Binding agent molecules with high affinity and / or specificity and methods for making and using same

Abstract

Provided herein, in some embodiments, are binding molecules that include co-binders that have high affinity and / or high specificity for a target. In other embodiments, provided herein are compositions, methods of making, and methods of using the binding molecules taught herein, including diagnostic and therapeutic uses that include the co-binders taught herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 133,005, filed December 31, 2020, and U.S. Provisional Patent Application No. 63 / 133,020, filed December 31, 2020, each of which is incorporated herein by reference in its entirety.

[0002] In some embodiments, the present application is directed to binding agent molecules, e.g., cobinders, that have high affinity and / or high specificity for target molecules. Also provided in other embodiments are methods of making, using, e.g., diagnostic and therapeutic methods, and compositions comprising the binding agent molecules, e.g., cobinders. [Background technology]

[0003] Antibodies and other binding molecules are useful in many fields, including those related to molecular detection, diagnosis, and therapy. Producing such binding molecules with desired properties, such as size and immunogenicity, let alone desired binding affinity and specificity, remains a challenge in the art. Summary of the Invention [Means for solving the problem]

[0004] In some aspects, provided are co-binding agents comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, optionally wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"), and wherein the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain, optionally via a linker. In some embodiments, the second binding moiety is a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). In some embodiments, the co-binding agent comprises a linker. In some embodiments, the co-binding agent comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"), and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain via a linker.

[0005] In some embodiments, the co-binding agent binds to the second target site with an affinity that is at least about three times that of a control co-binding agent, hi some embodiments, the control co-binding agent comprises an antibody variable domain without an N-terminal truncation (e.g., an N-terminally truncated antibody variable domain of a second binding moiety of a co-binding agent described herein).

[0006] In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule, hi some embodiments, the co-binder binds to the target molecule with an affinity that is at least about three times that of a control co-binder comprising an antibody variable domain without an N-terminal truncation.

[0007] In some embodiments, the first binding moiety is a first antibody moiety. In some embodiments, the first antibody moiety is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the first antibody moiety is a single domain antibody.

[0008] In some embodiments, the second antibody portion is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the N-terminal truncated antibody variable domain is a truncated VH or a truncated VL domain. In some embodiments, the second antibody portion is a single domain antibody. In some embodiments, the N-terminal truncated antibody variable domain is a truncated VH or VL domain. H It is the H domain.

[0009] In some embodiments, the first binding moiety is a first V H H domain, and the second binding moiety comprises a second V H domain having an N-terminal truncation. H H domain ("truncated V H the first V H domain) H The C-terminus of the H domain is the second V H It is connected to the N-terminus of the H domain via a linker.

[0010] In some embodiments, the N-terminal truncated antibody variable domain is truncated at the N-terminus by about 1 to about 25 amino acids, In some embodiments, the N-terminal truncated antibody variable domain is truncated at the N-terminus by 1 amino acid.

[0011] In some embodiments, the linker is a peptide linker. In some embodiments, the C-terminal amino acid of the peptide linker that directly connects the N-terminal truncated antibody variable domain is G.

[0012] In some embodiments, the C-terminal three amino acids of the peptide linker directly connected to the N-terminal truncated antibody variable domain are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G. In some embodiments, the C-terminal three amino acids of the peptide linker directly connected to the N-terminal truncated antibody variable domain are selected from the group consisting of GVG, DSG, LLG, VSG, PPG, SCG, TLG, and NPG.

[0013] In some embodiments, the linker is x S y ) n where x is 1 to 5, y is 0 to 5, and n is 1 or greater. In some embodiments, the linker comprises [EAAAK] n where n is 1 or greater. In some embodiments, the linker is about 40 amino acids or less in length. In some embodiments, the linker is [EEEEKKKK] n wherein n is 1 or greater. In some embodiments, the linker comprises [AP] n where n is 1 or greater.

[0014] In some embodiments, the truncated variable domain is derived from an antibody variable domain of any of the IgG, IgA, IgE, IgM, or IgD types.

[0015] In some embodiments, the co-binding agent further comprises a third binding moiety that specifically recognizes a third target site. In some embodiments, the third binding moiety is a third antibody moiety. In some embodiments, the third antibody moiety comprises an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). In some embodiments, the third antibody moiety is connected to the second antibody moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain of the third antibody moiety.

[0016] In some embodiments, the third antibody portion is connected to a fourth binding portion through the N-terminus of the N-terminally truncated antibody variable domain of the third antibody portion via a linker.

[0017] In some embodiments, the co-binding agent is an antibody comprising an Fc region.

[0018] In some embodiments, the co-binding agent is a chimeric antigen receptor (“CAR”).

[0019] In another embodiment, provided is a co-binding agent comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain via a peptide linker, and the C-terminal three amino acids of the peptide linker that directly connect the antibody variable domain of the second binding moiety are X1-X2-X3, where X1 is any amino acid, X2 is K, R, Y, M, G, or N, and X3 is R, G, Y, or P.

[0020] In some embodiments, the cobinder binds to the second target site with at least about three times the affinity of the linker control cobinder.

[0021] In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule.

[0022] In some embodiments, the cobinder binds to the target molecule with an affinity that is at least about three times that of a linker control cobinder.

[0023] In some embodiments, the first binding moiety is a first antibody moiety. In some embodiments, the first antibody moiety is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the first antibody moiety is a single domain antibody.

[0024] In some embodiments, the second antibody portion is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the antibody variable domain is a VH or VL domain. In some embodiments, the second antibody portion is a single domain antibody. In some embodiments, the antibody variable domain is a V H It is the H domain.

[0025] In some embodiments, the first binding moiety is a first V H H domain, and the second binding moiety comprises a second V H H domain, H The C-terminus of the H domain is the second V H It is connected to the N-terminus of the H domain via the peptide linker.

[0026] In some embodiments, the three C-terminal amino acids of the peptide linker directly connecting the N-terminal truncated antibody variable domain are selected from the group consisting of GVG, DSG, LLG, VSG, PPG, SCG, TLG, and NPG.

[0027] In some embodiments, the linker is x S y ) n where x is 1 to 5, y is 0 to 5, and n is 1 or greater. In some embodiments, the linker comprises [EAAAK] n where n is 1 or greater. In some embodiments, the linker is about 40 amino acids or less in length. In some embodiments, the linker is [EEEEKKKK] nwherein n is 1 or greater. In some embodiments, the linker comprises [AP] n where n is 1 or greater.

