity of all proteins are assessed by SDS-PAGE.Example 2: Assembly of Knob and Hole Heterodimeric Proteins

[0124] The knob-into-hole approach can be an effective way of preventing mispairing and promoting heterodimerization to produce a desired heterodimeric protein (bispecific antibodies).Assembly

[0125] Purified Knob and Hole protein fragments were incubated in Tris buffer (e.g., 10 mM Tris, pH 8, 0.1 M NaCl, 160 mM Arginine, and 2 mM Glutathione) at a ratio of about 3:4 (e.g., knob:hole ratio). Purified Knob and Hole protein fragments have also been incubated at a ratio of about 1:2 (e.g., knob:hole ratio) to produce the heterodimeric protein product (bispecific antibody). The incubation of the purified Knob and Hole protein fragments was fora period of about 16 hours to about 24 hours at 35°C with shaking at 150 RPM, resulting in the formation of the heterodimeric protein product (bi specific antibody).Example 3: Purification of Knob and Hole Heterodimeric Proteins by CHI Resin

[0126] The differing CHI domains of the heterodimeric protein product (bispecific antibody) facilitates a one-step purification protocol with anti-CHl resins as described below. This simplifies the purification process by removing knob-knob homodimers as well as other impurities in a single step.

[0127] Alternative bispecific purification methods (such as Protein A resin purification) often involve mutations and / or modifications in the Fc region, such as the FcRn binding site which can result in unfavorable pharmacokinetics. Alternatively, the CHI domain plays no role in the pharmacokinetics or activity of an antibody and modification of it is has not been found to have any impact on any critical function and maintains key portions of the Fc region, such as the FcRn binding site. Other alternative purification techniques (such as IEX, HIC, or MMC) typically bind to all species present in a sample and rely on salt gradients to achieve fine separation of target and off-target species. This type of separation must be run on dedicated equipment, such as FPLC or HPLC, and the method must be refined for each individual molecule, severely limiting the generalizability and throughput of these methods.Binding of CHI domains

[0128] The binding of a half antibody (100 nM) containing a CHI domain (wild-type IgGl, mouse-human chimeric, mouse, IgA, IgD, IgE, or IgM CHI domain) was assessed using CaptureSelect™ Biotin Anti-IgG-CHl ligand (10 nM) immobilized on a streptavidin BLI sensor. The half antibody containing the wild-type IgGl CHI domain shows strong binding with little dissociation observed over a 10-minute period (see FIG. 15, WT CHI). The half antibody containing the mouse-human chimeric CHI shows significantly weakened binding (see FIG. 15) and half antibodies containing the mouse, IgA, IgD, IgE, or IgM CHI domains fail to bind (see FIG. 15). These results demonstrates that bispecific antibodies containing wild-type IgGl CHI domain and a mouse-human chimeric, mouse, IgA, IgD, IgE, or IgM CHI domain will bind to an anti-CHl resin. However, a mis-paired bispecific antibody containing hole-hole homodimers (e.g., bispecific antibody containing two mouse-human chimeric, mouse, IgA, IgD, IgE, or IgM CHI domains) will not bind to an anti-CHl resin.CHI Resin Purification

[0129] The assembly reaction (see Example 2) was nutated for about one hour at room temperature (from about 21 °C to about 25°C) with CaptureSelect™ CHI -XL resin (ThermoScientific™) that has been pre-equilibrated with PBS. The resin was then added to a gravity column and washed 3X with PBS to remove everything except the heterodimeric protein (bispecific antibody), see FIG. 16, FIG. 19 (lanes 6-10), and FIG. 20 (lane 2) depicting gel images of the bispecific antibodies purified using CHI resin purification.

[0130] If no additional purification steps are needed following the CHI resin purification, the heterodimeric protein was eluted as follows. The column was washed 2X with 2.4 mM Histidine, pH 6, followed by a wash with 2.4 mM Histidine, pH 4. The heterodimeric protein (bispecific antibody) was eluted from the column by washing with 12 mM Histidine, pH 2.5 and neutralized with 60 mg / ml Arginine, pH 8.4. The final buffer conditions are 10 mM Histidine, 10 mg / ml Arginine, pH 6 to which 10 mM Histidine, 10 mg / ml Arginine, pH 6, and +25% (v / v) Trehalose (final concentration of Trehalose 5% (v / v)) were added. The desired final product (bispecific antibody) was then diluted to a target concentration in 10 mM Histidine, 10 mg / ml Arginine, pH 6, +5% (v / v) Trehalose. The concentration of the heterodimeric protein was determined using absorbance at 280 nm based on calculating the extinction coefficient.

