Bispecific screening compositions and methods

Abstract

Described herein are cognate pairs of fusion proteins and methods of screening multispecific antibodies.

Description

Atorney Docket No. DUA-OOIWO BISPECIFIC SCREENING COMPOSITIONS AND METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 557,100, filed on February 23, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submited electronically and is hereby incorporated by reference in its entirety. The XML copy of the Sequence Listing, created on February 2, 2025, is named DUA-001WO_SL.xml and is 130,062 bytes in size.BACKGROUND

[0003] Therapeutic antibodies are gaining clinical approval in record numbers. Bispecific antibodies, designed to engage two distinct targets or target epitopes, represent the next generation of antibody therapeutics. There are over 90 bispecific antibodies in development. Exemplary bispecifics include emicizumab-kxwh (Hemlibra®), co-targeting factor IX and factor X for hemophilia A, and blinatumomab (Blincyto®), a bispecific T cell engager (BiTE) cotargeting CD3 and CD 19 for acute lymphoblastic leukemia. Bispecific antibodies are an atractive candidate for clinical therapies, as they can uniquely influence cellular responses. However, selecting target combinations for optimal functional activity remains challenging.

[0004] Bispecific target selection to date has been largely driven by pre-selection of a defined antigen pair or a small number of functionally-related targets, based upon their known biological function. The functional activity of bispecific antibodies is difficult to predict from that of the individual parental antibodies, and a systematic, large-scale screening approach has not yet been fully exploited for the co-discovery of the optimal target antigen pairs for modulation of a given cellular response. Therefore, there exists an outstanding need for improved bispecific and multispecific antibody combinations screening methods.SUMMARY OF THE DISCLOSURE

[0005] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to hen egg white lysozyme; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-hen egg white lysozymeAtorney Docket No. DUA-OOIWO antibody or antibody fragment thereof configured to form a complex with the hen egg white lysozyme. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a hen egg white lysozyme and an anti-hen egg white lysozyme antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof is an anti-hen egg white lysozyme heavy chain variable antibody (VHH). In some embodiments, the anti -hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the hen egg white lysozyme. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the hen egg white lysozyme by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-hen egg white lysozyme antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti -hen egg white lysozyme antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody orAtorney Docket No. DUA-OOIWO antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the hen egg white lysozyme binds the anti -hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 10 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 1 nM.

[0006] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to ribonuclease A; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-ribonuclease A antibody or antibody fragment thereof configured to form a complex with the ribonuclease A. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a ribonuclease A and an anti-ribonuclease A antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof is an anti-ribonuclease A VHH. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the ribonuclease A. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the ribonuclease A by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-ribonuclease A antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-ribonuclease A antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).Atorney Docket No. DUA-OOIWO In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof bind with a KDICSS than about 10 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0007] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to maltose binding protein (MBP); and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-MBP antibody or antibody fragment thereof configured to form a complex with the MBP. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a MBP and an anti-MBP antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the MBP comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the anti-MBP antibody or antibody fragment thereof is an anti-MBP VHH. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the MBP. In some embodiments, theAtorney Docket No. DUA-OOIWO antibody or antibody fragment of the first fusion protein is operably linked to the MBP by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-MBP antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-MBP antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0008] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to dihydrofolate reductase; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-dihydrofolate reductaseAtorney Docket No. DUA-OOIWO antibody or antibody fragment thereof configured to form a complex with the dihydrofolate reductase. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of dihydrofolate reductase and an anti-dihydrofolate reductase antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof is an anti-dihydrofolate reductase VHH. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the dihydrofolate reductase. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the dihydrofolate reductase by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-dihydrofolate reductase antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-dihydrofolate reductase antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibodyAtorney Docket No. DUA-OOIWO fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0009] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-BC2 peptide antibody or antibody fragment thereof configured to form a complex with the BC2 peptide. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a BC2 peptide and an anti-BC2 peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof is an anti-BC2 peptide VHH. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the BC2 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the BC2 peptide by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-BC2 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-BC2 peptide antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In someAtorney Docket No. DUA-OOIWO embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0010] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide antibody or antibody fragment thereof configured to form a complex with the Alfa peptide. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of an Alfa peptide and an anti-Alfa peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof is an anti-Alfa peptide VHH. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the AlfaAtorney Docket No. DUA-OOIWO peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the Alfa peptide by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-Alfa peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-Alfa peptide antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the Alfa peptide and the anti-Alfa peptide antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0011] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide; and b) a second fusion protein comprising an antibody orAtorney Docket No. DUA-OOIWO antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof configured to form a complex with the GCN4 peptide. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a GCN4 peptide and an anti-GCN4 peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the GCN4 peptide comprises the amino acid sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises the amino acid sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises the amino acid sequence according to SEQ ID NO: 112. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof is a GCN4 peptide scFv. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the GCN4 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the GCN4 peptide by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-GCN4 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-GCN4 peptide antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34,Atorney Docket No. DUA-OOIWO 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0012] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide; and b)a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide antibody or antibody fragment thereof configured to form a complex with the Flag peptide. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a Flag peptide and an anti-Flag peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the Flag peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof is an anti-Flag peptide VHH. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the Flag peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the Flag peptide by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-Flag peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-Flag peptide antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97),Atorney Docket No. DUA-OOIWO GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0013] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a thermostable green fluorescent protein (TFP); and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-TFP antibody or antibody fragment thereof configured to form a complex with the TFP. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a thermostable green fluorescent protein (TFP) and an anti-TFP antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the TFP comprises the amino acid sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the anti-TFP antibody or antibody fragment thereof is an anti-TFP VHH. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20.Atorney Docket No. DUA-OOIWO In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the TFP. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the TFP by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-TFP antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-TFP antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0014] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a gp41 peptide; and b) a second fusion protein comprising an antibody orAtorney Docket No. DUA-OOIWO antibody fragment thereof operably linked to an anti-gp41 peptide antibody or antibody fragment thereof configured to form a complex with the gp41 peptide. Described herein, in certain embodiments, are compositions comprising a multispecific antibody, the improvement comprising a complex of a gp41 peptide and an anti-gp41 peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the gp41 peptide comprises the amino acid sequence according to SEQ ID NO: 21. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof is an anti-gp41 peptide VHH. In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the gp41 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the gp41 peptide by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-gp41 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-gp41 peptide antibody or antibody fragment thereof by at least one linker. In some embodiments, the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96). In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to theAtorney Docket No. DUA-OOIWO sequence according to any one of SEQ ID NOs: 42, 44, and 46. In some embodiments, the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof bind with a KD less than about 10 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof bind with a KD less than about 1 nM.

[0015] Described herein, in certain embodiments, are cognate pairs, comprising a hen egg white lysozyme comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 2 and an anti-hen egg white lysozyme antibody or antibody fragment thereof.

[0016] Described herein, in certain embodiments, are cognate pairs, comprising a ribonuclease A comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7 and an anti-ribonuclease A antibody or antibody fragment thereof.

[0017] Described herein, in certain embodiments, are cognate pairs, comprising a maltose binding protein (MBP) comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 9 and an anti-MBP antibody or antibody fragment thereof.

[0018] Described herein, in certain embodiments, are cognate pairs, comprising a dihydrofolate reductase comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13 and an anti-dihydrofolate reductase antibody or antibody fragment thereof.

[0019] Described herein, in certain embodiments, are cognate pairs, comprising a human [3-catenin (BC2) peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 25 and an anti-BC2 peptide antibody or antibody fragment thereof.

[0020] Described herein, in certain embodiments, are cognate pairs, comprising an Alfa peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 27 and an anti-Alfa peptide antibody or antibody fragment thereof.

[0021] Described herein, in certain embodiments, are cognate pairs, comprising a GCN4 peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 23 and an anti-GCN4 antibody or antibody fragment thereof. Described herein, in certain embodiments, are cognate pairs, comprising a GCN4 peptide comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 111 and 112 and an anti-GCN4 antibody or antibody fragment thereof.Atorney Docket No. DUA-OOIWO

[0022] Described herein, in certain embodiments, are cognate pairs, comprising a Flag peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 29 and an anti-Flag peptide antibody or antibody fragment thereof.

[0023] Described herein, in certain embodiments, are cognate pairs, comprising a thermostable green fluorescent protein (TFP) comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 16-18 and an anti-TFP antibody or antibody fragment thereof.

[0024] Described herein, in certain embodiments, are cognate pairs, comprising a gp41 peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 21 and an anti-gp41 peptide antibody or antibody fragment thereof.

[0025] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to hen egg white lysozyme, ribonuclease A, maltose binding protein (MBP), dihydrofolate reductase, a human [3-catenin (BC2) peptide, an Alfa peptide, a GCN4 peptide, a Flag peptide, thermostable green fluorescent protein (TFP), or a gp41 peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to a means for binding the hen egg white lysozyme, ribonuclease A, MBP, dihydrofolate reductase, a BC2 peptide, an Alfa peptide, a GCN4 peptide, a Flag peptide, TFP, or a gp41 peptide.

[0026] Described herein, in certain embodiments, are pluralities of cognate pairs, wherein the cognate pair is a cognate pair described herein.

[0027] Described herein, in certain embodiments, are mammalian expression vectors, comprising: a) a first nucleic acid encoding any one of SEQ ID NOs: 1-4, 6, 7, 9, 12, 13, 16-18, 21, 23, 25, 27, 29, 111, and 112; b) a second nucleic acid encoding any one of SEQ ID NOs: 5, 8, 10, 11, 14, 15, 19, 20, 22, 24, 26, 28, and 30; and c) a mammalian promoter.

[0028] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a hen egg white lysozyme and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-hen egg white lysozyme antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the hen egg white lysozyme joined with the anti-hen egg white lysozyme antibody or antibody fragment thereof.

[0029] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a ribonuclease A and a second fusion protein comprising an antibodyAtorney Docket No. DUA-OOIWO or antibody fragment thereof operably linked to an anti-ribonuclease A antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the ribonuclease A joined with the anti-ribonuclease A antibody or antibody fragment thereof.

[0030] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a maltose binding protein (MBP) and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-MBP antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the MBP joined with the anti-MBP antibody or antibody fragment thereof.

[0031] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a dihydrofolate reductase and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-dihydrofolate reductase antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the dihydrofolate reductase joined with the anti-dihydrofolate reductase antibody or antibody fragment thereof.

[0032] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a human [3-catenin (BC2) peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-BC2 peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the BC2 peptide joined with the anti-BC2 peptide antibody or antibody fragment thereof.

[0033] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the Alfa peptide joined with the anti-Alfa peptide antibody or antibody fragment thereof.

[0034] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide with an antibody or antibody fragment thereof comprising a BC2 peptide binding domain and an Alfa peptide binding domain; and b) identifying the cognate pair comprising the first fusion protein and the secondAtorney Docket No. DUA-OOIWO fusion protein joined by the antibody or antibody fragment thereof comprising the BC2 peptide binding domain and the Alfa peptide binding domain.

[0035] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the GCN4 peptide joined with the anti-GCN4 peptide antibody or antibody fragment thereof.

[0036] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the Flag peptide joined with the anti-Flag peptide antibody or antibody fragment thereof.

[0037] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide with an antibody or antibody fragment thereof comprising a GCN4 peptide binding domain and a Flag peptide binding domain; and b) identifying the cognate pair comprising the first fusion protein and the second fusion protein joined by the antibody or antibody fragment thereof comprising the GCN4 peptide binding domain and the Flag peptide binding domain.

[0038] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the GCN4 peptide joined with the anti-GCN4 peptide antibody or antibody fragment thereof.

[0039] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a thermostable green fluorescent protein (TFP) and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-TFP antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the TFP joined with the anti-TFP antibody or antibody fragment thereof.Atorney Docket No. DUA-OOIWO

[0040] Described herein, in certain embodiments, are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a gp41 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-gp41 peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the gp41 peptide joined with the anti-gp41 peptide antibody or antibody fragment thereof.

[0041] Described herein, in certain embodiments, is a hen egg white lysozyme comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 2-4.

[0042] Described herein, in certain embodiments, is a ribonuclease A comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 7.

[0043] Described herein, in certain embodiments, is a dihydrofolate reductase comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 13.

[0044] Described herein, in certain embodiments, is a thermostable green fluorescent protein (TFP) comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 17-18.