[0028] In some embodiments, the co-binding agent further comprises a third binding moiety that specifically recognizes a third target site. In some embodiments, the third binding moiety is a third antibody moiety. In some embodiments, the third antibody moiety comprises an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). In some embodiments, the third antibody moiety is connected to the second antibody moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain of the third antibody moiety. In some embodiments, the third antibody moiety is connected to a fourth binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain of the third antibody moiety.

[0029] In some embodiments, the co-binding agent is an antibody comprising an Fc region.

[0030] In some embodiments, the co-binding agent is a chimeric antigen receptor (“CAR”).

[0031] In another aspect, provided is a library comprising a plurality of co-binding agents or a plurality of polynucleotides encoding a plurality of co-binding agents, each co-binding agent comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, the second binding moiety being a second antibody moiety comprising an antibody variable domain, the first binding moiety being connected to the second binding moiety through the N-terminus of the antibody variable domain via a peptide linker, and at least two co-binding agents comprised in the library differ from each other in terms of the peptide linker sequence.

[0032] In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule.

[0033] In some embodiments, the antibody variable domain has an N-terminal truncation (an "N-terminally truncated antibody variable domain"). In some embodiments, at least two co-binders comprised in the library differ from each other by an N-terminal truncation of the antibody variable domain.

[0034] In some embodiments, the diversity of the library is at least about 5000.

[0035] In some embodiments, substantially all of the plurality of co-binding agents comprise the same first binding moiety and second binding moiety.

[0036] In some embodiments, at least two of the plurality of co-binding agents comprise different first binding moieties and / or second binding moieties.

[0037] In another aspect, provided is a method of screening for a co-binder that specifically binds to a second target site with a desired affinity, the method comprising: (1) contacting a library described herein with a target molecule comprising the second target site to form a complex between the target molecule and a co-binder that specifically binds to the target molecule; and (2) identifying a co-binder that binds to the second target site with the desired affinity.

[0038] In another aspect, provided is a method of screening for a cobinder that specifically binds to a target molecule with a desired affinity, the method comprising: (1) contacting a library described herein with the target molecule to form a complex between the target molecule and a cobinder that specifically binds to the target molecule; and (2) identifying a cobinder that binds to the target molecule with the desired affinity.

[0039] In another embodiment, provided is a method of increasing the binding affinity of a control cobinder that specifically binds to a target molecule, wherein the control cobinder comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second binding target site, the second binding moiety being a second antibody moiety comprising an antibody variable domain, the first binding moiety being connected to the second binding moiety through the N-terminus of the antibody variable domain via a linker, the control cobinder comprising a full-length antibody variable domain, and the binding affinity of the control cobinder to the second target site is lower than that of the second antibody moiety in the free state, the method comprising obtaining a cobinder having an N-terminal truncation in the antibody variable domain of the second antibody moiety compared to the control cobinder.

[0040] In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule.

[0041] All applications, publications, patents, and other references, including GenBank citations, cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. [Brief explanation of the drawings]

[0042] [Figure 1] 1 shows an exemplary algorithm for identifying truncations or deletions of N-terminal residues in antibody variable regions. [Figure 2] 1 shows another exemplary algorithm for identifying truncations or deletions of N-terminal residues in antibody variable regions. [Figure 3A] 1 illustrates exemplary sources of loss of binding energy upon linking two binding moieties. [Figure 3B] 1 illustrates exemplary sources of loss of binding energy upon linking two binding moieties. [Figure 3C] 1 illustrates exemplary sources of loss of binding energy upon linking two binding moieties. [Figure 3D] 1 illustrates exemplary sources of loss of binding energy upon linking two binding moieties. [Figure 3E] The crystal structures of 7D12 and 9G8 VHHs bound to EGFR are shown, with the crystal structure of cetuximab overlaid for comparison. [Figure 4A] 10 illustrates a strategy for improving the binding properties of cobinders by altering the linker attachment point between the linker and the antigen. [Figure 4B] SDS-PAGE gel of purified proteins from the N-terminally truncated HuL6-7D12 variant of 7D12. All variants, except for the non-cleavable cobinder, were expressed and purified in the same manner. [Figure 4C] SDS-PAGE gel of purified proteins from the N-terminally truncated HuL6-9G8 variant of 9G8. All variants, except for the non-cleavable co-binder, were expressed and purified in the same manner. [Figure 5] 1 shows the design of a cobinder library in which three amino acids were randomized at the C-terminus of the linker with or without the first amino acid of the second binder. [Figure 6] A shows the consensus sequence for each library as described in Table 15 and the accompanying text, and the top 20 most enriched sequences were subjected to motif analysis using WebLogo software (Crooks et al., Genome Res. 2004 Jun;14(6):1188-90). B shows yeast display and SPR measurements of affinity (KD) between selected constructs with linker terminal modifications and human EGFR. [Figure 7A] Figure 1 shows the design of a cobinder library with three randomized amino acids at the N-terminus and two randomized amino acids at the C-terminus of the linker, where the last amino acid at the C-terminus of the linker is glycine. The library utilizes four different linker motifs: EAAAK and E4K4 repeats, AP repeats, and G3-4S repeats. [Figure 7B]Consensus sequences for each library are shown in Table 16 and the accompanying text, and the top 20 most enriched sequences were subjected to motif analysis using WebLogo software (Crooks et al., Genome Res. 2004 Jun;14(6):1188-90). [Figure 7C] Enrichment of linker lengths from the screen described in Table 16 and accompanying text is shown. [Figure 8] 1 shows SPR affinity measurements of engineered cobinders against mouse EGFR-Fc and human EGFR-Fc mutant (L325V, S340A). [Figure 9] FIG. 1 shows a schematic diagram of a method for discovering cobinders with synergistic cobinding. [Figure 10A] 1 shows that an anti-EGFR VHH yeast surface display library SB0 was constructed and single binder selection was performed using FACS. [Figure 10B] 1 shows the selection of high affinity cobinders from a CB0 cobinder library using FACS. [Figure 11] 1 shows downregulation of EGF-induced EGFR signaling by cobinders. [Figure 12A] Sensorgrams are shown for immobilized EGFR-Fc injected with 81 nM of 1E10 EGFR binder followed by 81 nM of 15E2 EGFR binder. [Figure 12B] Sensorgrams are shown for immobilized EGFR-Fc injected with 81 nM of 7D12-9G8 EGFR binder followed by 81 nM of 15E2 EGFR binder. [Figure 12C] Sensorgrams are shown for immobilized EGFR-Fc injected with 81 nM of 7D12-9G8 EGFR cobinder followed by 81 nM of 1E10 EGFR binder. [Figure 13] A plot of the distance between the N-terminus of the VHH and Fab domains and the antigen surface is shown, with each dot representing a unique structure selected from the PDB. [Figure 14]Affinity plots of anti-EGFR (filled circles) and anti-HIV p24 (open squares) cobinders and single binders are shown. [Figure 15] 1 shows a plot of the affinity of cobinders to 14 different targets and a conventional antibody to the targets. DETAILED DESCRIPTION OF THE INVENTION

[0043] Provided herein are binder molecules comprising a second binding moiety that specifically recognizes a target site, the second binding moiety being a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). The disclosure of the present application is based on the inventors' unexpected discovery that such binder molecules, e.g., co-binders, comprising a second binding moiety with an N-terminally truncated antibody variable domain provide a platform technology for binder molecules with high affinity and specificity. Furthermore, the second binding moiety with an N-terminally truncated antibody variable domain can be combined with various other features, including linkers, first binding moieties, labels, and / or drugs, to generate desired binder molecules. Furthermore, the binder molecule designs encompassed herein allow for production, such as via polypeptide expression, without the need for post-production synthetic steps, which often result in yield loss and contamination.