[0131] If additional steps are needed following the CHI resin purification, the heterodimeric protein was eluted as follows. The heterodimeric protein was eluted from the column with 50 mM sodium acetate, pH 3.5 and neutralized with 1 M Tris, pH 8. The desired final product (bispecific antibody) was then concentrated to a target concentration using a 30K MWCO centrifugal filter.Protein A Resin Purification

[0132] To compare purification of the heterodimeric protein (bispecific antibody) to a non-CH1 resin, the heterodimeric protein was purified using MabSelect PrismA™ using similar techniques as described above, see FIG. 19 (lanes 1-5) and FIG. 20 (lane 1) depicting gel images of the bispecific antibodies purified using protein A resin purification.Example 4: Assessing Heterodimeric Protein Purity

[0133] Purification of bispecific antibodies using methods described herein differ from other purification methods, including affinity capture, ion exchange (IEX), hydrophobic interaction (HIC), and multi-mode chromatography (MMC), in that substitution of the WT IgG CHI domain with a mouse-human chimeric CHI domain, IgA, IgD, IgE, or IgM CHI domain, or CHI domain from a non-binding species, completely ablates or severely weakens binding to the CHI resin by one half of the bispecific antibody (see FIG. 15), providing binding of target species and little-to-no binding off-target species. The purification methods describedherein are run on a 96-well filter plate in a single purification step resulting in over 97% of purified bispecific antibodies with greater than 90% purity (as measured by SDS-PAGE, see description below) and greater than 90% monomer (as judged by analytical SEC, see description below). The purification methods described herein are highly generalizable and have been readily and successfully applied to the purification of nearly 2000 unique constructs.See Table 2 for examples of unique constructs comprising non-IgG CHI domains (as disclosed herein) and purified using methods disclosed herein.Table 2. Exemplary constructs comprising non-IgG CHI domains and purified using CHI resin.Knob Antigen Hole AntigenCD71 Amyloid BetaTREM2 Amyloid BetaCD 163 Amyloid BetaRSV F Protein Amyloid BetaCD206 Amyloid BetaMSR1 Amyloid BetaMUCI EGFR RNF43 EGFRCD71 EGFRMRC2 EGFRMET EGFR CD276 EGFRTROP2 EGFRRSV F Protein EGFRhuHER.2 EGFRCXCR7 EGFRRSV EGFR CD71 METRSV F Protein METACKR4 RSV F ProteinCD71 RSV F ProteinCD32b RSV F ProteinmuCD68 RSV F ProteinMUCI RSV F ProteinCXCR7 RSV F ProteinICAM1 RSV F ProteinLDLR RSV F ProteinMSR1 RSV F ProteinCXCR3 RSV F ProteinCD248 RSV F ProteinMRC2 RSV F ProteinANTXR1 RSV F ProteinCD8 RSV F ProteinSORT1 RSV F ProteinCD99 RSV F ProteinIL 1 IRA RSV F ProteinCD25 RSV F ProteinCD63 RSV F ProteinMET RSV F ProteinIGF2R RSV F ProteinCD19 RSV F ProteinEGFR RSV F ProteinITGB7 RSV F ProteinNECTIN2 RSV F ProteinENG RSV F ProteinASGPR RSV F ProteinSDC1 RSV F ProteinCD 146 RSV F ProteinCD 163 RSV F ProteinPDGFRA RSV F ProteinRNF43 RSV F ProteinCD45RO RSV F ProteinCD83 RSV F ProteinCD38 RSV F ProteinTROP2 RSV F ProteinGPR20 RSV F ProteinD0G1 RSV F ProteinIL6R RSV F ProteinCD20 RSV F ProteinCCR4 RSV F ProteinTREM2 RSV F ProteinCD206 RSV F ProteinLampl RSV F ProteinActivin RSV F ProteinRSV F Protein RSV F ProteinCD68 RSV F ProteinFcRn RSV F ProteinNon-Reducing SDS-PAGE