[0045] Described herein, in certain embodiments is a mammalian expression vector, comprising a nucleic acid encoding any one of SEQ ID NOs: 2-4, 7, 13, and 17-18; and a mammalian promoter.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The novel features of the disclosure are set forth with particularity in the appended claims. A beter understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0047] FIGs. 1A-1B are a set of schematics depicting cognate pair fusion proteins of the disclosure. FIG. 1A is a schematic depicting a cognate pair comprising a first heavy chain variable domain (VH), a first constant heavy chain 1 (CHI), a first light chain variable domain (VL), and a first constant light chain (CL) (i.e., a Fab antibody fragment) operably linked to a protein or peptide (“A”). A binds to a protein or peptide (“B”) that is operably linked to a second Fab antibody fragment. Binding of “A” and “B” mediates complex formation of the first and second fusion proteins. FIG. IB is a schematic depicting a cognate pair comprising a VHH (e.g., nanobody, left) or a VHH linked to a CHI and CL (right) operably linked to a protein or peptide (“A”), which can bind to a protein or peptide (“B”), as described in FIG. 1A.Atorney Docket No. DUA-OOIWO

[0048] FIGs. 2A-2B are a set of graphs depicting the production yield of various fusion proteins (as described in FIGs. 1A-1B). Tested fusion proteins comprised proteins / peptides (i.e., fusion component) that included a hen egg white lysozyme (HEL) protein (“CP1A(WT)”), a function-deficient HEL (“CPIA(KO)”), an anti -HEL VHH antibody fragment (referred to interchangeably throughout the disclosure as “CP1B(1)” or “CP1B”), a ribonuclease A (“CP2A(WT)”), a function-deficient ribonuclease A (“CP2A(KO)”), an anti-ribonuclease A VHH antibody fragment (“CP2B(1)”), a maltose binding protein (“CP3A(WT)”), an antimaltose binding protein VHH antibody fragment (“CP3B(1)” and “CP3B(2)”), a dihydrofolate reductase (“CP4A(WT)”), a function-deficient dihydrofolate reductase (“CP4A(KO)”), an anti-dihydrofolate reductase VHH antibody fragment (“CP4B(1)” and “CP4B(2)”), a thermostable green fluorescent protein (GFP; “CP5A(WT)), a function-deficient thermostable GFP (“CP5A(KO1)” and “CP5A(KO2)”), an anti-GFP VHH antibody fragment (“CP5B(1)” and “CP5B(2)”), a gp41 peptide (“CP6A”), an anti-gp41 peptide VHH antibody fragment (“CP6B”), a yeast transcription factor GCN4 peptide (“CP7A”), an anti-GCN4 peptide single chain variable domain (scFv) antibody fragment (“CP7B”), a human |3-catcnin peptide (“CP8A”), an Alfa peptide (“CP9A”), and a Flag peptide (“CP10A”). FIG. 2A is a graph depicting the production yield in micrograms of each fusion protein, calculated by multiplying the concentration postpurification by the total volume of purified sample, divided by the volume (3 mL) of expression culture. FIG. 2B is a graph depicting the same data as FIG. 2A but with the mass yield data converted to molar yield by adjusting for the calculated molecular weight of each construct.

[0049] FIG. 3 is a graph depicting the correlation between the molar production yield of fusion proteins from FIG. 2B as a function of the molecular weight of the given protein / peptide.

[0050] FIG. 4 is a graph depicting the percentage of monomeric species of the fusion proteins described in FIGs. 2A-2B as determined by size exclusion chromatography (SEC). The percentage of monomers reflects the area under the curve of the production yield following postaffinity purification.

[0051] FIG. 5 is a set of immunoblots from SDS polyacrylamide gels depicting the production and potential glycosylation (“*”) of fusion proteins described in FIGs. 2A-2B.

[0052] FIGs. 6A-6B are a set of graphs depicting the production yield of fusion proteins described in FIGs. 2A-2B each operably linked to a HER2 Fab antibody component. FIG. 6A is a graph depicting the expression in mass of the respective cognate pair in 3 mL Expi293 culture (n=8). FIG. 6B is a graph depicting the same data as FIG. 6A but with the expression depicted in a molar basis.Atorney Docket No. DUA-OOIWO

[0053] FIG. 7 is a graph depicting the percentage of bound cognate pair fusion proteins in an enzyme-linked immunosorbent assay (ELISA). As described in FIG. 2B, CP1A(WT), CPIA(KO), CP2A(WT), CP2A(KO), CP3A(WT), CP4A(WT), CP4A(KO), CP5A(WT), CP5A(KO1), CP5A(KO2), and CP7A were fused to an anti-HER2 Fab antibody fragment (“A fusion proteins”); while the CP1B(1), CP2B(1), CP3B(1), CP3B(2), CP4B(1), CP4B(2), CP5B(1), CP5B(2), and CP7B were fused to an anti-CD22 Fab antibody fragment (“B fusion proteins”). The x-axis reflects a dose range of A and B fusion proteins for each cognate pair preformed at 1: 1 molar ratio. The y-axis reflects absorbance at 450 nm normalized to the maximum absorbance across the experiment.

[0054] FIG. 8 is a graph depicting the percentage of bound cognate pair fusion proteins in an ELISA. The experiment is as described for FIG. 7 but in a replicate experiment only for the cognate pair fusion proteins CPIA(KO): CP1B and CP7A:CP7B.

[0055] FIG. 9 is a graph depicting the percentage of bound cognate pair fusion proteins in an ELISA. As described in FIGs. 2A-2B, CP1A(WT) and CPIA(KO) were fused to an anti-HER2 Fab antibody fragment (“A fusion proteins”); while the CP1B was fused to an anti-PD-1 Fab antibody fragment (“B fusion proteins”). PD-1 antigen protein was coated on plates, and complexes were detected with biotinylated HER2 protein and streptavidin fused to horseradish peroxidase. A plate not coated with PD-1 was used as a negative control. The x-axis reflects a dose range of A and B fusion proteins for each cognate pair preformed at a 1 : 1 molar ratio. The y-axis reflects absorbance at 450 nm.

[0056] FIG. 10 is a graph depicting the percentage of bound cognate pair fusion proteins in an ELISA. As described in FIGs. 2A-2B, CP1A(WT) and CPIA(KO) were fused to an anti-HER2 Fab antibody fragment (“A fusion proteins”); while the CP IB was fused to an anti-PD-1 Fab antibody fragment (“B fusion proteins”). PD-1 antigen protein was coated on plates, and complexes were detected with biotinylated HER2 protein and streptavidin fused to horseradish peroxidase. A fusion protein comprising an anti-PD-1 Fab as the antibody component and a nanobody control that does not bind CP1A(WT) or (KO) as the fusion component was used as a negative control. The x-axis reflects a dose range of HER2Fab-linker(“lnkl”)-CPlA(WT) and HER2Fab-lnkl-CPlA(KO). The y-axis reflects absorbance at 450 nm.

[0057] FIG. 11 is a graph depicting the stability of cognate fusion proteins of the disclosure comprising fusion components described in FIGs. 2A-2B each fused to an anti-HER2 Fab antibody component, as measured by differential scanning fluorimetry (DSF). The circles in the graph represent melting temperatures (Tm), with a higher Tm representing melting of the Fab, whereas a second, lower Tm represents fusion. The presence of only one higher Tm with anAtorney Docket No. DUA-OOIWO absence of a second Tm indicates that the fusion partner melting overlapped with the Tm of the Fab.

[0058] FIG. 12 is a graph depicting non-specific cell binding of cognate pair fusion proteins of the disclosure comprising proteins / peptides described in FIGs. 2A-2B each fused to an anti-HER2 Fab antibody fragment. Fusion proteins were tested for binding to activated primary human CD4-positive T cells, which do not express HER2. Anti-LAG3 and anti-PD-1 IgG antibodies were used as positive controls, as they do bind activated CD4-positive T cells. The percentage of positive cells for labeled fusion proteins and controls are depicted.

[0059] FIG. 13 is a graph depicting non-specific cell binding of cognate pair fusion proteins of the disclosure comprising proteins / peptides described in FIGs. 2A-2B each fused to an anti-PD- 1 Fab antibody fragment. Binding was measured in HEK293 cells transiently transfected with PD-1, and the respective proteins / peptides fused to an anti-HER2 Fab antibody fragment were tested as negative controls. The percentage of positive cells for labeled fusion proteins and controls are depicted.

[0060] FIG. 14 is a schematic depicting multispecific antibodies of the disclosure, including a bispecific antibody (left panel) and two exemplary trispecific antibody orientations (middle and right panels). Similar to FIG. 1A, the left panel depicts a cognate pair comprising a first VH, a first CHI, a first VL, and a first CL (i.e., a Fab antibody fragment) operably linked to a first protein or peptide. The first protein or peptides binds to a second protein or peptide that is operably linked to a second Fab antibody fragment. Binding of the first and second proteins or peptides, respectively, mediates complex formation of the first and second fusion proteins. The middle panel depicts a similar orientation as the left panel except that the first Fab antibody fragment is covalently linked to a third Fab antibody fragment. Meanwhile, the right panel depicts a similar orientation as the left panel except that the first protein or peptide is bound noncovalently by a counter-epitope binder that is itself covalently linked to a third Fab antibody fragment.

[0061] FIG. 15 is a graph depicting the percentage of B cell proliferation inhibition when cells are stimulated with anti-IgM and anti-CD40 and exposed to bispecific anti-CD22 / anti-CD79b antibodies having CD22(1) (“1”), CD22(2) (“2”), or CD22(3) (“3”) binders as well as CD79b(l) (“1”), CD79b(3) (“3”), or CD79b(4) (“4”) binders, when constructed using full-length knobs-into-hole bispecific IgG format (BsAb) or CPI or CP7 cognate pair fusion proteins described herein.

[0062] FIG. 16 is a set of schematics depicting bispecific antibodies of the disclosure, as described in FIG. 1A and FIG. 14, and four exemplary orientations of labeled (gray star)Atorney Docket No. DUA-OOIWO counter-epitope binders to enable detection of one of the proteins or peptides of the cognate pair fusion protein.DETAILED DESCRIPTION DEFINITIONS

[0063] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0064] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as a Fc fragment thereof of an antibody (e.g., an Fc fragment thereof of a monoclonal antibody), or an antigen-binding fragment thereof of an antibody (e.g., an antigen-binding fragment thereof of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated. In general, antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable domain (VL) and one constant region (CL). The heavy chain consists of one variable domain (VH) and at least three constant regions (CHI, CH2, and CH3). The variable domains determine the binding specificity of the antibody. Each variable domain contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The extent of the FRs and CDRs has been defined (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies. Examples of antibody-based antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. Antibodies or antibody fragments include single chain antibodies (e.g., single-chain fragment variable antibodies (scFvs)), single domain antibodies (e.g., heavy-chain-only antibodies (VHH), shark heavy-chain-only antibodies (VNAR)), nanobodies, diabodies, minibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, or Fv fragments. Examples of antibodies that have been modified or engineered includeAtorney Docket No. DUA-OOIWO chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific and trispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0065] As used herein, the term “multispecific antibody” refer to antibodies that bind to two or more distinct antigens or two or more distinct epitopes within the same antigen or a plurality of antigens. Multispecific antibodies may have cross-reactivity to other related antigens, for example to the different forms of an antigen from other species (i.e., homologs), such as human or monkey, for example Maccicci fasciculciris (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0066] The terms “variable domain” and “variable region” are used interchangeably and refer to the portions of the antibody or immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody. Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable domains. These sub-domains are called “hypervariable regions” or “complementarity determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called the “framework” regions (FRM or FR) and provide a scaffold for the six CDRs in three-dimensional space to form an antigen-binding surface. An antibody variable domain may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL), or it may comprise a single heavy chain variable domain (VHH), also referred to as a nanobody as is known in the art.

[0067] An “Fc polypeptide” of a dimeric Fc as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e., a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, an Fc polypeptide of a dimeric IgG Fc comprises an IgG CH2 and an IgG CH3 constant domain sequence. An Fc can be of the class IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 5, a, y, and p, respectively. Several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0068] As used herein, the term “complex” refers to a joining of at least two components. The two components may each retain the propertie s / activities they had prior to forming the complex or gain properties as a result of forming the complex. The joining includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bond), use of a linker, fusion (i.e., fused directly), or any other suitable method. Contemplated components of the complex includeAtorney Docket No. DUA-OOIWO polynucleotides, polypeptides, or combinations thereof. For example, a complex comprises a hen egg white lysozyme and an anti-hen egg white lysozyme antibody or fragment thereof.

[0069] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0070] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90%-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold includes 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, and 20-fold, etc., as well as 1.1-, 1.2-, 1.3-, 1.4-, and 1.5-fold, etc., 2.1-, 2.2-, 2.3-, 2.4-, and 2.5-fold, etc., and so forth.

[0071] “About” a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.“Percent (%) identity” refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al. , 1984).

[0072] As used herein, the terms “single domain antibodies (sdAb),” “nanobodies (Nb),” and “single heavy variable domains (VHH)” are used interchangeably and refer to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Nanobodies are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family. Single domain antibodies are fairly stable and easy to express as fusion partner with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). Appl. Microbiol Biotechnol. 77(1): 13-22).Atorney Docket No. DUA-OOIWO

[0073] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0074] As a general mater, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the definition known in the art of the variable controls.COGNATE PAIR FUSION PROTEINS

[0075] A barrier to screening large panels of multispecific e.g., bispecific antibodies is combinatorial scale. Number of pairwise combinations are calculated based on the equation n! / (2 ! (n-2) ! ), where n equals the number of antibodies in the set. As an illustration, a screen of 100 targets with a set of 5 antibodies per target (500 antibodies total), contains 124,750 pairwise combinations. Production of that number of individual covalently-linked bispecific antibodies in a reasonable timescale is presently intractable.

[0076] There is a need to address this by separating the production of the individual bispecific arms from the combinatorial scale of the screen. While production of 124,750 bispecific antibodies is intractable, the number of individual antibody arms that make up those bispecifics is, as stated, only 500. Production of 500 individual antibodies is experimentally manageable.