[0044] Thus, in some embodiments, provided herein are binding agent molecules that comprise a second binding moiety that specifically recognizes a target site, e.g., a target polypeptide, wherein the second binding moiety is a second antibody moiety that comprises an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain").

[0045] In other aspects, provided herein is a co-binding agent comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"), and wherein the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain via a linker.

[0046] In another aspect, provided herein is a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain via a peptide linker, and the C-terminal three amino acids of the peptide linker directly connecting the antibody variable domain of the second binding moiety are X1-X2-X3, where X1 is any amino acid, X2 is K, R, Y, M, G, or N, and X3 is R, G, Y, or P. In some embodiments, X3 of X1-X2-X3 is G.

[0047] In another aspect, provided herein is a library comprising a plurality of co-binding agents or a plurality of polynucleotides encoding a plurality of co-binding agents, each co-binding agent comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, wherein the first binding moiety is connected to the second binding moiety through the N-terminus of the antibody variable domain via a peptide linker, and wherein at least two co-binding agents comprised in the library differ from each other in terms of the peptide linker sequence.

[0048] In another aspect, provided herein is a method of screening for a cobinder that specifically binds to a second target site with a desired affinity, the method comprising: (1) contacting a library described herein with a target molecule comprising the second target site to form a complex between the target molecule and a cobinder that specifically binds to the target molecule; and (2) identifying a cobinder that binds to the second target site with the desired affinity.

[0049] In another aspect, provided herein is a method for screening for a cobinder that specifically binds to a target molecule with a desired affinity, the method comprising: (1) contacting a library described herein with the target molecule to form a complex between the target molecule and a cobinder that specifically binds to the target molecule; and (2) identifying a cobinder that binds to the target molecule with the desired affinity.

[0050] In another aspect, provided herein is a method of increasing the binding affinity of a control cobinder that specifically binds to a target molecule, wherein the control cobinder comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second binding target site, the second binding moiety being a second antibody moiety comprising an antibody variable domain, the first binding moiety being connected to the second binding moiety through the N-terminus of the antibody variable domain via a linker, the control cobinder comprising a full-length antibody variable domain, and the binding affinity of the control cobinder to the second target site is lower than that of the second antibody moiety in the free state, the method comprising obtaining a cobinder having an N-terminal truncation in the antibody variable domain of the second antibody moiety compared to the control cobinder.

[0051] I. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following explanations of terms shall apply, and whenever appropriate, terms used in the singular shall also include the plural, and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any explanation of a term set forth conflicts with any document incorporated herein by reference, the explanation of the term set forth below shall prevail.

[0052] The techniques and procedures described or referenced herein are generally well understood and / or commonly employed using conventional methods by those of skill in the art, including, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010).

[0053] The terms "co-binder," "co-binders," "cobinder," and "cobinders" are intended to refer to a molecule having at least two binding moieties (i.e., a first binding moiety comprising a first paratope and a second binding moiety comprising a second paratope) that bind to non-overlapping epitopes of a single target molecule or a single target complex (e.g., a protein complex). In some embodiments, the first and second binding moieties simultaneously bind to non-overlapping epitopes of a single target molecule or a single target complex (e.g., a protein complex). In some embodiments, the at least two binding moieties simultaneously bind to non-overlapping epitopes of a single target molecule or a single target complex (e.g., a protein complex). A co-binder described herein comprises at least two binding moieties, e.g., any of 2, 3, 4, 5, 6, or 7 or more binding moieties. In some embodiments, two or more binding moieties on a single binding molecule are the same. In some embodiments, two or more binding moieties on a single binding molecule are different. In some embodiments, a co-binding agent has two binding moieties, and the two epitopes recognized by the co-binding agent are non-overlapping and distinct. In some embodiments, a co-binding agent has two binding moieties, and the two epitopes recognized by the co-binding agent are located near each other but still spaced far enough apart to accommodate the co-binding agent's linker. In some embodiments, a co-binding agent has two binding moieties, and the first and second epitopes are spaced no more than 150 angstroms apart. In some embodiments, a co-binding agent has two binding moieties, and the first and second epitopes are spaced no more than 100 angstroms, 50 angstroms, 40 angstroms, 30 angstroms, 20 angstroms, 15 angstroms, 10 angstroms, or 5 angstroms apart. In the case of linear epitopes on a target peptide or protein, the spacing between any two epitopes can be within 200 amino acids of each other.In some embodiments, a co-binding agent has two binding moieties, and the two epitopes can be spaced within 200, 150, 100, 50, 40, 30, 20, 15, or 10 amino acids of each other. In some embodiments, a co-binding agent has two binding moieties, and the two epitopes recognized by the co-binding agent are selected so that the two binding interactions are cooperative and synergistic and do not interfere with each other. A co-binding agent has both higher binding affinity and higher binding specificity than a typical bivalent antibody, for example, due to the additive effect of the two paratope-epitope binding interactions.

[0054] As used herein, the term "binding moiety" refers to a molecule or a portion of a molecule that binds to a specific target molecule. Binding moieties can include proteins, peptides, nucleic acids, carbohydrates, lipids, or low molecular weight compounds. In some embodiments, a binding moiety includes an antibody. In some embodiments, a binding moiety includes an antigen-binding fragment of an antibody. In some embodiments, a binding moiety includes an antibody or an antigen-binding fragment thereof. In some embodiments, a binding moiety includes a heavy chain variable region of an antibody. In some embodiments, a binding moiety includes a light chain variable region of an antibody. In some embodiments, a binding moiety includes a variable region of an antibody. In some embodiments, a binding moiety includes an antibody mimetic. In some embodiments, a binding moiety includes a low molecular weight component. In some embodiments, a binding molecule has only one binding moiety. In some embodiments, a binding molecule has two binding moieties. In some embodiments, a binding molecule has three or more binding moieties. In some embodiments, two or more binding moieties on a single binding molecule are the same. In some embodiments, two or more binding moieties on a single binding molecule are different. For example, a binding molecule can have two binding moieties, both of which are antigen-binding fragments, e.g., VHHs. As another example, a binding molecule can also have two binding moieties, one of which is a VHH and the other of which is an scFv.