[0134] Non-Reducing SDS-PAGE was used to separate protein subunits not linked by disulfide bonds (e.g., noncovalently linked proteins) to analyze the purity of the heterodimeric protein (bispecific antibody). Briefly, a sample of the purified heterodimeric protein wasdiluted to 10 uM in PBS then added to 4x Laemmli Sample Buffer (Bio-Rad). The resulting mixture was incubated from about 68°C to about 72°C (e.g., 70°C) for 5 minutes to allow for denaturation to occur. The incubated sample is then loaded onto a 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (along with a reference standard) and run at about 200 V for about 35 minutes. The purity of the heterodimeric protein (bispecific antibody) was analyzed by imaging the resulting gel using GelDoc Go Imaging System (Bio-Rad). For example, see FIG. 16, FIG. 19 (lanes 6-10), and FIG. 20 (lane 2) depicting gel images of the bispecific antibodies purified using CHI resin purification which show a single purified band (the purified bispecific antibodies) indicating high yield and homogeneity of the purified product. For bispecific antibodies purified using MabSelect PrismA™, see FIG. 19 (lanes 1-5), and FIG.20 (lane 1), results show multiple purified bands (the purified bispecific antibodies and impurities) indicating low yield and heterogeneity of the purified product. Percent purity reported at the bottom of each lane was quantified by integrating intensity of all detectable bands that could be accurately integrated.Size Exclusion Chromatography (SEC)

[0135] Size exclusion chromatography (SEC) was used to determine the purity and assess aggregation of the purified heterodimeric protein (bispecific antibody). Briefly, the SEC column (AdvanceBIO™ SEC 300A 2.7 um 4.6x300 mm column, MabPAC™ SEC-1 4x150 mm column) was equilibrated with 150 mM sodium phosphate, pH 7. A sample of the purified heterodimeric protein was loaded onto the column and run at a flow-rate according to manufacturer instructions. SEC output was analyzed for monomeric content and retention time of the target species (purified heterodimeric protein) according to manufacturer instructions. For example, see FIG. 1, FIG. 3, FIG. 5, FIG. 7, FIG. 9, FIG. 11, and FIG. 13 for SEC chromatograms of the purified heterodimeric protein (bi specific antibody) which show a single purified peak (the purified heterodimeric protein) indicating high yield and homogeneity of the purified product.Example 5: Heterodimeric Protein Functionality

[0136] A binding assay (e.g., Octet® binding assay) was used to assess the functionality of the purified heterodimeric protein by monitoring the binding between the heterodimeric protein (bispecific antibody) and corresponding antigens.Measuring Binding A ffinity

[0137] Octet® AHC2 biosensors were equilibrated in binding buffer (PBS and 0.1% Casein, pH 7.4) for at least 10 minutes and then regenerated using the standard low pH protocolaccording to manufacturer instructions. A sample of the purified heterodimeric protein was diluted to a concentration of 10 nM in binding buffer. The corresponding antigens were also diluted in binding buffer. Diluted heterodimeric protein samples, a corresponding antigen, and binding buffer were loaded onto a 384-well plate and run with step times according to manufacturer instructions. The Octet® binding assay output was analyzed for affinity measurements and epitope binning of the purified heterodimeric protein (bispecific antibody) according to manufacturer instructions. For example, see the binding curves depicted in FIG.2, FIG. 4, FIG. 6, FIG. 8, FIG. 10, FIG. 12, FIG. 14, FIG. 17A, FIG. 17B, FIG. 17C, FIG.17D, FIG. 17E, FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, and FIG. 18E showing binding kinetics of the purified heterodimeric protein (bispecific antibody) to a corresponding antigen, indicating binding functionality of the purified heterodimeric protein. Table 3 and Table 4 below show the KD (nM) of bispecific antibodies containing non-IgG CHI domains to target antigens.Table 3. KD (nM) of bispecific antibodies containing non-IgG CHI domains to RSV F protein.Non-IgG CHI domain KD(nM)Mouse-Human Chimeric 0.142Mouse <0.5IgA <0.5IgD 0.146IgE 0.237IgM 0.130Table 4. KD (nM) of bispecific antibodies containing non-IgG CHI domains to human CD71.Non-IgG CHI domain KD(nM)Mouse-Human Chimeric 0.420Mouse 0.292IgA 0.172IgD 0.389IgE 0.490IgM 0.379