[0077] Provided herein are compositions and methods relating to improving screening of multispecifics. For example, cognate pair fusion proteins described herein can bring together two antibodies in a manner that approximates a covalently-linked bispecific IgG (FIG. 1A). Fusion proteins can comprise an antibody or antibody fragment component that is operably linked to a fusion component (e.g., a protein or a peptide). Antibodies or antibody fragments (e.g., Fabs, scFvs, VHH, nanobodies, or single domain antibodies) can then be fused to a set of fusion components that are themselves a cognate binding pair, referred to herein as fusion components “A” and “B.” Mixture of any fusion protein comprising component A with any fusion protein comprising component B produces a noncovalent bispecific antibody that can then be investigated in the context of large scale screens. Meanwhile, as one of skill in the art will understand, cognate pair fusion proteins described herein can also bring together three antibodies (FIG. 14, middle and right panels).Atorney Docket No. DUA-OOIWO

[0078] Described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to hen egg white lysozyme, ribonuclease A, maltose binding protein (MBP), dihydrofolate reductase, a human [3-catenin (BC2) peptide, an Alfa peptide, a GCN4 peptide, a Flag peptide, thermostable green fluorescent protein (TFP), or a gp41 peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to a means for binding the hen egg white lysozyme, the ribonuclease A, the MBP, the dihydrofolate reductase, the BC2 peptide, the Alfa peptide, the GCN4 peptide, the Flag peptide, TFP, or the gp41 peptide.

[0079] Further described herein, in certain embodiments, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to hen egg white lysozyme, ribonuclease A, maltose binding protein (MBP), dihydrofolate reductase, a human [3-catenin (BC2) peptide, an Alfa peptide, a GCN4 peptide, a Flag peptide, thermostable green fluorescent protein (TFP), or a gp41 peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an antibody or antibody fragment thereof selected from the group consisting of an anti-hen egg white lysozyme antibody or antibody fragment thereof, anti-ribonuclease A antibody or antibody fragment thereof, anti-MBP antibody or antibody fragment thereof, anti -dihydrofolate reductase antibody or antibody fragment thereof, anti-BC2 peptide antibody or antibody fragment thereof, anti-Alfa peptide antibody or antibody fragment thereof, anti-GCN4 peptide antibody or antibody fragment thereof, anti-Flag peptide antibody or antibody fragment thereof, anti-TFP antibody or antibody fragment thereof, and anti-gp41 peptide antibody or antibody fragment thereof.

[0080] In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to hen egg white lysozyme. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to ribonuclease A. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to MBP. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked dihydrofolate reductase. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to a BC2 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to an Alfa peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to a GCN4 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to a Flag peptide. In some embodiments, the antibody or antibodyAtorney Docket No. DUA-OOIWO fragment of the first fusion protein is operably linked to a TFP. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to a gp41 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to an antibody or antibody fragment thereof comprising a BC2 peptide binding domain and an Alfa peptide binding domain or an antibody or antibody fragment thereof comprising a GCN4 peptide binding domain and a Flag peptide binding domain. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to an antibody or antibody fragment thereof comprising a BC2 peptide binding domain and an Alfa peptide binding domain. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to an antibody or antibody fragment thereof comprising a GCN4 peptide binding domain and a Flag peptide binding domain.

[0081] In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-hen egg white lysozyme antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-ribonuclease A antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-MBP antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-dihydrofolate reductase antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-BC2 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-Alfa peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-Flag peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-TFP antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to an anti-gp41 peptide antibody or antibody fragment thereof.

[0082] In some embodiments, the first fusion protein comprises a protein or peptide (e.g., hen egg white lysozyme) comprising an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a sequence disclosed in Table 1 and Table 5, below. In some embodiments, the second fusion proteinAtorney Docket No. DUA-OOIWO comprises an antibody or antibody fragment thereof (e.g., anti -hen egg white lysozyme antibody or antibody fragment thereof) comprising an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a sequence disclosed in Table 1 and Table 5, below.Hen Egg White Lysozyme

[0083] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to hen egg white lysozyme; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-hen egg white lysozyme antibody or antibody fragment thereof configured to form a complex with the hen egg white lysozyme.

[0084] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a hen egg white lysozyme and an anti-hen egg white lysozyme antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0085] For example, a composition comprising a bispecific antibody comprising a complex of a hen egg white lysozyme and an anti-hen egg white lysozyme antibody or antibody fragment thereof can be constructed by mixing a Fab fused to the anti-hen egg white lysozyme antibody or antibody fragment thereof with a stacked Fab fused to hen egg white lysozyme, in which the stacked Fab is a Fab at the N-terminus and the other Fab is at the C-terminus followed by hen egg white lysozyme. As one of skill in the art will understand, the reverse orientation of hen egg white lysozyme and the anti -hen egg white lysozyme antibody or antibody fragment thereof could also be generated. One of skill in the art will understand that the sortase-based coupling approach described in Example 8 is one of several methods that could be employed to link antibodies in tandem, including chemical coupling, for example using disulfide chemistry and / or click chemistry, as well as using recombinant linkage, including but not limited to recombinant fusion of Fabs, Nbs, or scFvs.

[0086] Further described herein, are cognate pairs, comprising a hen egg white lysozyme comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 1-4 and an anti-hen egg white lysozyme antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 5).

[0087] In some embodiments, the hen egg white lysozyme is wild-type. In some embodiments, the hen egg white lysozyme comprises one or more modifications. In someAtorney Docket No. DUA-OOIWO embodiments, the modifications are selected from the group consisting of: E35Q, E35D, and D52N.

[0088] In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 85% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 90% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 91% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 92% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 93% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 94% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 95% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 96% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 97% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 98% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence at least 99% identical to the sequence according to any one of SEQ ID NOs: 1-4. In some embodiments, the hen egg white lysozyme comprises an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 1-4.

[0089] In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof is a single chain antibody (e.g., a single-chain fragment variable antibody (scFvs)), a single domain antibody (e.g., a heavy-chain-only antibody (VHH), a shark heavychain-only antibody (VNAR)), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof is an anti-hen egg white lysozyme heavy chain variable antibody (VHH). In some embodiments, the anti -hen egg white lysozyme antibody or antibody fragment thereof is an anti-hen egg white lysozyme nanobody. In some embodiments,Atorney Docket No. DUA-OOIWO the anti-hen egg white lysozyme antibody or antibody fragment thereof is an anti-hen egg white lysozyme Fab.

[0090] In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the antihen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 5. In some embodiments, the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises a sequence 100% identical to the amino acid sequence according to SEQ ID NO: 5.

[0091] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the hen egg white lysozyme. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the hen egg white lysozyme by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused toAtorney Docket No. DUA-OOIWO the anti-hen egg white lysozyme antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-hen egg white lysozyme antibody or antibody fragment thereof by at least one linker.

[0092] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0093] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0094] In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD lower than or equal to 10 nanomolar (nM), 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM,Atorney Docket No. DUA-OOIWO about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.8 nM. In some embodiments, the hen egg white lysozyme binds the anti -hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the hen egg white lysozyme binds the anti -hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by surface plasmon resonance (SPR). In some embodiments, the KD is measured by Biolayer Interferometry (BLI).RnaseA

[0095] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to ribonuclease A; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-ribonuclease A antibody or antibody fragment thereof configured to form a complex with the ribonuclease A.

[0096] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a ribonuclease A and an anti-ribonuclease A antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0097] Further described herein, are cognate pairs, comprising a ribonuclease A comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 6 or SEQ IDAtorney Docket No. DUA-OOIWO NO: 7 and an anti-ribonuclease A antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 8).

[0098] In some embodiments, the ribonuclease A is wild-type. In some embodiments, the ribonuclease A comprises one or more modifications. In some embodiments, the modifications are selected from the group consisting of: H12A and Hl 19A.

[0099] In some embodiments, the ribonuclease A comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the ribonuclease A comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7.

[0100] In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g., a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof is an anti-ribonuclease A VHH. In some embodiments, the anti-ribonuclease A antibody orAtorney Docket No. DUA-OOIWO antibody fragment thereof is an anti-ribonuclease A nanobody. In some embodiments, the antiribonuclease A antibody or antibody fragment thereof is an anti-ribonuclease A Fab.

[0101] In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 8. In some embodiments, the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 8.

[0102] In some embodiments, the anti-ribonuclease A antibody is CAB-RN05 (see e.g., Absolute Antibody, Ab03287-1.159 and Ab03287-23.159 or Creative Biolabs, Cat#:MDWJ5860).

[0103] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the ribonuclease A. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the ribonuclease A by at least one linker. In someAtorney Docket No. DUA-OOIWO embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-ribonuclease A antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-ribonuclease A antibody or antibody fragment thereof by at least one linker.

[0104] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0105] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0106] In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM toAtorney Docket No. DUA-OOIWO about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the ribonuclease A binds the antiribonuclease A antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD less than about 0.8 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a Koless than about 0.7 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a Koless than about 0.2 nM. In some embodiments, the ribonuclease A binds the anti-ribonuclease A antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.MBP

[0107] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to maltose binding protein (MBP); and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-MBP antibody or antibody fragment thereof configured to form a complex with the MBP.

[0108] Described herein, in a composition comprising a multispecific antibody, the improvement comprising a complex of a MBP and an anti-MBP antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0109] In some embodiments, the MBP comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 90% identicalAtorney Docket No. DUA-OOIWO to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 9. In some embodiments, the MBP comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 9.

[0110] In some embodiments, the anti-MBP antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g, a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-MBP antibody or antibody fragment thereof is an anti-MBP VHH. In some embodiments, the anti-MBP antibody or antibody fragment thereof is an anti-MBP nanobody. In some embodiments, the anti-MBP antibody or antibody fragment thereof is an anti-MBP Fab.

[0111] In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identicalAtorney Docket No. DUA-OOIWO to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11.

[0112] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the MBP. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the MBP by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-MBP antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-MBP antibody or antibody fragment thereof by at least one linker.

[0113] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequenceAtorney Docket No. DUA-OOIWO GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0114] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0115] In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 0.8 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KDICSS than about 0.4 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the MBP binds the anti-MBP antibody or antibody fragment thereof with aAtorney Docket No. DUA-OOIWO KDICSS than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.Dihydrofolate Reductase

[0116] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to dihydrofolate reductase; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-dihydrofolate reductase antibody or antibody fragment thereof configured to form a complex with the dihydrofolate reductase.

[0117] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of dihydrofolate reductase and an anti-dihydrofolate reductase antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0118] Further described herein, are cognate pairs, comprising a dihydrofolate reductase comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13 and an anti-dihydrofolate reductase antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 14 or SEQ ID NO: 15).

[0119] In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 96%Atorney Docket No. DUA-OOIWO identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the dihydrofolate reductase comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13.

[0120] In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g., a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof is an anti-dihydrofolate reductase VHH. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof is an anti-dihydrofolate reductase nanobody. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof is an anti-dihydrofolate reductase Fab.

[0121] In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody orAtorney Docket No. DUA-OOIWO antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15.

[0122] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the dihydrofolate reductase. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the dihydrofolate reductase by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-dihydrofolate reductase antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-dihydrofolate reductase antibody or antibody fragment thereof by at least one linker.

[0123] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acidAtorney Docket No. DUA-OOIWO sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0124] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0125] In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.8 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, theAtorney Docket No. DUA-OOIWO dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the dihydrofolate reductase binds the anti-dihydrofolate reductase antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.BC2

[0126] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a human |3-catenin (BC2) peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-BC2 peptide antibody or antibody fragment thereof configured to form a complex with the BC2 peptide.

[0127] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a BC2 peptide and an anti-BC2 peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0128] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide; and b) an antibody or antibody fragment comprising a BC2 peptide binding domain and an Alfa peptide binding domain; and c) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide, wherein the antibody or antibody fragment comprising the BC2 peptide binding domain and the Alfa peptide binding domain joins the first fusion protein and the second fusion protein.

[0129] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a BC2 peptide and an Alfa peptide and an antibody or antibody fragment comprising a BC2 peptide binding domain and an Alfa peptide binding domain. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0130] Further described herein, are cognate pairs, comprising a BC2 peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 25 and an anti-BC2 peptide antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 26).

[0131] In some embodiments, the BC2 peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2Atorney Docket No. DUA-OOIWO peptide comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 25. In some embodiments, the BC2 peptide comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 25.

[0132] In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g., a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof is an anti-BC2 peptide VHH. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof is an anti-BC2 peptide nanobody. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof is an anti-BC2 peptide Fab.

[0133] In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereofAtorney Docket No. DUA-OOIWO comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 26. In some embodiments, the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 26.

[0134] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the BC2 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the BC2 peptide by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-BC2 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-BC2 peptide antibody or antibody fragment thereof by at least one linker.

[0135] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ IDAtorney Docket No. DUA-OOIWO NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0136] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0137] In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.8 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragmentAtorney Docket No. DUA-OOIWO thereof with a KD less than about 0.3 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the BC2 peptide binds the anti-BC2 peptide antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.A Ifa

[0138] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide antibody or antibody fragment thereof configured to form a complex with the Alfa peptide.

[0139] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of an Alfa peptide and an anti-Alfa peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0140] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide; and b) an antibody or antibody fragment comprising a BC2 peptide binding domain and an Alfa peptide binding domain; and c) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide, wherein the antibody or antibody fragment comprising the BC2 peptide binding domain and the Alfa peptide binding domain joins the first fusion protein and the second fusion protein.

[0141] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a BC2 peptide and an Alfa peptide and an antibody or antibody fragment comprising a BC2 peptide binding domain and an Alfa peptide binding domain. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0142] Further described herein, are cognate pairs, comprising an Alfa peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 27 and an anti-Alfa peptide antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 28).