[0055] As used herein, the term "paratope" refers to the portion of a binding moiety that recognizes and binds to a target molecule. The paratope of an antibody is also called the "antigen-binding site." The epitope and paratope of a given target molecule / binding molecule (e.g., Ag / Ab) pair can be identified by routine methods. For example, the target molecule and binding moiety can be combined to form a complex, which can be crystallized. The crystal structure of the complex can be determined, for example, by X-ray diffraction, and can be used to identify the specific site of interaction between the target molecule and the binding molecule, i.e., the epitope / paratope.

[0056] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds, e.g., a localized region on the surface of an antigen (e.g., EGFR), that can bind to one or more antigen-binding regions of an antibody and has antigenic or immunogenic activity in an animal, e.g., a mammal (e.g., a human), capable of eliciting an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody binds and can be identified by any method known in the art, including, for example, immunoassays. An antigenic epitope is not necessarily immunogenic. Epitopes often consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids within a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but come together after the protein folds into a three-dimensional structure. Induced epitopes are formed when the three-dimensional structure of the protein is in an altered conformation, for example, after activation or binding of another protein or ligand. Generally, an antigen has several or many different epitopes and can react with many different antibodies.

[0057] The term "binding protein" refers to a protein comprising a portion (e.g., one or more binding regions, such as CDRs) that binds to a target antigen (e.g., EGFR) and, optionally, a scaffold or framework portion (e.g., one or more scaffold or framework regions) that allows the binding portion to adopt a conformation that promotes binding of the binding protein to a target polypeptide, fragment, or epitope thereof. Examples of such binding proteins include antibodies, e.g., human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single-chain antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(ab')2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies, and fragments thereof. The binding proteins can comprise alternative protein scaffolds or artificial scaffolds, e.g., with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to stabilize the three-dimensional structure of the binding protein, and fully synthetic scaffolds, e.g., comprising biocompatible polymers. See, e.g., Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics 53(1):121-29, and Roque et al., 2004, Biotechnol. Prog. 20:639-54. Additionally, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimetics that utilize fibronectin components as the scaffold. In the context of the present disclosure, a binding protein is one that is characterized by, for example, a dissociation constant (K D ) is 10 -5 In some embodiments, the binding proteins (e.g., cobinders and antibodies) are said to specifically or selectively bind to a target if their binding affinity to the target is less than or equal to K D about 10 -7 M ~ about 10 -12 In certain embodiments, the binding proteins (e.g., co-binders and antibodies) can specifically bind at K D is 10 -8 M or below or K D is 10-9 In one embodiment, the binding proteins (e.g., cobinders and antibodies) can specifically bind to a target with high affinity when the K is less than or equal to 1 M as measured by Biacore® against a purified human target. D 1×10 -9 M~10×10 -9 In another embodiment, the binding proteins (e.g., co-binders and antibodies) can specifically bind to purified human targets at a K D 0.1×10 -9 M~1×10 -9 In yet another embodiment, the binding proteins (e.g., co-binders and antibodies) can specifically bind to targets expressed on cells at K D 0.1×10 -9 M~10×10 -9 In certain embodiments, the binding proteins (e.g., co-binders and antibodies) specifically bind to targets expressed on cells at K D 0.1×10 -9 M~1×10 -9 In some embodiments, the binding proteins (e.g., co-binders and antibodies) specifically bind to targets expressed on cells at K D 1×10 -9 M~10×10 -9 In certain embodiments, the binding proteins (e.g., co-binders and antibodies) specifically bind to targets expressed on cells at K D Approximately 0.1×10 -9 M, approx. 0.5×10 -9 M, about 1 x 10 -9 M, about 5 x 10 -9 M, about 10 x 10 -9 M, or any range or interval thereof.

[0058] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense to specifically refer to individual monoclonal antibodies (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), single-chain antibodies, and antibody fragments, e.g., as described below. Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies derived from other species, such as mouse and rabbit. Thus, the term "antibody" encompasses a variety of antibody structures, including, but not limited to, polyclonal antibodies, recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, biparatopic antibodies, bispecific antibodies, multispecific antibodies, diabodies, tribodies, tetrabodies, single-chain Fv (scFv) antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides, which are capable of binding to a specific molecular antigen and are composed of a pair of two identical polypeptide chains, each pair having one heavy chain (approximately 50-70 kDa) and one light chain (approximately 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). The term "intact antibody" or "full-length antibody" refers to an antibody having a structure substantially similar to that of a native antibody. This includes, for example, antibodies comprising two light chains, each comprising a variable region and a light chain constant region (CL), and two heavy chains, each comprising a variable region and at least heavy chain constant regions CH1, CH2, and CH3. In certain embodiments, the specific molecular antigen can be bound by the antibodies provided herein.Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelid antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above. A functional fragment refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), disulfide-linked scFvs (dsscFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that contain an antigen-binding site (e.g., one or more CDRs of an antibody) that bind to an antigen. Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies can be agonist or antagonist antibodies. Provided herein are antagonist antibodies against target antigens such as EGFR.

[0059] An "antigen" is a predetermined antigen to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.

[0060] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," "antibody fragment," and similar terms refer to the portion of an antibody that contains amino acid residues that interact with an antigen and confer its specificity and affinity for the antigen (e.g., CDRs). Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab'), Fv, single-chain antibody molecules (e.g., scFv), disulfide-linked Fv (dsFv), disulfide-linked scFv (dsscFv), Fd fragments, diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), single variable domains of heavy chain antibodies (VHHs), and multispecific antibodies formed from antibody fragments.

[0061] Unless otherwise indicated herein, light chain variable region (VLAb) as used herein encompasses all light chain variable region subtypes, including, for example, kappa (κ) light chain variable regions (κVLAb) and / or lambda (λ) light chain variable regions (λVLAb). Unless otherwise indicated herein, heavy chain variable region (VHAb) as used herein encompasses all heavy chain variable region subtypes, including, for example, gamma, delta, alpha, mu, and / or epsilon heavy chain variable regions. In some embodiments, VLAb is followed by an Arabic numeral to indicate different VLAbs. In some embodiments, VHAb is followed by an Arabic numeral to indicate different VHAbs.