[0138] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the instant disclosure. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the embodiments disclosed herein, and that methods and structures within the scope of these claims and their equivalents be covered thereby.SEQUENCESSEQ ID NO. Sequence Annotation 1 NHKPSN human iggl epitope 2 AHPASS mouse epitope 3 KHPASS mod mouse epitope 4 KHYTNP iga epitope 5 QHTASK igd epitope 6 AHTPSS ige epitope 7 QHPNGN igm epitope 8 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD h iggl to mouse YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLS S V VT VP S S SLGTQT YICNVAHP AS STK V DKKV9 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD h iggl to mod mouse YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLS SV VT VP S S SLGTQT YICNVKHP AS STK V DKKV10 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD h_iggl to iga YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVKHYTNPTK VDKKV11 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD h_iggl to igd YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVQHTASKTK VDKKV12 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD h_iggl to ige YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVAHTPSSTKV DKKV13 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD h_iggl to igM YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVQHPNGNTK VDKKV14 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD h_iggl wt YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV15 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP human fc knob KDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP human fc hole KDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY human cl kappa PREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC GQPKANPTVTLFPPSSEELQANKATLVCLISD human cl lambda 1 FYPGAVTVAWKADGSPVKAGVETTKPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS GQPKAAPSVTLFPPSSEELQANKATLVCLISD human cl lambda 2 FYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS GQPKAAPSVTLFPPSSEELQANKATLVCLISD human cl lambda 3 FYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHKSYSCQVTHE GSTVEKTVAPTECS GQPKAAPSVTLFPPSSEELQANKATLVCLVS human cl lambda 7 DFNPGAVTVAWKADGSPVKVGVETTKPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

Claims

CLAIMS1. A composition comprising a heterodimeric protein, wherein the heterodimeric protein comprises a first heavy chain (HC) constant region and a second HC constant region, wherein:(i) the first HC constant region comprises a first CHI domain comprising a first CHI amino acid sequence; and(ii) the second HC constant region comprises a second CHI domain, wherein the second CHI domain comprises a second CHI amino acid sequence that is different from the first CHI amino acid sequence.

2. The composition of claim 1, wherein the first CHI domain comprises a human CHI amino acid sequence and the second CHI domain comprises a non-human CHI amino acid sequence.

3. The composition of claim 1, wherein the first CHI domain comprises an IgG CHI amino acid sequence and the second CHI domain comprises a non-IgG CHI amino acid sequence.

4. The composition of claim 1, wherein the first CHI domain comprises an IgGl CHI amino acid sequence and the second CHI domain comprises a non-IgGl CHI amino acid sequence.

5. The composition of any one of claims 1-4, wherein the first CHI domain comprises an IgGl CHI amino acid sequence.

6. The composition of any one of claims 1-5, wherein the second CHI domain exhibits at a 2-fold or greater reduction in binding to a resin having a ligand that specifically binds the first CHI domain.

7. The composition of claim 6, wherein the second CHI domain is not retained by a column comprising a resin having a ligand that specifically binds the first CHI domain.

8. The composition of any one of claims 1-7, wherein:the first CHI domain comprises a first FG-loop structure and the first CHI amino acid sequence comprises a first FG-loop amino acid sequence; andthe second CHI domain comprises a second FG-loop structure and the second CHI amino acid sequence comprises a second FG-loop amino acid sequence that is different from the first FG-loop sequence.

9. The composition of claim 8, wherein the first FG-loop structure and the second FG- loop structure comprises CHI domain amino acid positions 203-208 per EU numbering.

10. The composition of any one of claims 8-9, wherein:(i) the first FG-loop structure comprises a human FG-loop amino acid sequence and the second FG-loop structure comprises a non-human FG-loop amino acid sequence;(ii) the first FG-loop structure comprises an IgG FG-loop amino acid sequence and the second FG-loop structure comprises a non-IgG FG-loop amino acid sequence; or (iii) the first FG-loop structure comprises an IgGl FG-loop amino acid sequence and the second FG-loop structure comprises a non-IgGl FG-loop amino acid sequence.

11. The composition of any one of claims 8-10, wherein the first FG-loop structure comprises the amino acid sequence of SEQ ID NO: 1.