[0143] In some embodiments, the Alfa peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the AlfaAtorney Docket No. DUA-OOIWO peptide comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 27. In some embodiments, the Alfa peptide comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 27.

[0144] In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g., a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof is a anti-Alfa peptide VHH. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof is a anti-Alfa peptide nanobody. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof is a anti-Alfa peptide Fab.

[0145] In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereofAtorney Docket No. DUA-OOIWO comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 28. In some embodiments, the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 28.

[0146] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the Alfa peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the Alfa peptide by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-Alfa peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-Alfa peptide antibody or antibody fragment thereof by at least one linker.

[0147] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ IDAtorney Docket No. DUA-OOIWO NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0148] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0149] In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.8 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less thanAtorney Docket No. DUA-OOIWO about 0.3 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the Alfa peptide binds the anti-Alfa peptide antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.GCN4

[0150] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof configured to form a complex with the GCN4 peptide.

[0151] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a GCN4 peptide and an anti-GCN4 peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0152] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide; and b) an antibody or antibody fragment comprising a GCN4 peptide binding domain and a Flag peptide binding domain; and c) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide, wherein the antibody or antibody fragment comprising the GCN4 peptide binding domain and the Flag peptide binding domain joins the first fusion protein and the second fusion protein.

[0153] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a GCN4 peptide and a Flag peptide and an antibody or antibody fragment comprising a GCN4 peptide binding domain and the Flag peptide binding domain. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0154] In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an aminoAtorney Docket No. DUA-OOIWO acid sequence at least 92% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 23. In some embodiments, the GCN4 peptide comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 23.

[0155] In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 111. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 111. In some embodiments, theAtorney Docket No. DUA-OOIWO GCN4 peptide comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 111.

[0156] In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 112. In some embodiments, the GCN4 peptide comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 112.

[0157] In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g., a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab’ fragment, F(ab’)2 fragment, or a Fv fragment. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof is an anti-GCN4 peptide scFv. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof is an anti-GCN4 peptide VHH. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof is an anti-GCN4 peptide nanobody. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof is an anti-GCN4 peptide Fab.

[0158] In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ IDAtorney Docket No. DUA-OOIWO NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 24. In some embodiments, the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 24.

[0159] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the GCN4 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the GCN4 peptide by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-GCN4 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-GCN4 peptide antibody or antibody fragment thereof by at least one linker.

[0160] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptideAtorney Docket No. DUA-OOIWO or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0161] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0162] In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.8 nM. InAtorney Docket No. DUA-OOIWO some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the GCN4 peptide binds the anti-GCN4 peptide antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.Flag

[0163] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide antibody or antibody fragment thereof configured to form a complex with the Flag peptide.

[0164] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a Flag peptide and an anti-Flag peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0165] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide; and b) an antibody or antibody fragment comprising a GCN4 peptide binding domain and a Flag peptide binding domain; and c) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide, wherein the antibody or antibody fragment comprising the GCN4 peptide binding domain and the Flag peptide binding domain joins the first fusion protein and the second fusion protein.

[0166] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a GCN4 peptide and a Flag peptide and an antibody or antibody fragment comprising a GCN4 peptide binding domain and the Flag peptide bindingAtorney Docket No. DUA-OOIWO domain. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0167] Further described herein, are cognate pairs, comprising a Flag peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 29 and an antiFlag peptide antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 30).

[0168] In some embodiments, the Flag peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 29. In some embodiments, the Flag peptide comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 29.

[0169] In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g., a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof is an anti-Flag peptide VHH. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof is an anti-Flag peptide nanobody. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof is an anti-Flag peptide Fab.Atorney Docket No. DUA-OOIWO

[0170] In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 30. In some embodiments, the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 30.

[0171] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the Flag peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the Flag peptide by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-Flag peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-Flag peptide antibody or antibody fragment thereof by at least one linker.Atorney Docket No. DUA-OOIWO

[0172] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n (SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0173] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0174] In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments,Atorney Docket No. DUA-OOIWO the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.8 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the Flag peptide binds the anti-Flag peptide antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.GFP

[0175] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a green fluorescent protein (GFP, e.g., a thermostable green fluorescent protein (TFP)); and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-TFP antibody or antibody fragment thereof configured to form a complex with the TFP.

[0176] Described herein, in a composition comprising a multispecific antibody, is an improvement comprising a complex of a green fluorescent protein (e.g., a thermostable green fluorescent protein (TFP)) and an anti-TFP antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0177] Further described herein, are cognate pairs, comprising a TFP comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 16-18 and an anti-TFP antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 19 or SEQ ID NO: 20). Described herein, is a plurality of cognate pairs of fusion proteins, wherein the cognate pairs of fusion proteins are a cognate pairs of fusion proteins of any of the foregoing.

[0178] In some embodiments, the green fluorescent protein is a superfolder GFP (sfGFP).

[0179] In some embodiments, the TFP comprises an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments,Atorney Docket No. DUA-OOIWO the TFP comprises an amino acid sequence at least 85% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 90% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 91% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 92% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 93% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 94% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 95% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 96% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 97% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 98% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence at least 99% identical to the sequence according to any one of SEQ ID NOs: 16-18. In some embodiments, the TFP comprises an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 16-18.

[0180] In some embodiments, the anti-TFP antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g, a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-TFP antibody or antibody fragment thereof is an anti-TFP VHH. In some embodiments, the anti-TFP antibody or antibody fragment thereof is an anti-TFP nanobody. In some embodiments, the anti-TFP antibody or antibody fragment thereof is an anti-TFP Fab.

[0181] In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibodyAtorney Docket No. DUA-OOIWO fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20.

[0182] In some embodiments, the anti -green fluorescent protein (e.g., TFP) antibody or antibody fragment thereof is anti-sfGFP VHH or Nb2 (Twair et al., 2014, Molecular Biology Reports 41: 6887-6898; Zhong et al., 2021, Biochem Biophys Res Commun 565: 57-63; Protein Data Bank accession 7E53, each of which is incorporated herein by reference in its entirety).

[0183] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the TFP. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the TFP by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-TFP antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-TFP antibody or antibody fragment thereof by at least one linker.

[0184] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic toAtorney Docket No. DUA-OOIWO avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0185] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0186] In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.8 nM. In someAtorney Docket No. DUA-OOIWO embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.6 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.4 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.2 nM. In some embodiments, the TFP binds the anti-TFP antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.

[0187] Described herein, are cognate pairs of fusion proteins, comprising: a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a gp41 peptide; and b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-gp41 peptide antibody or antibody fragment thereof configured to form a complex with the gp41 peptide.

[0188] Described herein, in a composition comprising a multispecific antibody, the improvement comprising a complex of a gp41 peptide and an anti-gp41 peptide antibody or antibody fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0189] Further described herein, are cognate pairs, comprising a gp41 peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 21 and an anti-gp41 peptide antibody or antibody fragment thereof (e.g., the amino acid sequence according to SEQ ID NO: 22). Described herein, is a plurality of cognate pairs of fusion proteins, wherein the cognate pairs of fusion proteins are a cognate pairs of fusion proteins of any of the foregoing.

[0190] In some embodiments, the gp41 peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequenceAtorney Docket No. DUA-OOIWO at least 92% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 94% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 21. In some embodiments, the gp41 peptide comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 21.

[0191] In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof is a single chain antibody (e.g., a scFvs, a single domain antibody (e.g., a VHH, a VNAR), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, F(ab')2 fragment, or a Fv fragment. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof is an anti-gp41 peptide VHH. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof is an anti-gp41 peptide nanobody. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof is an anti-gp41 peptide Fab.

[0192] In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 85% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 90% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 91% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 92% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 93% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 94% identical to the sequence according to SEQ IDAtorney Docket No. DUA-OOIWO NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 95% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 96% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 97% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 99% identical to the sequence according to SEQ ID NO: 22. In some embodiments, the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 22.

[0193] In some embodiments, the antibody or antibody fragment of the first fusion protein is fused to the gp41 peptide. In some embodiments, the antibody or antibody fragment of the first fusion protein is operably linked to the gp41 peptide by at least one linker. In some embodiments, the antibody or antibody fragment of the second fusion protein is fused to the anti-gp41 peptide antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment of the second fusion protein is operably linked to the anti-gp41 peptide antibody or antibody fragment thereof by at least one linker.

[0194] In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize instability of the cognate pair. In some embodiments, the linker is sufficiently hydrophilic to avoid or minimize insolubility of the cognate pair. In some embodiments, the linker is a peptide or polypeptide. In some embodiments, the linker comprises hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n(SEQ ID NO: 99 or (GGGGS)n (SEQ ID NO: 100), where n is 1-5. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 97). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 101). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker comprises the amino acid sequence EPKSS (SEQ ID NO: 103). Additional exemplary linkerAtorney Docket No. DUA-OOIWO sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0195] In some embodiments, the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

[0196] In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD lower than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD within the range of about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, or about 0.1 nM to about 0.5 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD less than about 1 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD less than about 0.9 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KDICSS than about 0.8 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD less than about 0.7 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KDICSS than about 0.6 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD less than about 0.5 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KDICSS than about 0.4 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD less than about 0.3 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KDICSS than about 0.2 nM. In some embodiments, the gp41 peptide binds the anti-gp41 peptide antibody or antibody fragment thereof with a KD less than about 0.1 nM. In some embodiments, the KD is measured by SPR. In some embodiments, the KD is measured by BLI.Atorney Docket No. DUA-OOIWO NUCLEIC ACIDS AND VECTORS

[0197] Provided herein, in certain embodiments, are isolated nucleic acids (e.g., polynucleotide) encoding the cognate pairs of fusion proteins and multispecific antibodies (e.g., bispecific antibodies or trispecific antibodies) described herein, vectors comprising the nucleic acids, and host cells comprising the vectors and nucleic acids.

[0198] Further described herein, are mammalian expression vectors, comprising a) a first nucleic acid encoding any one of SEQ ID NOs: 1-4, 6, 7, 9, 12, 13, 16-18, 21, 23, 25, 27, 29, 111, and 112; b) a second nucleic acid encoding any one of SEQ ID NOs: 5, 8, 10, 11, 14, 15, 19, 20, 22, 24, 26, 28, and 30; and c) a mammalian promoter.

[0199] Further described herein, in certain embodiments, are isolated nucleic acids (e.g., polynucleotides) encoding a protein described herein (e.g., a hen egg white lysozyme comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 2-4, a ribonuclease A comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 7, a dihydrofolate reductase comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 13, and a thermostable green fluorescent protein (TFP) comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 17-18), vectors comprising the nucleic acids, and host cells comprising the vectors and nucleic acids. In certain embodiments, provided herein are mammalian expression vectors comprising a nucleic acid encoding any one of SEQ ID NOs: 2-4, 7, 13, and 17-18 and a mammalian promoter.

[0200] In some embodiments, the nucleic acids are DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), either single- and / or double-stranded, or native or stabilized forms of polynucleotides, such as, e.g., polynucleotides with a phosphonothioate backbone, or combinations thereof and it may or may not contain introns.

[0201] In some embodiments, the nucleic acids are recombinant. The recombinant nucleic acids may be constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or replication products thereof. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0202] In some embodiments, the nucleic acids encoding the cognate pairs of fusion proteins and multispecific antibodies described herein are delivered by a nucleic acid-based vector. In some embodiments, the nucleic acids encoding the cognate pairs of fusion proteins and multispecific antibodies described herein are delivered by a plasmid (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomesAtorney Docket No. DUA-OOIWO (YAC), bacterial artificial chromosome (BAC), Pl -derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus. In some embodiments, the nucleic acid is comprised in a vector selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFlag, pSF-CMV-NEO-COOH-3XFlag, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-IH-TEV-Flag(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-Nl vector, pEFla-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal,pSF-OXB20-Fluc, pSF-OXB20, pSF-Tac, pRI lOl-AN DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0203] In some embodiments, the vector comprises one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, tip, Ptac, p5, pl 9, p40, Synapsin, CaMKII, GRK1, and derivatives thereof.

[0204] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0205] In some embodiments, the nucleic acid encoding the cognate pairs of fusion proteins and multispecific antibodies described herein is delivered by a non-nucleic acid-based delivery system (e.g., anon-viral delivery system). In some embodiments, the nucleic acid is comprised in a liposome. In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, atached to a liposome via a linkingAtorney Docket No. DUA-OOIWO molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0206] In some embodiments, the nucleic acid encoding the cognate pairs of fusion proteins and multispecific antibodies described herein is introduced into the cell in any suitable way, either stably or transiently. For example, a cell is transduced (e.g., with a virus encoding the cognate pairs of fusion proteins or multispecific antibodies), or transfected (e.g., with a plasmid encoding the cognate pairs of fusion proteins or multispecific antibodies) with a nucleic acid that encodes the cognate pairs of fusion proteins or multispecific antibodies, or the translated cognate pairs of fusion proteins or multispecific antibodies. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, a plasmid expressing the cognate pairs of fusion proteins or multispecific antibodies is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction (for example lentivirus or AAV) or other methods known to those of skill in the art. In some embodiments, the cognate pairs of fusion proteins or multispecific antibodies is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and / or RNPs are known in the art, for example by electroporation or by cell squeezing.MULTISPECIFIC ANTIBODIES

[0207] Described herein are multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies). Bispecific antibodies (e.g., comprising a first binding domain and a second binding domain) are engineered to recognize two different epitopes either on the same or different target antigens. Such antibodies can be operably linked to each other with or without a linker via conventional conjugation chemistries, or, as described herein, indirectly by a cognate pair comprising a first fusion protein and a second fusion protein or a first fusion protein, alone.