[0062] As used herein, the term "antibody mimetic" refers to a molecule that, like an antibody, can specifically bind to an antigen but is not structurally related to an antibody. Antibody mimetics are typically artificial peptides with a molar mass of approximately 2-20 kDa. Nucleic acids and small molecules may also be considered antibody mimetics. Antibody mimetics known in the art include affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and nanoCLAMPs.

[0063] As used herein, the term "antagonist," when used in reference to antigen function, is intended to mean a molecule capable of inhibiting, reducing, attenuating, diminishing, or completely blocking one or more biological activities or functions of an antigen. Antigen function antagonists include molecules capable of blocking, inhibiting, attenuating, or reducing antigen-mediated or antigen-dependent signal transduction in cells expressing the antigen. Antigen function antagonists also include molecules capable of blocking, inhibiting, attenuating, or reducing antigen signal transduction, including downstream signal transduction induced by ligation or engagement between an antigen and its ligand. In some examples, antigen antagonists further include molecules capable of blocking, inhibiting, attenuating, or reducing antigen binding to a natural antigen-binding molecule. In other examples, antigen antagonists further include molecules capable of blocking, inhibiting, or reducing antigen binding to its ligand. An antigen "antagonist" is "antagonistic" with respect to the function of the antigen. In some embodiments, provided herein are competitive co-binding agents. In some embodiments, provided herein are co-binding agents that are EGFR antagonists.

[0064] A "blocking" cobinder, a "neutralizing" cobinder, or an "antagonist" cobinder, when used in reference to the function of an antigen, is intended to mean a cobinder that binds to the antigen and acts as an antagonist to the activity or function of the antigen. For example, a blocking cobinder or antagonist cobinder can substantially or completely inhibit the biological activity of an antigen or the binding of the antigen to its ligand. In some embodiments, provided herein are blocking cobinders. In some embodiments, provided herein are EGFR-blocking cobinders.

[0065] The term "binding" or "binding" refers to the interaction between a molecule comprising a binding molecule (e.g., a cobinder or binding moiety) and a target molecule, for example, to form a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of all non-covalent interactions between a single binding molecule and a single epitope on a target molecule is the affinity of the binding molecule or binding moiety for that epitope. The dissociation rate (k off ) and association rate (k on ) ratio (k off / k on ) is the dissociation constant K D which correlates inversely with affinity. D The lower the value, the higher the affinity of the antibody. D The value of k depends on the complex between the binding molecule and the target molecule. on and k off The dissociation constant K of the binding molecules provided herein depends on both Dcan be determined using any method provided herein or any other method known to those skilled in the art. Affinity at one binding site does not always reflect the true strength of the interaction between a binding molecule and a target molecule. When a target molecule containing multiple epitopes comes into contact with a binding molecule containing multiple binding moieties that bind to the target molecule, interaction of the binding molecule with one site on the target molecule increases the probability of reaction at a second site. The strength of such multiple interactions between a multivalent binding molecule and a target molecule is called avidity. The avidity of a binding molecule can be a more appropriate measure of its binding ability than the affinity of each individual binding site. For example, high avidity can compensate for low affinity and allow effective antigen binding, as may be found in pentameric IgM antibodies, which, due to their multivalency, may have lower affinity than IgG but the high avidity of IgM.

[0066] As used herein, the term "specifically binds" refers to a binding molecule or binding moiety that interacts more frequently, rapidly, for a long period of time, with a particular epitope or target molecule than another substance, or that interacts with the target molecule more rapidly, with a long period of time, with a high affinity, with a strong affinity, or that dissociates less frequently, dissociates slowly, or dissociates for a short period of time, or some combination or permutation of the above. Binding molecules (e.g., cobinders, binding moieties, antibodies, or antigen-binding fragments thereof) that specifically bind to a target molecule (e.g., an antigen) can be identified, for example, by immunoassay, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a specific response is at least twice the background signal or noise, and may exceed 10 times the background signal or noise. For a discussion of antibody specificity, see, for example, Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York, at pages 332-336. A binding molecule (e.g., a cobinder, binding moiety, antibody, or antigen-binding fragment thereof) that specifically binds to a target molecule can bind to the target molecule with higher affinity than the affinity for another molecule. In some embodiments, a binding molecule (e.g., a cobinder, binding moiety, antibody, or antigen-binding fragment thereof) that specifically binds to a target molecule can bind to the target molecule with an affinity that is at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times greater than the affinity for another molecule. In some embodiments, a binding agent that specifically binds to a particular target molecule binds to another molecule with such low affinity that binding cannot be detected using the assays described herein or known in the art. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., excess unlabeled target.In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. As used herein, the terms "specific binding" or "specifically binds" or "is specific" for a particular target molecule or an epitope on a particular target molecule means, for example, at least about 10 for the target. -5 M, alternatively at least about 10 -6 M, alternatively at least about 10 -7 M, alternatively at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 M, alternatively at least about 10 -13 M, alternatively at least about 10 -14 M, alternatively at least about 10 -15 M or less K D In one embodiment, the term "specific binding" refers to binding of a binding molecule to a particular target molecule or an epitope on a particular target molecule without substantially binding to any other polypeptides or polypeptide epitopes.

[0067] As used herein, the term "bispecific antibody" refers to an antibody that is at least bispecific, i.e., capable of binding to two different antigens or target molecules. A bispecific antibody has at least two different antigen-binding sites, where a first antigen-binding site binds to a first antigen or target molecule and a second antigen-binding site binds to a second antigen or target molecule. In particular, a bispecific antibody can bind to different surface molecules on two different cells, bringing these cells into close proximity. For example, a bispecific antibody that recognizes both an antigen on a target cell (e.g., FLT3 or CD19 on leukemia cells, CSPG4 antigen on melanoma cells, or EGFR on glioblastoma cells) and an antigen-specific T cell receptor (TCR) / CD3 complex can target tumor cells for T cell-mediated lysis.