12. The composition of any one of claims 8-11, wherein the second FG-loop structure comprises the amino acid sequence represented by the formula (N- to C-terminus):- XI - His - X2 - X3 - X4 - X5 - (Formula 1)wherein:XI is Ala, Lys, or Gin;X2 is Pro, Tyr, or Thr;X3 is Ala, Thr, Asn, or Pro;X4 is Asn or Ser; andX5 is Asn, Ser, Pro, or Lys.

13. The composition of any one of claims 8-12, wherein the second FG-loop structure comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.

14. The composition of any one of claims 8-13, wherein the first CHI domain comprises:(i) the first FG-loop structure comprising the amino acid sequence of SEQ ID NO: 1; and(ii) an amino acid sequence having at least 75% or greater sequence identity to SEQ ID NO: 14.

15. The composition of any one of claims 8-13, wherein the second CHI domain comprises:(i) the second FG-loop structure comprising the amino acid sequence of Formula 1 or any one SEQ ID NOs: 2-7; and(ii) an amino acid sequence having at least 30% or greater sequence identity to SEQ ID NO: 14.

16. The composition of claim 15, wherein the second CHI domain comprises:(i) the second FG-loop structure comprising the amino acid sequence of Formula 1 or any one SEQ ID NOs: 2-7; and(ii) an amino acid sequence having at least 75% or greater sequence identity to SEQ ID NO: 14.

17. The composition of any one of claims 1-16, wherein:(i) the first HC constant region further comprises a first CH2 domain and a first CH3 domain; and(ii) the second HC constant region further comprises a second CH2 domain and a second CH3 domain.

18. The composition of claim 17, wherein:(i) the first CH3 domain comprises a first CH3 domain amino acid sequence comprising a knob modification; and(ii) the second CH3 domain comprises a second CH3 domain amino acid sequence comprising a hole modification.

19. The composition of claim 17, wherein:(i) the first CH3 domain comprises a first CH3 domain amino acid sequence comprising a hole modification; and(ii) the second CH3 domain comprises a second CH3 domain amino acid sequence comprising a knob modification.

20. The composition of any one of claims 1-19, wherein the heterodimeric protein is an antibody.

21. The composition of any one of claims 1-20, wherein the heterodimeric protein is a bispecific antibody.

22. The composition of claim 21, wherein the bispecific antibody further comprises:a first light chain variable domain and a first heavy chain variable domain; and a second light chain variable domain and a second heavy chain variable domain, wherein, optionally, the first light chain variable domain and the second light chain variable domain are different.

23. The composition of claim 22, wherein the first heavy chain variable domain is covalently linked to the first CHI domain and the second heavy chain variable domain is heavy chain variable domain is linked to the first CHI domain linked to the second CHI domain.

24. The composition of claim 23, wherein the bispecific antibody further comprises first light chain constant domain covalently linked to the first light chain variable domain, and a second light chain constant domain covalently linked to the second light chain variable domain.

25. A polypeptide comprising 75% to 95% sequence identity to SEQ ID NO: 14 wherein polypeptide at positions 84-89 does not comprise SEQ ID NO: 1.

26. The polypeptide of claim 25, wherein the polypeptide exhibits at a 2-fold or greater reduction in binding to a resin having a ligand that binds a CHI domain comprising SEQ ID NO: 14.

27. The polypeptide of claim 26, wherein the polypeptide is not retained by a column comprising a resin having a ligand that specifically binds a CHI domain comprising SEQ ID NO: 14.

28. The polypeptide of any one of claims 24-27, wherein the amino acid sequence at positions 84-89 of SEQ ID NO: 14 represented by the formula:- XI - His - X2 - X3 - X4 - X5 - (Formula 1)wherein:XI is Ala, Lys, or Gin;X2 is Pro, Tyr, or Thr;X3 is Ala, Thr, Asn, or Pro;X4 is Asn or Ser; andX5 is Asn, Ser, Pro, or Lys.

29. A polypeptide comprising the amino acid sequence at positions 84-89 of SEQ ID NO:14 represented by the formula:- XI - His - X2 - X3 - X4 - X5 - (Formula 1)wherein:XI is Ala, Lys, or Gin;X2 is Pro, Tyr, or Thr;X3 is Ala, Thr, Asn, or Pro;X4 is Asn or Ser; andX5 is Asn, Ser, Pro, or Lys; andwherein the polypeptide comprises at least 75% sequence identity to SEQ ID NO: 14.