[0208] Trispecific antibodies (e.g., comprising a first binding domain, a second binding domain, and a third binding doming) are engineered to recognize three different epitopes either on the same or different target antigens. Such antibodies can be operably linked to each other with or without a linker via conventional conjugation chemistries, or, as described herein, indirectly by a cognate pair comprising a first fusion protein and a second fusion protein.Atorney Docket No. DUA-OOIWO

[0209] Tetraspecific antibodies (e.g., comprising a first binding domain, a second binding domain, a third binding doming, and a fourth binding domain) are engineered to recognize four different epitopes either on the same or different target antigens. Such antibodies can be operably linked to each other with or without a linker via conventional conjugation chemistries, or, as described herein, indirectly by a cognate pair comprising a first fusion protein and a second fusion protein.

[0210] Described herein, are cognate pairs of fusion proteins comprising a first fusion protein comprising a first and / or second antibody or antibody fragment thereof and a second fusion protein comprising a first and / or second antibody or antibody fragment thereof (See e.g., FIG.14).

[0211] In some embodiments, the antibody or antibody fragment thereof of the first fusion protein and / or the second fusion protein is a HER2 binding protein.

[0212] In some embodiments, the HER2 binding protein comprises a VH that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an HER2 binding protein disclosed in Table 2, below, and a VL that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of the same HER2 binding protein disclosed in Table 2, below.

[0213] In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 31 ; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprisingAtorney Docket No. DUA-OOIWO an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 32. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to SEQ ID NO: 31; and a light chain variable domain comprising an amino acid sequence according to SEQ ID NO: 32.

[0214] In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO:Atorney Docket No. DUA-OOIWO 116; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 117. In some embodiments, the HER2 binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to SEQ ID NO: 116; and a light chain variable domain comprising an amino acid sequence according to SEQ ID NO: 117.

[0215] In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 41. In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 41. In some embodiments, theAtorney Docket No. DUA-OOIWO HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 41. In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 41. In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 41. In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 41. In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 41. In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 41. In some embodiments, the HER2 binding protein comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 42; and a light chain comprising an amino acid sequence according to SEQ ID NO: 41.

[0216] Additional exemplary HER2 binding proteins can include those described in U.S.P.N 9,518,118, incorporated herein by reference in its entirety, such as antibody 7C2.

[0217] In some embodiments, the antibody or antibody fragment thereof of the first fusion protein and / or the second fusion protein is a PD-1 binding protein.

[0218] In some embodiments, the PD-1 binding protein comprises a VH that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an PD-1 binding protein disclosed inAtorney Docket No. DUA-OOIWO Table 2, below, and a VL that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of the same PD-1 binding protein disclosed in Table 2, below.

[0219] In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprisingAtorney Docket No. DUA-OOIWO an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 34. In some embodiments, the PD-1 binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 33; and a light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 34.

[0220] In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 98% sequence identity with an amino acidAtorney Docket No. DUA-OOIWO sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 43. In some embodiments, the PD-1 binding protein comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 44; and a light chain comprising an amino acid sequence according to SEQ ID NO: 43.

[0221] In some embodiments, the antibody or antibody fragment thereof of the first fusion protein and / or the second fusion protein is a CD22 binding protein.

[0222] In some embodiments, the CD22 binding protein comprises a VH that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an CD22 binding protein disclosed in Table 2, below, and a VL that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of the same CD22 binding protein disclosed in Table 2, below.

[0223] In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at leastAtorney Docket No. DUA-OOIWO 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 36. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 35; and a light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 36.

[0224] In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variableAtorney Docket No. DUA-OOIWO domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 119. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 118; and a light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 119.

[0225] In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with anAtorney Docket No. DUA-OOIWO amino acid sequence according to any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 120; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 121. In some embodiments, the CD22 binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 120; and aAtorney Docket No. DUA-OOIWO light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 121.

[0226] In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 45. In some embodiments, the CD22 binding protein comprises a heavy chain comprising an amino acidAtorney Docket No. DUA-OOIWO sequence according to SEQ ID NO: 46; and a light chain comprising an amino acid sequence according to SEQ ID NO: 45.

[0227] Additional exemplary CD22 binding proteins can include those described in U.S.P.N.10,370,447, incorporated herein by reference in its entirety, such as antibody 4130; and m971, See Protein Data Bank accession 7052, incorporated herein by reference in its entirety.

[0228] In some embodiments, the antibody or antibody fragment thereof of the first fusion protein and / or the second fusion protein is a CD79 or CD79b binding protein.

[0229] In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQAtorney Docket No. DUA-OOIWO ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 123. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 122; and a light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 123.

[0230] In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variableAtorney Docket No. DUA-OOIWO domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 125. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 124; and a light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 125.

[0231] In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acidAtorney Docket No. DUA-OOIWO sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 127. In some embodiments, the CD79b binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 126; and a light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 127.

[0232] Exemplary CD79b binding proteins can include those described in U.S.P.N.10,370,447, incorporated herein by reference in its entirety, such as Ab4447; those described in Doman et al., 2009, Blood, The Journal of the American Society of Hematology 114.13 (2009): 2721-2729. incorporated herein by reference in its entirety, such as SN8.v28; and those described in U.S.P.N. 9,845,355, incorporated herein by reference in its entirety, such as hu2F2.D7.

[0233] In some embodiments, the antibody or antibody fragment thereof of the first fusion protein and / or the second fusion protein is a CD3 binding protein.Atorney Docket No. DUA-OOIWO

[0234] In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO:Atorney Docket No. DUA-OOIWO 128; and a light chain variable domain comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NO: 129. In some embodiments, the CD3 binding protein comprises a heavy chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 128; and a light chain variable domain comprising an amino acid sequence according to any one of SEQ ID NO: 129.

[0235] Exemplary CD3 binding proteins can include those described in Protein Data Bank accession 1SY6, incorporated herein by reference in its entirety.

[0236] The multispecific antibodies described herein can be conjugated with or without a linker and / or via conventional conjugation chemistries or conditions.SCREENING METHODS

[0237] The present disclosure, in certain embodiments, provides methods of using the cognate pairs of fusion proteins described herein for screening for multispecific antibodies and antibody fragments thereof. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0238] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a hen egg white lysozyme and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-hen egg white lysozyme antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the hen egg white lysozyme joined with the anti-hen egg white lysozyme antibody or antibody fragment thereof.

[0239] Described herein are methods for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a ribonuclease A and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-ribonuclease A antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the ribonuclease A joined with the anti-ribonuclease A antibody or antibody fragment thereof.

[0240] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a MBP and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-MBP antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the MBP joined with the anti-MBP antibody or antibody fragment thereof.

[0241] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to aAtorney Docket No. DUA-OOIWO dihydrofolate reductase and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-dihydrofolate reductase antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the dihydrofolate reductase joined with the anti-dihydrofolate reductase antibody or antibody fragment thereof.

[0242] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-BC2 peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the BC2 peptide joined with the anti-BC2 peptide antibody or antibody fragment thereof.

[0243] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the Alfa peptide joined with the anti-Alfa peptide antibody or antibody fragment thereof.

[0244] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide with an antibody or antibody fragment thereof comprising a BC2 peptide binding domain and an Alfa peptide binding domain; and b) identifying the cognate pair comprising the first fusion protein and the second fusion protein joined by the antibody or antibody fragment thereof comprising the BC2 peptide binding domain and the Alfa peptide binding domain.

[0245] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the GCN4 peptide joined with the anti-GCN4 peptide antibody or antibody fragment thereof.

[0246] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide antibody or antibody fragment thereof; and b) identifyingAtorney Docket No. DUA-OOIWO the cognate pair comprising the Flag peptide joined with the anti-Flag peptide antibody or antibody fragment thereof.

[0247] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide with an antibody or antibody fragment thereof comprising a GCN4 peptide binding domain and a Flag peptide binding domain; and b) identifying the cognate pair comprising the first fusion protein and the second fusion protein joined by the antibody or antibody fragment thereof comprising the GCN4 peptide binding domain and the Flag peptide binding domain.

[0248] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a TFP and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-TFP antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the TFP joined with the anti-TFP antibody or antibody fragment thereof.

[0249] Described herein are methods for identifying a cognate pair, comprising: a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a gp41 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-gp41 peptide antibody or antibody fragment thereof; and b) identifying the cognate pair comprising the gp41 peptide joined with the anti-gp41 peptide antibody or antibody fragment thereof.

[0250] Identification of a cognate pair can be determined by measuring the concentration or relative abundance of a corresponding protein product of interest. Protein levels can be assessed using standard detection techniques known in the art. Examples of protein expression analysis that generate data suitable for use with the methods described herein include, without limitation, proteomics approaches, immunohistochemical and / or western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assay (ELIFA), mass spectrometry, mass spectrometric immunoassay, and biochemical enzymatic activity assays.

[0251] In some embodiments, a sample comprising a cognate pair of the disclosure is contacted with an antibody specific for a component of the cognate pair (e.g., a first fusion protein or a second fusion protein) under conditions sufficient for an antibody-protein complex to form. In some embodiments, the antibody-complex is detected in a number of ways, such as by an label (e.g., an enzyme, a fluorescent label, a chromogenic label, a radionuclide containingAtorney Docket No. DUA-OOIWO molecule (i.e., a radioisotope), or a chemiluminescent molecule), Western bloting, or ELISA procedures using any of a wide variety of tissues or samples, including plasma or serum. A wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653. These include both single-site and two-site or “sandwich” assays of the noncompetitive types, as well as traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target protein.

[0252] Another method involves immobilizing the target protein (e.g., on a solid support) and then exposing the immobilized target to a specific antibody, which may or may not contain a label. Depending on the amount of target and the strength of the label’s signal, a bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emited by a label, e.g., an enzyme, a fluorescent label, a chromogenic label, a radionuclide containing molecule (i.e., a radioisotope), or a chemiluminescent molecule.

[0253] Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to a bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In a typical forward sandwich assay, a first antibody having specificity for the biomarker is either covalently or passively bound to a solid surface (e.g., a glass or a polymer surface, such as those with solid supports in the form of tubes, beads, discs, or microplates), and a second antibody is linked to a label that is used to indicate the binding of the second antibody to the molecular marker.

[0254] In alternative methods, the expression of a protein in a sample may be examined using immunohistochemistry (“IHC”) and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of assessing or detecting presence of proteins in a sample. IHC and immunofluorescence techniques use an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. The tissue sample may be fixed (i.e., preserved) by conventional methodology (see, e.g., “Manual of Histological Staining Method of the Armed Forces Institute of Pathology,” 3rd edition (1960) Lee G. Luna, HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, New York; The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, D.C.). One of skill in the art will appreciate that the choice of a fixative is determined by the purpose for which the sample is to be histologically stained or otherwise analyzed. By way ofAtorney Docket No. DUA-OOIWO example, neutral buffered formalin, Bouin’s, or formaldehyde, may be used to fix a sample. Generally, the sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated and embedded with paraffin or other sectioning media so that the tissue sample may be sectioned. Alternatively, one may section the tissue and fix the sections obtained. The primary and / or secondary antibody used for immunohistochemistry, typically, will be labeled with a detectable moiety, such as a radioisotope, a colloidal gold particle, a fluorescent label, a chromogenic label, or an enzyme-substrate label.

[0255] Exemplary peptide microarrays have a substrate-bound plurality of polypeptides, the binding of an oligonucleotide, a peptide, or a protein to each of the plurality of bound polypeptides being separately detectable. Alternatively, the peptide microarray may include a plurality of binders, including but not limited to monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins.

[0256] Alternatively, the levels of proteins of interest may be detected without the use of binding agents. In some instances, a sample is analyzed, for example, by one or more enzymatic methods, chromatographic methods, mass spectrometry (MS) methods, chromatographic methods followed by MS, electrophoretic methods, electrophoretic methods followed by MS, nuclear magnetic resonance (NMR) methods, and combinations thereof. Exemplary chromatographic methods include, but are not limited to, Strong Anion Exchange chromatography using Pulsed Amperometric Detection (SAX-PAD), liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (U PLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof. Exemplary MS include, but are not limited to, tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), Multiple Reaction Monitoring (MRM), and combinations thereof. Exemplary electrophoretic methods include, but are not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western bloting using antibodies that recognize specific glycan structures, and combinations thereof. Exemplary nuclear magnetic resonance (NMR) include, but are not limited to, onedimensional NMR (1 D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic -angle spinning NMR (COSY -NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NM R), heteronuclear multipleAtorney Docket No. DUA-OOIWO quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY -NMR), nuclear overhauser effect spectroscopy (NOESY -NMR), and combinations thereof.