[0068] As used herein, the term "linker" refers to a molecule that connects two binding moieties via either a covalent or non-covalent bond. Thus, a peptide linker is an intervening peptide sequence that does not include any amino acid residues from either the C-terminus of the variable region (e.g., variable light chain or variable heavy chain) of a first binding moiety or the N-terminus of the variable region (e.g., variable light chain or variable heavy chain) of a second binding moiety. Because a linker "links" two binding moieties, the connection to each binding moiety can be covalent or non-covalent. Specifically, the connection between the linker and the two binding moieties can be covalent and covalent, covalent and non-covalent, or non-covalent and non-covalent. In some embodiments, the linker of the co-binder facilitates the co-binder achieving a binding interaction with its target molecule. In some embodiments, the linker does not interfere with the binding interaction of the first and second binding moieties to their respective epitopes contained in the antigen. In some embodiments, the length of the linker is minimized to reduce or minimize entropy loss during binding. In some embodiments, the rigidity of the linker is enhanced or maximized to reduce or minimize entropy loss upon binding. The linker may be a "non-cleavable" linker. The linker may be a "cleavable linker" that can be cleaved under various physiological or non-physiological conditions. Such cleavable linkers include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., Chari et al., 1992, Cancer Res. 52:127-31, and U.S. Patent No. 5,208,020), thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance (see, e.g., Kovtun et al., 2010, Cancer Res. 70:2528-37). The linkers can be made with different compositions or chemistries.In some embodiments, the linker is a polypeptide linker, a nucleic acid linker, and / or a chemical linker. In some embodiments, the linker is non-antigenic and does not induce an immune response. The linker can connect the variable region of a first antibody that is part of the first binding moiety to the variable region of a second antibody that is part of the second binding moiety through a covalent bond. The linker can also connect the variable region of a first antibody that is part of the first binding moiety to the variable region of a second antibody that is part of the second binding moiety through a non-covalent bond. Some examples of polypeptide linkers are described in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15;65(10):1357-1369, which is incorporated herein by reference in its entirety.

[0069] An "isolated" antibody is substantially free of cellular material or other contaminating proteins and / or components from the cell or tissue source from which it is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibodies having less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein"). In certain embodiments, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1% of the volume of the protein preparation. In certain embodiments, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, e.g., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such antibody preparations have less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminant components can also include, but are not limited to, substances that interfere with the therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody, for example, 96%, 97%, 98%, or 99%, as measured by the Lowry method (Lowry et al., 1951, J. Bio. Chem. 193:265-75); (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. However, isolated antibodies are usually prepared by at least one purification step.In certain embodiments, the antibodies provided herein are isolated.

[0070] A four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its opposite end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains. The pairing of a VH and a VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994).

[0071] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody light or heavy chain, generally located at the amino terminus of the light or heavy chain, approximately 110-140 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, that is used to determine the binding and specificity of each particular antibody for a particular antigen. The variable region of the heavy chain may be referred to as "VH." The variable region of the light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary extensively in sequence among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acids of the variable region. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches of about 15 to 30 amino acids called framework regions (FRs), separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions," each about 9 to 12 amino acids long. The heavy and light chain variable regions each contain four FRs, adopting a primarily β-sheet structure connected by three hypervariable regions, which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions of each chain are held in close proximity by the FRs and are linked to the hypervariable regions from other chains, contributing to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding of the antibody to the antigen but are involved in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary significantly in sequence among different antibodies, hi certain embodiments, the variable regions are human variable regions.

[0072] The phrases "Kabat variable region residue numbering" or "Kabat amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the "standard" Kabat numbering sequence with the homologous regions of that antibody's sequence. The Kabat numbering system is typically used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is typically used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index according to Kabat" refers to the numbering of residues in a human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHo.

[0073] An "intact" antibody is one that comprises an antigen-binding site, as well as a CL and at least a heavy chain constant region, CH1, CH2, and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In some embodiments, an intact antibody has one or more effector functions.

[0074] An "antibody fragment" includes a portion of an intact antibody, e.g., the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies and di-diabodies (e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2005, J. Biol. Chem. 280:19665-72). al., 2003, Nat. Med. 9:129-34, WO93 / 11161, and U.S. Pat. Nos. 5,837,242 and 6,492,123), single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,482,858, and 5,476,786), dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181), single variable domain antibodies (sdAbs) (see, e.g., Woolven et al., 1999, Immunogenetics 50:98-101, and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49), and multispecific antibodies formed from antibody fragments.

[0075] A "functional fragment," "binding fragment," or "antigen-binding fragment" of a therapeutic antibody exhibits at least one, if not some or all, of the biological functions attributable to an intact antibody, said function including at least binding to a target antigen.

[0076] The term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five different types (e.g., isotypes), termed alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. Different heavy chains vary in size; i.e., α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains give rise to the five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including four IgG subclasses: IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0077] The term "light chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

[0078] As used herein, the term "host" refers to an animal, for example, a mammal (e.g., a human).

[0079] As used herein, the term "host cell" refers to the particular subject cell that can be transfected with a nucleic acid molecule, and to the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur later, or due to integration of the nucleic acid molecule into the genome of the host cell.

[0080] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody typically recognizes a single epitope of an antigen. In certain embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single hybridoma or other cell, and the antibody binds only to a target epitope identified, for example, by ELISA or other antigen-binding or competitive binding assays known in the art. The term "monoclonal" is not limited to any particular method for producing the antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or may be produced using recombinant DNA methods in bacteria or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002). Exemplary methods for producing monoclonal antibodies are provided in the Examples herein.

[0081] The term "naturally occurring," when used in reference to biological material, such as a nucleic acid molecule, polypeptide, or host cell, refers to one that is found in nature and has not been manipulated, modified, and / or altered (e.g., isolated, purified, selected) by human beings.

[0082] The antibodies provided herein can include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55).

[0083] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies comprising a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from the corresponding CDR of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and capacity (e.g., donor antibody). In some cases, residues from one or more FR regions of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of a humanized antibody can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0084] A "human antibody" is one that has an amino acid sequence corresponding to that of an antibody produced by a human and / or is produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68). Similarly applicable to the preparation of human monoclonal antibodies are the methods described by Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., 1991, J. Immunol. 147(1):86-95, and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-74. Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, whose endogenous gene loci have been disabled but which have been engineered to produce such antibodies in response to antigenic challenge (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Patent Nos. 6,075,181 and 6,150,584, relating to XENOMOUSE™ technology). Similarly, see, e.g., Li et al., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62, regarding human antibodies generated by human B-cell hybridoma technology.

[0085] "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) in the non-framework region of the β-sheet framework of the VH of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of the β-sheet framework of the VL of the antibody. Thus, CDR is a variable region sequence interspersed within the sequence of the framework region. CDR regions are well known to those skilled in the art, and are defined, for example, by Kabat as the most hypervariable region within the antibody variable (V) domain (Kabat et al., 1997, J. Biol. Chem. 252:6609-16; Kabat, 1978, Adv. Prot. Chem. 32:1-75). The sequences of CDR regions have also been structurally defined by Chothia as residues that are not part of the conserved β-sheet framework and can therefore adopt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). Both terms are well recognized in the art. The sequences of CDR regions have also been defined by AbM, Contact, and IMGT. The positions of CDRs within standard antibody variable regions have been identified by comparison of multiple structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Because the number of residues within hypervariable regions varies between antibodies, additional residues relative to the standard positions are conventionally numbered a, b, c, etc., next to the residue number in the standard variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is likewise well known to those skilled in the art.