30. The polypeptide of claim 29, wherein the amino acid sequence at positions 84-89 of SEQ ID NO: 1 comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.

31. The polypeptide of claim 29 or claim 30, wherein the polypeptide is an immunoglobulin heavy chain constant region CHI domain.

32. One or more polynucleotides encoding the heterodimeric protein of any one of claims 1-23 or the polypeptide of any one of claims 24-31.

33. The one or more polynucleotides of claim 32, wherein the one or more polynucleotides are one or more expression vectors.

34. A host cell comprising the one or more polynucleotide of claim 32 or claim 33.

35. One or more host cells comprising the one or more polynucleotide of claim 32 or claim 33.

36. A method of isolating a bispecific antibody comprising the heterodimeric protein of any one of claims 1-23 or the polypeptide of any one of claims 24-31, the method comprising:(a) contacting the bispecific antibody with a support that binds first CHI domain; (b) washing the support; and(c) detaching the bispecific antibody from the support.

37. A method of separating a bispecific antibody comprising the heterodimeric protein of any one of claims 1-23 or the polypeptide of any one of claims 24-31 from a monomeric antibody component thereof or mono-specific antibody, the method comprising: (a) contacting the bispecific antibody with a support that binds first CHI domain; (b) washing the support; and(c) detaching the bispecific antibody from the support.

38. The method of any of claims 36-37, wherein the support exhibits at least a 2-fold reduction in binding to the second CHI domain compared to binding to the first CHI.

39. The method of any one of claims 36-38, wherein the support comprises a resin having a ligand that specifically binds the first CHI domain.

40. The method of claim 39, wherein the ligand specifically binds a human IgG CHI domain.

41. A method of isolating a bispecific antibody comprising a first CHI domain and a second CHI domain, wherein the second CHI domain comprises a different sequence from the first CHI domain, the method comprising:(a) contacting the bispecific antibody with a support that binds the first CHI domain, wherein the support does not bind the second CHI domain; and(b) eluting the bispecific antibody comprising the first CHI domain from the support.

42. A method of separating a bispecific antibody comprising a first CHI domain and a second CHI domain from a monomeric antibody component thereof or monovalent antibody, wherein the second CHI domain comprises a different sequence from the first CHI domain, the method comprising:(a) contacting the bispecific antibody with a support that binds the first CHI domain, wherein the support does not bind the second CHI domain; and (b) eluting the bispecific antibody comprising the first CHI domain from the support.

43. The method of claim 41 or claim 42, wherein the first CHI domain comprises SEQ ID NO: 1.

44. The method of claim 43, wherein the different sequence has less than 90%, less than 80%, less than 60%, or less than 50% sequence identity to SEQ ID NO:1.

45. The method of claim 43, wherein the second CHI does not comprise SEQ ID NO: 1.

46. The method of claims 43, wherein the second CHI domain at amino acid positions corresponding to 84-89 of SEQ ID NO: 14 comprises the amino acid sequence represented by the formula:- XI - His - X2 - X3 - X4 - X5 - (Formula 1)wherein:XI is Ala, Lys, or Gin;X2 is Pro, Tyr, or Thr;X3 is Ala, Thr, Asn, or Pro;X4 is Asn or Ser; andX5 is Asn, Ser, Pro, or Lys.

47. The method of claims 46, wherein the second CHI domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-7 at amino acid positions corresponding to 84- 89 of SEQ ID NO: 14.

48. The method of any one of claims 42-47, wherein the support comprises a resin having a ligand that specifically binds the first CHI domain.

49. The method of claim 48, wherein the ligand specifically binds a human IgG CHI domain.

50. The method of any one of claims 42-49, wherein after (a) and before (b), the method comprises washing the support.

51. The method of any one of claims 42-50, wherein the contacting is performed on a column comprising the support that binds the first CHI domain.

52. A method of purifying an unpurified composition comprising one or more bispecific antibodies, wherein each of the one or more bispecific antibodies comprises a heterodimer formed by a first heavy chain (HC) and a second HC, wherein the method comprises:(a) contacting the one or more bispecific antibodies with a support that binds the first HC;(b) washing the support; and(c) detaching the one or more bispecific antibodies from the support,wherein the purifying results in a purified composition comprising one or more bispecific antibodies at a purity of greater than about 95%.