[0257] Any of the methods herein that rely upon protein measurement can also be adapted for use with the measurement of mRNA levels for the protein. The level of mRNA can be determined using methods known in the art. Methods to measure mRNA levels generally include, but are not limited to, sequencing, northern bloting, RT-PCR, gene array technology, and RNAse protection assays.EQUIVALENTS

[0258] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.EXAMPLES

[0259] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are, therefore, to be considered in all respects illustrative rather than limiting the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.Example 1: Cognate Pair Fusion Proteins, Construction, Expression, and Purification Thereof

[0260] This Example describes the design of cognate pair fusion proteins. This Example further describes the DNA construction, protein expression, and protein purification of the fusion proteins described herein. For a given cognate pair, one fusion protein included an antibody or antibody fragment thereof operably linked to a first fusion component “A,” and the other fusion protein included an antibody or antibody fragment thereof operably linked to a second fusion component “B.” Proteins A and B bound each other to promote complex formation between the first and second protein fusions (FIGs. 1A-1B). Antibodies or antibodyAtorney Docket No. DUA-OOIWO fragments were linked to proteins A and B directly, or separated by a polypeptide linker (e.g., Inkl), as described herein.Materials and MethodsCognate Pair Fusion Proteins

[0261] For a first cognate pair (CP), referred to herein as CPI, a hen egg white lysozyme (HEL) protein (“CP1A(WT)”) was utilized as fusion component A. The sequence of residues K19-L147 of wild-type (WT) HEL are shown in Table 1. Tested knockout (KO) variants thereof included E35Q (referred to interchangeably throughout as “CPIA(KO)” or “CPIA(KOI)”), D52N (“CP1A(KO2)”), and E35D / D52N (“CP1A(KO3)”). For CPI, an anti-HEL VHH (nanobody e.g., see De Genst et al., 2006, PNAS 103 (12): 4586-4591; and Protein Data Bank accession 1ZVY, each of which is incorporated herein by reference in their entirety) was utilized as fusion component B. The sequence of D3-L11 is shown in Table 1.

[0262] For a second cognate pair, referred to as CP2, a globular ribonuclease A (RNase A) protein (“CP2A(WT)”) was utilized as fusion component A. The sequence of WT bovine RNase A is shown in Table 1. A tested KO variant thereof included H12A (“CP2A(KO)”). For CP2, an anti -RNase A VHH (“CP2B”) was utilized as fusion component B. The sequence of CAB-RN05 is shown in Table 1. As used herein, the anti-RNase VHH CAB-RN05 component is referred to as “CP2B” (Table 1).

[0263] For a third cognate pair, referred to as CP3, a maltose binding protein (MBP) was utilized as fusion component A. The sequence of WT Escherichia coli (E. coli) MBP (“CP3A(WT)”) is shown in Table 1. For CP3, an anti-MBP VHH (“CP3B(2)”) was utilized as fusion component B. Anti-MBP VHH referred to as Sb_MBP#l, Sb_MBP#2, and Sb_MBP#3 have been described (Zimmermann et al., 2018, Elife 7:e34317, Protein Data Bank accessions 5M13, 5M14, and 5M15, respectively, each of which is incorporated herein by reference in their entirety), and can bind in the absence of maltose. The sequences of Sb_MBP#l and Sb_MBP#2 are shown in Table 1. As used herein, the anti-MBP VHH Sb_MBP#l and Sb_MBP#2 components are referred to as “CP3B(1)” and “CP3B(2)” respectively (Table 1).

[0264] For a fourth cognate pair, referred to as CP4, a dihydrofolate reductase (DHFR) protein (“CP4A(WT)”) was utilized as fusion component A. The sequence of WT E. coli DHFR is shown in Table 1. A tested KO variant thereof included D27F (“CP4A(KO)”); Table 1). For CP4, the anti-DHFR VHH CA1698 (“CP4B(1)”) and Nbl 13 (“CP4B(2)”) (Oyen et al., 2013, Biochim Biophys Acta 1834: 2147-2157, Protein Data Bank accession 4EIG and 4EIZ, respectively, each of which is incorporated herein by reference in their entirety) were tested as fusion component B. The sequence of the tested anti-DHFR VHH are shown in Table 1.Atorney Docket No. DUA-OOIWO

[0265] For a fifth cognate pair, referred to as CP5, a green fluorescent protein (GFP) was utilized as fusion component A. The sequence of WT TFP is shown in Table 1. Tested KO variants thereof included TFP KO variant, Y66L (“CP5A(KO1)”) and Y66H (“CP5A(KO2)”) (Table 1). For CP5, two anti-TFP VHH were utilized as fusion component B, including Sb44 (“CP5B(1)”) and Sb92 (“CP5B(2)”) (Cai et al., 2020, Commun Biol 3: 753-753, Protein Data Bank accessions 6LZ2 and 7CZ0, respectively, each of which is incorporated herein by reference in their entirety). The sequence of Sb44 and Sb92 are shown in Table 1.

[0266] For a sixth cognate pair, referred to as CP6, an HIV gp41 peptide (“CP6A”) was utilized as fusion component A. The sequence of the gp41 peptide is shown in Table 1. For CP6, the anti-gp41 peptide VHH, 2E7 (Strokappe et al., 2019, Antibodies 8(2):38; Protein Data Bank accession 5HM1, each of which is incorporated herein by reference in their entirety), (“CP6B”) was utilized as fusion component B, though other known anti-gp41 peptide antibodies or fragments thereof may be used interchangeably. The sequence of VHH 2E7 is shown in Table 1.

[0267] For a seventh cognate pair, referred to as CP7, a yeast transcription factor GCN4 peptide (“CP7A”) was utilized as fusion component A. The sequence of the GCN4 peptide is shown in Table 1. For CP7, the anti-GCN4 peptide single chain variable domain (scFv), 52SR4 (Zahnd et al., 2004, J Biol Chem 279: 18870-18877), (“CP7B”)was utilized as fusion component B.

[0268] Also tested as individual components for putative cognate pairs were a human [3-catenin peptide referred to as BC2 (“CP8A”) ,an Alfa peptide (“CP9A”), and a Flag peptide (“CP10A”), the sequences of which are shown in Table 1.Table 1. Exemplary fusion componentsAtorney Docket No. DUA-OOIWOAtorney Docket No. DUA-OOIWOAtorney Docket No. DUA-OOIWOAttorney Docket No. DUA-OOIWO Operably linked antibodies or fragments thereof

[0269] Table 2 shows the sequences of antibody variable domains that were operably linked to the tested proteins described supra, including the variable heavy (VH) and variable light (VL) regions of the anti-HER2 antibody trastuzumab (“HER2”), the VH and VL regions of the anti-PD-1 antibody pembrolizumab (“PD1”), and the VH and VL regions of the anti-CD22 antibody epratuzumab (“CD22”). Table 2 also shows the sequences of antibody constant chains, including the human IgGl constant heavy domain 1 (CHI or Cyl), the human light chain kappa constant domain (Ck or CK), and the human light chain lambda constant domain (Cl or CX), which, in some embodiments, were operably linked to the tested proteins described supra. A DKTGS linker (“Inkl”; SEQ ID NO: 93) was used to connect antibody components to fusion components.Table 2. Exemplary antibody componentsAtorney Docket No. DUA-OOIWOAtorney Docket No. DUA-OOIWOAttorney Docket No. DUA-OOIWO

[0270] Based on the components described supra, a series of fusion proteins were constructed that, in some embodiments, operably linked antibody VH regions to CHI regions to linkers to fusion components (FIG. 1A). The sequences for the designed fusions are provided in Table 3.In these experiments, heavy chains were linked to fusion components, though linkage of light chains to fusion components is contemplated as well. Further, in these experiments antibodyAttorney Docket No. DUA-OOIWO fragments were to fused at their C-terminus (i.e., fusion components were fused at their N-terminus), though the present disclosure further contemplates fusion of antibody components at their N-terminus to the C-terminus of fusion components.Table 3. Exemplary fusion protein sequencesAtorney Docket No. DUA-OOIWOAtorney Docket No. DUA-OOIWOAtorney Docket No. DUA-OOIWO <<<Atorney Docket No. DUA-OOIWOAtorney Docket No. DUA-OOIWOAttorney Docket No. DUA-001WOFusion Protein Construction

[0271] DNA fragments of genes were synthesized and fusion proteins were assembled into a pcDNA3.4 expression vector. All constructs were operably linked at the N-terminus to a leader sequence (also referred to as a signal sequence) (MGWSCIILFLVATATGVHS (SEQ ID NO: 104)) to promote secretion. All DNA was sequenced to confirm fidelity to the designed sequence.

[0272] For the heavy chain fusions described above, co-expression of the heavy chain fusion DNA with the corresponding light chain of the antibody component produced a fusion protein that comprised a Fab antibody component linked to a fusion component. Table 4 lists examples of exemplary fusion proteins tested in the present study, though others were tested as well. Table 4. Exemplary fusion proteinsAtorney Docket No. DUA-OOIWOExpression and Purification of Fusion Proteins

[0273] Purified DNA for heavy and light chains of anti-HER2 fusion proteins and parental Fab control were co-transfected using polyethylenimine (PEI) into 3 mL of Expi293 cell culture. Cell cultures were harvested 6 days post-transfection by centrifugation and supernatants were transferred to deep 24-well plates. CH1-XL affinity resin was added, supernatants were shaken for 2 hours, and resins were transferred to 96 x 2 mL filter blocks with a 96-well deep well block stacked underneath. Resins were washed 3x with PBS, then bound proteins were eluted with phosphoric acid pH 3.0. Samples were neutralized with 20X PBS pH 10.5 to a final pH of about 6.0. All protein fusions were run in duplicate (n=2).Purification

[0274] The purification quality of produced protein fusion samples was investigated using analytical size exclusion chromatography (SEC). Affinity-purified samples were run over a Superdex S200 5 / 150 column using a Vanquish UHPLC System in phosphate buffered solution (PBS) at a flow rate of 0.2 mL / min. The elution of protein was monitored by absorbance at 280 nm. Peaks were integrated to calculate the percentage of monomeric versus higher-MW-species of each protein fusion.

[0275] Purity was also investigated by running purified samples on an SDS polyacrylamide gel followed by Coomassie staining.Results

[0276] Production '. The concentration of protein was quantitated by absorbance at 280 nm using a nanodrop instrument together with the calculated extinction coefficient for each protein.FIG. 2A presents the production yield in pg of each cognate pair component, calculated by multiplying the concentration post-purification by the total volume of purified sample, divided by the volume (3 mL) of expression culture. The mass yield data were converted to molar yield by adjusting for the calculated molecular weight (MW) of each construct (FIG. 2B). FIG. 3 shows the correlation between molar production yield of protein fusions ( see FIG. 2B) as aAtorney Docket No. DUA-OOIWO function of the MW of the fusion component. The results indicated that fusion to Fab generally resulted in lower molar production, with a negative slope (m = -0.09) reflecting an ~1 mol loss per 10 KDa of fusion component. This analysis highlighted the value of fusion components that are small. For example, the favorable molar production yield of some of the globular proteins tested, such as lysozyme and nanobodies, are a result of their high stability and favorable solution properties relative to their small size.

[0277] Production was repeated to compare the hen egg white lysozyme:anti-lysozyme VHH cognate pair (CPI) to the GCN4 peptide: anti -GCN4 peptide scFv cognate pair (CP7). This experiment was executed similarly as described above with all fusions comprising the HER2 Fab component, with expressions done in 3 mb Expi293 culture with n=8. Mass and molar basis production yields post-affinity purification are shown in FIGs. 6A-6B.

[0278] Purity. Regarding the purity, the percentage of monomeric species was ploted for each fusion, as depicted in FIG. 4. The percent monomer reflects the area under the curve (AUC) of the main peak by analytical SEC post-affinity purification. Purity, as shown by Coomassie staining, is depicted in FIG. 5.Example 2: Cognate Pair Fusion Proteins, Construction

[0279] This Example describes the design of cognate pair fusion proteins. For a given cognate pair, one fusion protein comprised an antibody or antibody fragment thereof operably linked to a first fusion component “A,” and the other fusion protein comprised an antibody or antibody fragment thereof operably linked to a second fusion component “B.” Proteins A and B bind each other to promote complex formation between the first and second protein fusions (FIGs. 1A-1B). Antibodies or antibody fragments are linked to proteins A and B directly, or separated by a polypeptide linker (e.g., Inkl), as described herein.Materials and MethodCognate Pair Fusion Proteins

[0280] A cognate pair, referred to as CP8, is generated comprising a human [3-catenin peptide (BC2; “CP8A”) as fusion component A. The sequence of the BC2 peptide is shown in Table 5.For CP8, an anti-BC2 peptide VHH (“CP8B”) is utilized as fusion component B. An anti-BC2 VHH has been described (Braun et al., 2016, Sci Rep 6: 19211; Protein Data Bank accessions 5IVN and 5IVO, each of which is incorporated herein by reference in their entirety) and is used, though other anti-BC2 antibody fragments known in the art can be used. The sequence of the anti-BC2 peptide VHH is shown in Table 5.Atorney Docket No. DUA-OOIWO

[0281] Another cognate pair, referred to as CP9, is generated comprising an Alfa peptide (“CP9A”) as fusion component A. The sequence of the Alfa peptide is shown in Table 5. For CP9, an anti -Alfa peptide VHH (“CP9B”) is utilized as fusion component B. An anti -Alfa peptide VHH referred to as NbAlfa has been described (Gotzke et al., 2019, Nat Commun 10: 4403-4403; Protein Data Bank accession 6I2G, each of which is incorporated herein by reference in their entirety) and is used, though other anti-Alfa peptide antibody fragments known in the art can be used. The sequence of the anti-Alfa peptide VHH NbAlfa is shown in Table 5.