[0086] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally distinct loops. Antibodies generally contain six hypervariable regions: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). Multiple hypervariable region delineations are in use and are encompassed herein. The Kabat complementarity-determining regions (CDRs) are based on sequence diversity and are the most commonly used (see, e.g., Kabat et al., supra). Chothia instead refers to the location of the structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When numbered using the Kabat numbering convention, the terminus of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent the intermediate Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2nd ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Residues from each of these hypervariable regions or CDRs are listed below.

[0087] Recently, a universal numbering system, the ImMunoGeneTics (IMGT) Information System®, has been developed and widely adopted (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system dedicated to immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides in structures called loops, a numbering system that aligns variable domain sequences according to structural features allows for easy identification of CDR and framework residues. This information can be used to graft and replace CDR residues from one immunoglobulin into an acceptor framework, typically a human antibody. An additional numbering system (AHo) was developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT-specific numbering system, is well known to those of skill in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). [Table 1]

[0088] The hypervariable regions may include the following "extended hypervariable regions": in VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and in VH, 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). As used herein, the terms "HVR" and "CDR" are used interchangeably.

[0089] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains, which are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as interaction with Fc receptors. The term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than other portions of the immunoglobulin, i.e., the variable region containing the antigen-binding site. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0090] The term "framework" or "FR" refers to variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain, diabodies, linear, and bispecific antibodies. FR residues are those variable domain residues other than those of the hypervariable regions or CDRs.

[0091] An "affinity matured" antibody is one that has one or more alterations (e.g., variations including amino acid sequence changes, additions, and / or deletions) in one or more of its HVRs that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not possess those alteration(s). Affinity matured antibodies may have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For reviews, see Hudson and Souriau, 2003, Nature Medicine 9:129-34; Hoogenboom, 2005, Nature Biotechnol. 23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51.

[0092] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. Certain exemplary embodiments include the following. In one embodiment, "K D " or "K D The "K value" can be measured by assays known in the art, for example, binding assays. D can be measured, for example, by RIA performed using the Fab form of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). D or K D The value may also be measured by Biacore®, for example, by using a surface plasmon resonance assay using a Biacore®-2000 or Biacore®-3000, or by biolayer interferometry, for example, using an Octet® QK384 system. "On-rate" or "rate of association" or "association rate" or "k on" may also be measured by the same surface plasmon resonance or biolayer interferometry techniques as above, for example, using a Biacore®-2000 or Biacore®-3000, or Octet® QK384 system. "Off-rate" or "rate of dissociation" or "dissociation rate" or "k off " can also be measured by the same surface plasmon resonance or biolayer interferometry techniques as above, for example using a Biacore®™-2000 or Biacore®™-3000, or Octet® QK384 system.

[0093] As used herein, the term "effective amount" refers to an amount of a cobinding agent or pharmaceutical composition provided herein sufficient to produce a beneficial or desired result. An effective amount can be provided in one or more administrations, applications, or dosages. Such delivery depends on several variables, including the duration for which the individual dosage units are used, the bioavailability of the agent, the route of administration, etc.

[0094] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., a cobinding agent provided herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease and / or its associated symptoms. A therapeutically effective amount of a therapeutic agent can be the amount necessary to reduce or ameliorate the progression or exacerbation of a given disease, reduce or ameliorate the recurrence, onset, or occurrence of a given disease, and / or improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administration of a cobinding agent provided herein).

[0095] The term "variant," when used with respect to a polypeptide, refers to a polypeptide that contains one or more (e.g., about 1 to about 50, about 1 to about 45, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 18, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and / or additions compared to the native or unmodified sequence of the polypeptide. For example, a variant of a cobinder can result from one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 18, about 1 to about 15, about 1 to about 10, or about 1 to about 5) alterations to the amino acid sequence of a native or previously unmodified cobinder. Variants can be constructed by molecular cloning techniques known to those of skill in the art, such as random mutagenesis or site-directed mutagenesis. Variants can also be prepared from the corresponding nucleic acid molecule encoding the variant. In certain embodiments, the variants of the cobinder retain the functional property or activity of the cobinder (e.g., binding, agonist, antagonist, blocking, neutralizing, and / or activating activity / property). In certain embodiments, the variants are encoded by nucleic acid molecules that contain one or more single nucleotide polymorphisms (SNPs) in one or more regions or subregions of the cobinder, such as one or more CDRs.

[0096] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, such as a nucleic acid sequence encoding a cobinding agent described herein, for introducing a nucleic acid sequence into a host cell. Vectors that can be used include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selection sequences or markers that function for stable integration into a host cell chromosome. Furthermore, the vector may contain one or more selection marker genes and appropriate expression control sequences. Selection marker genes that can be included, for example, confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply important nutrients not contained in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, all of which are well known in the art. When two or more nucleic acid molecules are coexpressed (e.g., antibody heavy and light chains, or both antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acids can be operably linked to a common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods for examining the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the nucleic acid molecule will be expressed in an amount sufficient to produce the desired product (e.g., a cobinding agent described herein), and further understand that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0097] As used herein, the term "conservative substitution" refers to an amino acid substitution known to those skilled in the art and may generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide will not substantially alter biological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions can be made in accordance with those described in Table 1 and the following description. In a conservative amino acid substitution, an amino acid residue is replaced with an amino acid residue containing a side chain with a similar charge or a side chain with similar properties. Families of amino acid residues containing side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acids can also be classified based on similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).Alternatively, naturally occurring residues may be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0098] For example, any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may also be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. In certain embodiments, conservative substitutions include replacing any of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids, aspartic acid (D) with glutamic acid (E) and vice versa, glutamine (Q) with asparagine (N) and vice versa, and serine (S) with threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) may be interchangeable, as may alanine (A) and valine (V). Methionine (M) is relatively hydrophobic and may be interchangeable with leucine and isoleucine, and in some cases with valine. Lysine (K) and arginine (R) are interchangeable where the important feature of the amino acid residue is its charge and the difference in pK between these two amino acid residues is not significant. Still other changes can be considered "conservative" in certain circumstances (see, e.g., Table 1 herein, pages 13-15 of "Biochemistry," 2nd ed., Lubert Stryer ed. (Stanford University); Henikoff et al., PNAS 1992 Vol. 89, 10915-10919; Lei et al., J. Biol. Chem. 1995 May 19;270(20):11882-11886). Other substitutions are permissible and can be determined empirically or in accordance with known conservative substitutions. [Table 2]