53. A method of purifying an unpurified composition comprising one or more bispecific antibodies, wherein each of the one or more bispecific antibodies comprises a heterodimer formed by a first heavy chain (HC) and a second HC, wherein the method comprises:(a) contacting the one or more bispecific antibodies with a support that binds the first HC;(b) washing the support; and(c) detaching the one or more bispecific antibodies from the support,wherein the purifying results in a purified composition comprising greater than 95% of the one or more bispecific antibodies with less than 10% protein impurity.

54. A method of purifying an unpurified composition comprising one or more bispecific antibodies, wherein each of the one or more bispecific antibodies comprises a heterodimer formed by a first heavy chain (HC) and a second HC, wherein the method comprises:(a) contacting the one or more bispecific antibodies with a support that binds the first HC;(b) washing the support; and(c) detaching the one or more bispecific antibodies from the support,wherein purifying the one or more bispecific antibodies takes less than about 2 hours.

55. The method of any one of claims 52-54, wherein:(i) the first HC comprises a first domain, or portion thereof, comprising a first amino acid sequence; and(ii) the second HC comprises a second domain, or portion thereof, wherein the second domain comprises a second amino acid sequence that is different from the first amino acid sequence.

56. The method of claim 55, wherein the first domain comprises a first CHI domain and the second domain comprises a second CHI domain.

57. The method of claim 55, wherein the first domain comprises a first CH2 domain and the second domain comprises a second CH2 domain.

58. The method of claim 55, wherein the first domain comprises a first CH3 domain and the second domain comprises a second CH3 domain.

59. The method of claim 58, wherein the one or more bispecific antibodies comprises a knob and hole bispecific IgG antibody.

60. The method of claim 55, wherein the first domain comprises a first VH domain and the second domain comprises a second VH domain.

61. The method of any one of claims 52-54, wherein each of the one or more bispecific antibodies comprises the heterodimeric protein of any one of claims 1-23 or the polypeptide of any one of claims 24-31.

62. The method of any one of claims 52-61, wherein purifying the one or more bispecific antibodies is performed in the absence of a protein tag.

63. The method of any one of claims 52-62, wherein each of the one or more bispecific antibodies does not comprise a protein tag.

64. The method of any one of claims 52-63 wherein purifying the one or more bispecific antibodies is performed in the absence of a pH gradient.

65. The method of any one of claims 52-64, wherein purifying the one or more bispecific antibodies is performed in the absence of a buffer gradient.

66. The method of any one of claims 52-65, wherein purity is determined by SDS-PAGE or size-exclusion chromatography.

67. The method of any one of claims 52-66, wherein the purified composition comprises monovalent antibodies at a concentration of less than 5%.

68. The method of any one of claims 52-67, wherein the purified composition comprises from about 0.05 mgs to about 50 kgs of the one or more bispecific antibodies.

69. The method of any one of claims 52-68, wherein purifying the one or more bispecific antibodies takes less than about 1 hour.

70. The method of any one of claims 52-69, wherein 1.35 equivalents of the one or more bispecific antibodies are produced from 1 equivalent of the first HC and 1.25 equivalents of the second HC.

71. The method of any one of claims 52-70, wherein the purified composition comprises greater than 80% of the one or more bispecific antibodies from the unpurified composition.

72. A method of producing a composition comprising about 50 kgs or more of a heterodimeric protein, wherein the composition comprises less than 5% protein impurity, and wherein the heterodimeric protein comprises a first heavy chain (HC) constant region and a second HC constant region, wherein the first HC constant region comprises:(i) a first CHI domain comprising a first CHI amino acid sequence,(ii) a first CH2 domain comprising a first CH2 amino acid sequence,(iii) a first CH3 domain comprising a first CH3 amino acid sequence, or(iv) a first VH domain comprising a first VH amino acid sequence; andthe second HC constant region comprises:(i) a second CHI domain, wherein the second CHI domain comprises a second CHI amino acid sequence that is different from the first CHI amino acid sequence,(ii) a second CH2 domain, wherein the second CH2 domain comprises a second CH2 amino acid sequence that is different from the first CH2 amino acid sequence,(iii) a second CH3 domain, wherein the second CH3 domain comprises a second CH3 amino acid sequence that is different from the first CH3 amino acid sequence, or(iv) a second VH domain, wherein the second VH domain comprises a second VH amino acid sequence that is different from the first VH amino acid sequence.

Images