[0282] Another cognate pair, referred to as CP 10, is generated comprising a Flag peptide (“CP10A”) as fusion component A. The sequence of the Flag peptide is shown in Table 5. For CP 10, an anti -Flag peptide scFv (“CPI 0B”) is utilized as fusion component B. An anti -Flag peptide antibody referred to as M2 has been described (Protein Data Bank accession 2G60, Roosild et al., 2006, Acta Crystallogr Sect F Struct Biol Cryst Commun 62: 835-839, each of which is incorporated herein by reference in their entirety) and is used, though other anti-Flag peptide antibody fragments can be used. The sequence of an exemplary anti-Flag peptide scFv of the M2 antibody in the VH-VL orientation is shown in Table 5. Other scFv formats are contemplated, including, for example, VL-VH orientations and different linker lengths and compositions (Zhang et al., 2015, Mabs 7(1): 42-52).

[0283] Additional peptide fusions and peptide tags are contemplated as fusion components for cognate pairs. For example an antibody or antibody fragment may be fused to a His tag (10X = HHHHHHHHHH (SEQ ID NO: 105)), Flag peptide (DYKDDDK (SEQ ID NO: 29)), Myc tag (CEQKLISEEDL (SEQ ID NO: 106)), HA tag (YPYDVPDYA (SEQ ID NO: 107)), HSV epitope tag (QPELAPED (SEQ ID NO: 108)), SI tag (NANNPDWDF (SEQ ID NO: 109)), VSV-G tag (YTDIEMNRLGK (SEQ ID NO: 110)), BC2 peptide (PDRKAAVSHWQQ (SEQ ID NO: 25)), or Alfa peptide (PSRLEEELRRRLTEP (SEQ ID NO: 27)). VHH, scFv, and other antibodies and antibody fragments that bind these peptide tags are known in the art and can be operably linked to the fusion proteins described herein.Table 5. Exemplary fusion componentsAttorney Docket No. DUA-OOIWO

[0284] In some embodiments, a DKTGS linker (“Inkl”; SEQ ID NO: 93) is used to connect antibody components to fusion components, though other linkers can be used. Linkers may be of any length and any sequence composition. For example, antibody and fusion components are linked with a linker that comprises part or all of a hinge region of a natural antibody (e.g., an IgGl, IgG2, IgG3, or IgG4 see e.g., Table 6), i.e., the region that connects an antibody Fab to the Fc region. In another example, one or more residues of the upper hinge of a human IgGl, comprising the sequence DKTHT (SEQ ID NO: 94), is used as a linker. A modified version of this linker, referred to as Inkl, DKTGS (SEQ ID NO: 93), can also be used. In this linker, the mutation of HT to GS keeps the linker the same length as the upper hinge but enables a convenient and unique restriction site for DNA cloning. Additionally, abbreviated versions of this linker may be used, such as DKT. Alternative linkers include DKTHT (SEQ ID NO: 94). Other linkers may utilize sequences containing small and / or flexible amino acids, e.g., glycine, serine, alanine, Gly-Ser, and Gly-Ala linkers. Linkers such as GGGGS (SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), and GGAGG (SEQ ID NO: 96) are examples of Gly-Ser and Gly-Ala linkers. In some embodiments, no linker is used to connect antibody components and fusion components. Examples of linkers are listed in Table 7.Attorney Docket No. DUA-001WO Table 6. Exemplary IgG hinge sequencesTable 7. Exemplary linker componentsExpression and Purification of Fusion Proteins

[0285] Fusion proteins are constructed and can be tested for expression and / or purified, as described in Example 1.Example 3: Cognate Pair Binding and Complex Formation

[0286] Fusion proteins described in Example 1 were expressed, purified, and their binding properties were examined. In addition to fusion proteins comprising HER2 Fab antibody components, fusion proteins were produced for each cognate pair that comprised a Fab of the anti-PD-1 antibody pembrolizumab (“PD1”) as well as a Fab of the anti-CD22 antibody epratuzumab (“CD22”). Fusion proteins comprising PD-1 and CD22 Fabs were expressed and purified as described in Example 1. In addition, C-terminally His-tagged versions of CP IB and CP2B fusion components were expressed and purified as test reagents.Atorney Docket No. DUA-OOIWO

[0287] Binding of cognate pairs was investigated by Biolayer Interferometry (BLI) using a Gator Prime (Gator Bio). In this experiment, B of the cognate pair was captured to a probe, followed by analyte binding by the other side of each pair. PIB-His or P2B-His proteins were loaded at 15 pg / mL to NiNTA probe (Gator) in Gator K buffer. Fusion proteins comprising A of the cognate pair were loaded as analyte at concentrations of 0.12, 0.37, 1.11, 3.33, 10, and 30 nM with 300 seconds for association, followed by 600 seconds for disassociation in K buffer. Sensorgram data were fit to a 1 : 1 kinetic binding model. Fited kinetics and affinities are shown in Table 8.Table 8. Summary of cognate pair affinities by Gator using NiNTA capture

[0288] In a subsequent experiment, binding of cognate pairs was further investigated with the Gator instrument using a method where a target antigen was captured to the probe, followed by binding of a fusion protein comprising one side of the cognate pair that binds that target, followed by analyte binding by the other side of each cognate pair. Biotinylated HER2 was captured to a streptavidin (SA) probe. Fusion proteins comprising an anti-HER2 antibody component and A of the cognate pair were loaded onto the HER2-captured probe at 10 pg / mL in K buffer. Fusion proteins comprising the corresponding B of the cognate pair were loaded as analyte at concentrations of 0.14, 0.41, 1.24, 3.74, 11.11, and 33.33 nM with 180 seconds for association, followed by 180 seconds for disassociation in K buffer. Sensorgram data were fit to a 1:1 kinetic binding model. Fited kinetics and affinities are shown in Table 9.Table 9. Summary of cognate pair affinities by Gator using biotin / streptavidin captureAtorney Docket No. DUA-OOIWO

[0289] CPI was investigated further for any impact of cognate fusion protein (A:B) binding on the binding to antigen by the antibody components. As described above, biotinylated HER2 was first captured to a streptavidin (SA) probe. A concentration series of analyte was then bound. In a first experiment, only HER2Fab-lnkl-CPlA(WT) was loaded as analyte. In a second experiment, a pre-formed complex of HER2Fab-lnkl-CPlA(WT) bound to PD1 Fab-lnkl -CP IB was loaded as analyte. Sensorgram data were fit to a 1 : 1 kinetic binding model. The resulting fited affinities for the first and second experiments were 0.19 nM and 0.25 nM, respectively. These results indicated that the anti-HER2 side of the cognate pair binds its target antigen equivalently in isolation or in complex with a non-HER2 binding cognate pair, such that CPI complex formation does not affect the capacity of the antibody component Fab to bind its target antigen.

[0290] An enzyme-linked immunosorbent assay (ELISA) was executed to investigate the ability of cognate pairs to bridge targets in solution. Cognate fusion pair complexes were formed by mixing A and B components at a 1: 1 molar ratio and incubated overnight at 4 °C. Cognate fusion pairs tested in this experiment comprised HER2 Fab fused to A fusion components and CD22 Fab fused to B fusion components. For example, CPI complexes were formed between HER2Fab-lnkl-CPlA(WT) and CD22Fab-lnkl-CPlB. After overnight incubation, 96-well plates were coated with 1 pg / m L human CD22 antigen, plates were blocked, and washed once with PBS buffer. 2x-diluted A / B complexes were added and plates were washed. 0.1 pg / mL biotinylated HER2 was added and plates were washed. Horseradish Peroxidase (HRP)-fiised streptavidin was added, followed by washing. Absorbance at 450 nm was read on a plate reader. Results are shown in FIG. 7. A repeat bridging experiment was executed as described above with HER2 and CD22 fusion proteins to directly compare CPI and CP7 fusion proteins (FIG.8).Atorney Docket No. DUA-OOIWO

[0291] In a subsequent experiment, target bridging of cognate pairs was further investigated by ELISA using cognate fusion pairs comprising HER2 Fab fused to A fusion components and PD-1 Fab fused to B fusion components. Cognate fusion pair complexes were formed by mixing A and B components at a 1 : 1 molar ratio and incubated overnight at 4 °C. After overnight incubation, 96-well plates were coated with 1 pg / mL human PD-1 antigen, plates were blocked (1% BSA), and plates were wash once with PBS buffer. Plates in which PD-1 was not coated were used as negative controls. 3x-diluted A / B complexes were added and plates were washed.0.1 pg / mL biotinylated HER2 was added and plates were washed. HRP -fused streptavidin was added, plates were washed, and absorbance at 450 nm was read on a plate reader. Results are shown in FIG. 9.

[0292] In a subsequent ELISA experiment, binding of CPI cognate pairs (A and B) was investigated by direct coating of B component fusion protein to a plate. 96-well plates were coated with 1 pg / mL PD1 Fab-lnkl -CP IB, plates were blocked, and washed with PBS buffer. As a negative control, plates were coated with 1 pg / mL PD 1 Fab fused to a nanobody that does not bind CP1A. HER2Fab-lnkl-CPlA(WT) or HER2Fab-lnkl-CPlA(KO) were added in a concentration series and plates were washed. 0.1 pg / mL biotinylated HER2 was added and plates were washed. HRP-fused streptavidin was added, plates were washed, and absorbance at 450 nm was read on a plate reader. Results are shown in FIG. 10.

[0293] Similar experiments can be conducted for the cognate pair fusion proteins described in Example 2.Example 4: Stability of Cognate Fusion Proteins

[0294] Stability of the cognate fusion proteins described throughout Examples 1 and 3 was investigated using differential scanning fluorimetry (DSF). DSF uses a hydrophobic fluorescent dye that binds to proteins as they unfold, thus enabling measurement of protein unfolding by monitoring fluorescence change as a function of temperature. For this experiment, all cognate pair fusion proteins contained the HER2 Fab as the antibody component. All fusion protein samples were exchanged into 10 mM phosphate buffer at pH 7.2 (no salt) and concentrated to 1 mg / mL. 100 mM buffer solutions were prepared for each pH (glycine pH 3.0, acetate pH 5.5, and phosphate pH 7.2). To each well of a PCR plate, 12.5 pL buffer stock, 10 pL protein, and 2.5 pL 100-fold diluted SYPRO Orange Stain were added and mixed. Thermal shift assay was run on a CFX96 qPCR machine with atemperature gradient from 10.0 °C to 95.0 °C in 10 second increments of 0.5 °C. Melting temperatures (Tm’s) were determined based on the negative peaks of the melt curve derivative. Results are shown in FIG. 11. The circles representAtorney Docket No. DUA-OOIWO Tm’s. Based on comparison with unfused Fab (referred to as “Fab” in FIG. 11), the higher Tm represents melting of the Fab, whereas a second lower Tm represents fusion. The presence of only one (higher) Tm (absence of a second Tm) indicates that the fusion partner melting overlaps with Tm of the Fab. The results indicate that fusion proteins do not generally impact Fab stab...

Claims

Atorney Docket No. DUA-OOIWO CLAIMS1. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to hen egg white lysozyme; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-hen egg white lysozyme antibody or antibody fragment thereof configured to form a complex with the hen egg white lysozyme.

2. In a composition comprising a multispecific antibody, the improvement comprising a complex of a hen egg white lysozyme and an anti-hen egg white lysozyme antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

3. The cognate pair of claim 1 or 2, wherein the hen egg white lysozyme comprises an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 1-4.

4. The cognate pair of any one of claims 1-3, wherein the anti-hen egg white lysozyme antibody or antibody fragment thereof is an anti-hen egg white lysozyme heavy chain variable antibody (VHH).

5. The cognate pair of any one of claims 1-4, wherein the anti-hen egg white lysozyme antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 5.

6. The cognate pair of any one of claims 1-5, wherein the antibody or antibody fragment of the first fusion protein is fused to the hen egg white lysozyme.

7. The cognate pair of any one of claims 1-5, wherein the antibody or antibody fragment of the first fusion protein is operably linked to the hen egg white lysozyme by at least one linker.

8. The cognate pair of claim 7, wherein the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

9. The cognate pair of any one of claims 1-7, wherein the antibody or antibody fragment of the second fusion protein is fused to the anti-hen egg white lysozyme antibody or antibody fragment thereof.

10. The cognate pair of any one of claims 1-7, wherein the antibody or antibody fragment of the second fusion protein is operably linked to the anti-hen egg white lysozyme antibody or antibody fragment thereof by at least one linker.Atorney Docket No. DUA-OOIWO 11. The cognate pair of claim 10, wherein the linker comprises the amino acid sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

12. The cognate pair of any one of claims 1-11, wherein the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody.

13. The cognate pair of any one of claims 1-11, wherein the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab.

14. The cognate pair of any one of claims 1-13, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128.

15. The cognate pair of any one of claims 1-14, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129.

16. The cognate pair of any one of claims 1-15, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46.

17. The cognate pair of any one of claims 1-16, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45.

18. The cognate pair of any one of claims 1-17, wherein the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 10 nM.

19. The cognate pair of any one of claims 1-17, wherein the hen egg white lysozyme binds the anti-hen egg white lysozyme antibody or antibody fragment thereof with a KD less than about 1 nM.

20. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to ribonuclease A; andAtorney Docket No. DUA-OOIWO b) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-ribonuclease A antibody or antibody fragment thereof configured to form a complex with the ribonuclease A.

21. In a composition comprising a multispecific antibody, the improvement comprising a complex of a ribonuclease A and an anti-ribonuclease A antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

22. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to maltose binding protein (MBP); andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-MBP antibody or antibody fragment thereof configured to form a complex with the MBP.

23. In a composition comprising a multispecific antibody, the improvement comprising a complex of a MBP and an anti-MBP antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

24. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to dihydrofolate reductase; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-dihydrofolate reductase antibody or antibody fragment thereof configured to form a complex with the dihydrofolate reductase.

25. In a composition comprising a multispecific antibody, the improvement comprising a complex of dihydrofolate reductase and an anti-dihydrofolate reductase antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

26. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-BC2 peptide antibody or antibody fragment thereof configured to form a complex with the BC2 peptide.

27. In a composition comprising a multispecific antibody, the improvement comprising a complex of a BC2 peptide and an anti-BC2 peptide antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

28. A cognate pair of fusion proteins, comprising:Atorney Docket No. DUA-OOIWO a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide antibody or antibody fragment thereof configured to form a complex with the Alfa peptide.

29. In a composition comprising a multispecific antibody, the improvement comprising a complex of an Alfa peptide and an anti-Alfa peptide antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

30. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof configured to form a complex with the GCN4 peptide.

31. In a composition comprising a multispecific antibody, the improvement comprising a complex of a GCN4 peptide and an anti-GCN4 peptide antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

32. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide antibody or antibody fragment thereof configured to form a complex with the Flag peptide.

33. In a composition comprising a multispecific antibody, the improvement comprising a complex of a Flag peptide and an anti-Flag peptide antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

34. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a thermostable green fluorescent protein (TFP); andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-TFP antibody or antibody fragment thereof configured to form a complex with the TFP.Atorney Docket No. DUA-OOIWO 35. In a composition comprising a multispecific antibody, the improvement comprising a complex of a thermostable green fluorescent protein (TFP) and an anti-TFP antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

36. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a gp41 peptide; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-gp41 peptide antibody or antibody fragment thereof configured to form a complex with the gp41 peptide.

37. In a composition comprising a multispecific antibody, the improvement comprising a complex of a gp41 peptide and an anti-gp41 peptide antibody or antibody fragment thereof, wherein the multispecific antibody is a bispecific or trispecific antibody.

38. The cognate pair of any one of claims 20-37, wherein:(a) the ribonuclease A comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7;(b) the MBP comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 9;(c) the dihydrofolate reductase comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13;(d) the BC2 peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 25;(e) the Alfa peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 27;(f) the GCN4 peptide comprises the amino acid sequence according to SEQ ID NO: 23; (g) the Flag peptide comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 29(h) the TFP comprises an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 16-18; or(i) the gp41 peptide comprises the amino acid sequence according to SEQ ID NO: 21.

39. The cognate pair of any one of claims 20-38, wherein:(a) the anti-ribonuclease A antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 8;(b) the anti-MBP antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11;Atorney Docket No. DUA-OOIWO (c) the anti -dihydrofolate reductase antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 14 or SEQ ID NO: 15;(d) the anti-BC2 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 26;(e) the anti-Alfa peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 28;(f) the anti-GCN4 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 24;(g) the anti-Flag peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 30;(h) the anti-TFP antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 19 or SEQ ID NO: 20; or(i) the anti-gp41 peptide antibody or antibody fragment thereof comprises an amino acid sequence at least 80% identical to the sequence according to SEQ ID NO: 22.

40. The cognate pair of any one of claims 20-39, wherein the anti -ribonuclease A, anti-MBP, anti-dihydrofolate reductase, anti-BC2 peptide, anti-Alfa peptide, anti-GCN4 peptide, anti-Flag peptide, anti-TFP, or anti-gp41 peptide antibody or antibody fragment thereof is an antiribonuclease A VHH, an anti-MBP VHH, an anti-dihydrofolate reductase VHH, anti-BC2 peptide VHH, anti-Alfa peptide VHH, a GCN4 peptide scFv, anti-Flag peptide VHH, anti-TFP VHH, or an anti-gp41 peptide VHH, respectively.

41. The cognate pair of any one of claims 20-40, wherein the antibody or antibody fragment of the first fusion protein is fused to the ribonuclease A, MBP, dihydrofolate reductase, BC2 peptide, Alfa peptide, GCN4 peptide, Flag peptide, TFP, or gp41 peptide, respectively.

42. The cognate pair of any one of claims 20-41, wherein the antibody or antibody fragment of the first fusion protein is operably linked to the ribonuclease A, MBP, dihydrofolate reductase, BC2 peptide, Alfa peptide, GCN4 peptide, Flag peptide, TFP, or gp41 peptide, respectively, by at least one linker.

43. The cognate pair of claim 42, wherein the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).Atorney Docket No. DUA-OOIWO 44. The cognate pair of any one of claims 20-43, wherein the antibody or antibody fragment of the second fusion protein is fused to the anti-gp41 peptide antibody or antibody fragment thereof.

45. The cognate pair of any one of claims 20-43, wherein the antibody or antibody fragment of the second fusion protein is operably linked to the anti-ribonuclease A, anti-MBP, anti-dihydrofolate reductase, anti-BC2 peptide, anti-Alfa peptide, anti-GCN4 peptide, anti-Flag peptide, anti-TFP, or anti-gp41 peptide antibody or antibody fragment thereof, respectively, by at least one linker.

46. The cognate pair of claim 45, wherein the linker comprises the sequence DKTGS (SEQ ID NO: 93), DKTHT (SEQ ID NO: 94), DKT, GGGGS (SEQ ID NO: SEQ ID NO: 97), GGSGG (SEQ ID NO: 95), GGGGA (SEQ ID NO: 98), or GGAGG (SEQ ID NO: 96).

47. The cognate pair of any one of claims 20-45, wherein the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a nanobody.

48. The cognate pair of any one of claims 20-47, wherein the antibody or antibody fragment thereof of the first fusion protein, the second fusion protein, or both the first and second fusion proteins is a Fab.

49. The cognate pair of any one of claims 20-48, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain variable domain (VH) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 31, 33, 35, 116, 118, 120, 122, 124, 126, and 128.

50. The cognate pair of any one of claims 20-49, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain variable domain (VL) comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 32, 34, 36, 117, 119, 121, 123, 125, 127, and 129.

51. The cognate pair of any one of claims 20-50, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a heavy chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 42, 44, and 46.

52. The cognate pair of any one of claims 20-51, wherein the antibody or antibody fragment thereof of the first fusion protein or the second fusion protein comprises a light chain comprising an amino acid sequence at least 80% identical to the sequence according to any one of SEQ ID NOs: 41, 43, and 45.Atorney Docket No. DUA-OOIWO 53. The cognate pair of any one of claims 20-52, wherein the ribonuclease A, MBP, dihydrofolate reductase, BC2 peptide, Alfa peptide, GCN4 peptide, Flag peptide, TFP, or gp41 peptide binds the anti-ribonuclease A, anti-MBP, anti-dihydrofolate reductase, anti-BC2 peptide, anti -Alfa peptide, anti-GCN4 peptide, anti -Flag peptide, anti -TFP, or anti-gp41 peptide antibody or antibody fragment thereof, respectively bind with a KD less than about 10 nM.

54. The cognate pair of any one of claims 20-53, wherein the ribonuclease A, MBP, dihydrofolate reductase, BC2 peptide, Alfa peptide, GCN4 peptide, Flag peptide, TFP, or gp41 peptide binds the anti-ribonuclease A, anti-MBP, anti-dihydrofolate reductase, anti-BC2 peptide, anti -Alfa peptide, anti-GCN4 peptide, anti -Flag peptide, anti -TFP, or anti-gp41 peptide antibody or antibody fragment thereof, respectively, bind with a KD less than about 1 nM.

55. A cognate pair, comprising a hen egg white lysozyme comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 2 and an anti-hen egg white lysozyme antibody or antibody fragment thereof.

56. A cognate pair, comprising a ribonuclease A comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 7 and an anti-ribonuclease A antibody or antibody fragment thereof.

57. A cognate pair, comprising a maltose binding protein (MBP) comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 9 and an anti-MBP antibody or antibody fragment thereof.

58. A cognate pair, comprising a dihydrofolate reductase comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 12 or SEQ ID NO: 13 and an anti-dihydrofolate reductase antibody or antibody fragment thereof.

59. A cognate pair, comprising a human -catenin (BC2) peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 25 and an anti-BC2 peptide antibody or antibody fragment thereof.

60. A cognate pair, comprising an Alfa peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 27 and an anti-Alfa peptide antibody or antibody fragment thereof.

61. A cognate pair, comprising a GCN4 peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 23 and an anti-GCN4 antibody or antibody fragment thereof.

62. A cognate pair, comprising a Flag peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 29 and an anti-Flag peptide antibody or antibody fragment thereof.Atorney Docket No. DUA-OOIWO 63. A cognate pair, comprising a thermostable green fluorescent protein (TFP) comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 16- 18 and an anti -TFP antibody or antibody fragment thereof.

64. A cognate pair, comprising a gp41 peptide comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 21 and an anti-gp41 peptide antibody or antibody fragment thereof.

65. A cognate pair of fusion proteins, comprising:a) a first fusion protein comprising an antibody or antibody fragment thereof operably linked to hen egg white lysozyme, ribonuclease A, maltose binding protein (MBP), dihydrofolate reductase, a human p-catenin (BC2) peptide, an Alfa peptide, a GCN4 peptide, a Flag peptide, thermostable green fluorescent protein (TFP), or a gp41 peptide; andb) a second fusion protein comprising an antibody or antibody fragment thereof operably linked to a means for binding the hen egg white lysozyme, ribonuclease A, MBP, dihydrofolate reductase, a BC2 peptide, an Alfa peptide, a GCN4 peptide, a Flag peptide, TFP, or a gp41 peptide.

66. A plurality of cognate pairs, wherein the cognate pair is a cognate pair of any one of claims 1-64.

67. A mammalian expression vector, comprisinga) a first nucleic acid encoding any one of SEQ ID NOs: 1-4, 6, 7, 9, 12, 13, 16-18, 21, 23, 25, 27, and 29;b) a second nucleic acid encoding any one of SEQ ID NOs: 5, 8, 10, 11, 14, 15, 19, 20, 22, 24, 26, 28, and 30; andc) a mammalian promoter.

68. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a hen egg white lysozyme and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-hen egg white lysozyme antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the hen egg white lysozyme joined with the anti-hen egg white lysozyme antibody or antibody fragment thereof.

69. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a ribonuclease A and a second fusion protein comprising an antibodyAtorney Docket No. DUA-OOIWO or antibody fragment thereof operably linked to an anti-ribonuclease A antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the ribonuclease A joined with the anti-ribonuclease A antibody or antibody fragment thereof.

70. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a maltose binding protein (MBP) and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-MBP antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the MBP joined with the anti-MBP antibody or antibody fragment thereof.

71. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a dihydrofolate reductase and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-dihydrofolate reductase antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the dihydrofolate reductase joined with the anti-dihydrofolate reductase antibody or antibody fragment thereof.

72. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a human p-catenin (BC2) peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-BC2 peptide antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the BC2 peptide joined with the anti-BC2 peptide antibody or antibody fragment thereof.

73. A method for identifying a cognate pair, comprising:a) Contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to an Alfa peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the Alfa peptide joined with the anti-Alfa peptide antibody or antibody fragment thereof.

74. A method for identifying a cognate pair, comprising:Atorney Docket No. DUA-OOIWO a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a BC2 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Alfa peptide with an antibody or antibody fragment thereof comprising a BC2 peptide binding domain and an Alfa peptide binding domain; andb) identifying the cognate pair comprising the first fusion protein and the second fusion protein joined by the antibody or antibody fragment thereof comprising the BC2 peptide binding domain and the Alfa peptide binding domain.

75. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the GCN4 peptide joined with the anti-GCN4 peptide antibody or antibody fragment thereof.

76. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a Flag peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the Flag peptide joined with the anti-Flag peptide antibody or antibody fragment thereof.

77. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-Flag peptide with an antibody or antibody fragment thereof comprising a GCN4 peptide binding domain and a Flag peptide binding domain; andb) identifying the cognate pair comprising the first fusion protein and the second fusion protein joined by the antibody or antibody fragment thereof comprising the GCN4 peptide binding domain and the Flag peptide binding domain.

78. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a GCN4 peptide and a second fusion protein comprising an antibodyAtorney Docket No. DUA-OOIWO or antibody fragment thereof operably linked to an anti-GCN4 peptide antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the GCN4 peptide joined with the anti-GCN4 peptide antibody or antibody fragment thereof.

79. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a thermostable green fluorescent protein (TFP) and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-TFP antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the TFP joined with the anti-TFP antibody or antibody fragment thereof.

80. A method for identifying a cognate pair, comprising:a) contacting a first fusion protein comprising an antibody or antibody fragment thereof operably linked to a gp41 peptide and a second fusion protein comprising an antibody or antibody fragment thereof operably linked to an anti-gp41 peptide antibody or antibody fragment thereof; andb) identifying the cognate pair comprising the gp41 peptide joined with the anti-gp41 peptide antibody or antibody fragment thereof.

81. A hen egg white lysozyme comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 2-4.

82. A ribonuclease A comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 7.

83. A dihydrofolate reductase comprising an amino acid sequence 100% identical to the sequence according to SEQ ID NO: 13.

84. A thermostable green fluorescent protein (TFP) comprising an amino acid sequence 100% identical to the sequence according to any one of SEQ ID NOs: 17-18.

Images