[0099] The term "homology" or "homologous" is intended to refer to the sequence similarity between two polynucleotides or two polypeptides. Similarity can be determined by comparing positions within each sequence aligned for comparison purposes. If a given position in two polypeptide sequences is not identical, the similarity or conservation of that position can be determined by assessing the similarity of the amino acids at that position, for example, according to Table 1. The similarity of the amino acids at that position can be determined according to the similarity of the side chain charges described above or the similarity of the side chain properties described above. The degree of similarity between sequences is a function of the number of matching (identical) or homologous positions shared by the sequences. Alignment of two sequences to determine percent sequence similarity can be performed using software programs known in the art, such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Preferably, default parameters are used for the alignment, an example of which is provided below. One alignment program known in the art that can be used is BLAST, set to default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: Genetic Code=Standard, Filter=None, Strand=Both, Cutoff=60, Expectation=10, Matrix=BLOSUM62, Display=50 sequences, Sort=High-score, Database=Non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information.

[0100] The term "homolog" with respect to a given amino acid or nucleic acid sequence is intended to indicate that the corresponding sequence of the "homolog" has substantial identity or homology to the given amino acid or nucleic acid sequence.

[0101] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage (%) of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0102] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264 2268, as modified by Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed, for example, using the NBLAST nucleotide program parameters set to score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameters set, for example, to score 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0103] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0104] As used herein, the term "truncation," when used in the context of a polypeptide / protein, refers to a truncation of the amino acid sequence of a polypeptide from either end of the polypeptide sequence; algorithms for identifying this are further provided below, in the paragraphs following the paragraph beginning with the sentence, "In certain embodiments of the cobinders provided herein, the present disclosure identifies truncations or deletions in VR2, VLAb2, VHAb2, or second binding moieties, for example, by the following exemplary process." Similarly, the term "truncation," when used in the context of a nucleic acid, refers to a truncation of the nucleotide sequence of a nucleic acid from either the 5'-end or 3'-end of the nucleotide sequence. An N-terminal truncation or truncation from the N-terminus of a polypeptide / protein truncation refers to a truncation of the polypeptide / protein sequence from the N-terminal end, i.e., the N-terminus, of the polypeptide / protein. Similarly, a C-terminal truncation or truncation from the C-terminus of a polypeptide / protein refers to a truncation of the polypeptide / protein sequence from the C-terminal end, i.e., the C-terminus, of the polypeptide / protein. The truncation may be a shortening of one or more amino acids from either or both ends of the polypeptide / protein. For example, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids from either the N-terminus or the C-terminus of the polypeptide / protein. For example, the truncation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids from both the N-terminus and the C-terminus of the polypeptide / protein.Protein truncation can be the result of truncation of the nucleic acid sequence encoding the protein, substitutions or other mutations that create a premature stop codon without shortening the nucleic acid sequence, or alternative splicing of RNA where substitutions or other mutations that do not themselves cause truncation result in aberrant RNA processing. "Truncation mutant" or "truncation mutation," in the context of a polypeptide / protein, refers to a variant with a truncation of one or more amino acids, and in the context of a nucleic acid, refers to a variant with a truncation of one or more nucleotides.

[0105] As used herein, the term "deletion," when used in the context of a polypeptide / protein, refers to the removal of one or more amino acids from the sequence of the polypeptide / protein. The removed one or more amino acids may be a contiguous sequence, i.e., a continuous portion, of the polypeptide / protein, or may be dispersed throughout the sequence of the polypeptide / protein. A deletion may be an internal deletion, in which the removed one or more amino acids are not amino acids at the N- or C-terminus of the polypeptide / protein sequence. A deletion may also be from the N-terminus (N-terminal deletion) or the C-terminus (C-terminal deletion), in which case a sequence of one or more contiguous amino acids is removed from the N- or C-terminus of the polypeptide / protein. A deletion may also include an internal deletion, an N-terminal deletion, and / or a C-terminal deletion. As will be apparent from the present description, an N-terminal deletion is also an N-terminal truncation, and a C-terminal deletion is also a C-terminal truncation. A sequence that meets the definition of an internal deletion may also be considered an N-terminal truncation as described herein if the criteria for an N-terminal truncation are met by applying the algorithm described herein.

[0106] An "alteration" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, resulting from a mutation in the sequence involving that amino acid residue / position. For example, typical alterations include substitution of that residue with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (e.g., usually less than 5, 4, or 3) amino acids adjacent to that residue / position, and / or deletion of that residue / position.

[0107] When two antibodies recognize the same, overlapping, or adjacent epitopes in three-dimensional space, an antibody binds to the "epitope," "essentially the same epitope," or "same epitope" as a reference antibody.The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competitive assay, which can be configured in several different formats, for example, using either labeled antigen or labeled antibody.In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and the ability of unlabeled antibody to block the binding of labeled antibody is measured using radioactive, fluorescent, or enzyme labeling.

[0108] "Epitope mapping" is the process of identifying the binding site, or epitope, of an antibody on a target antigen. "Epitope binning" is the process of classifying antibodies based on the epitope they recognize. More specifically, epitope binning includes methods and systems for clustering antibodies based on their epitope recognition characteristics and identifying the epitope recognition characteristics of different antibodies using competitive assays combined with computational processes to identify antibodies with different binding specificities.

[0109] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are not toxic to cells or mammals exposed thereto at the dosages and concentrations used. Often, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a typical carrier when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.Oral compositions, including formulations, can contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990). The composition, including the pharmaceutical compound, can contain a co-binder, for example, in isolated or purified form, together with an appropriate amount of a carrier.

[0110] As used herein, the phrase "pharmaceutically acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans.

[0111] As used herein, "polyclonal antibody" refers to an antibody population generated in an immunogenic response to a protein with many epitopes, and thus includes a variety of different antibodies directed to the same or different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (see, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002)).

[0112] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acids, that has been substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the naturally occurring sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or may be substantially free of chemical precursors or other chemicals if chemically synthesized. In certain embodiments, one or more nucleic acid molecules encoding the antibodies described herein are isolated or purified. The term encompasses nucleic acid sequences that have been removed from their naturally occurring environment, including recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule can include isolated forms of the molecule.

[0113] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, generally single-stranded, synthetic polynucleotide that is generally, although not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The preceding description of polynucleotides applies equally and fully to oligonucleotides. Cells producing the cobinding agents of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Suitable host cells are disclosed below.

[0114] Unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction, and the region of the DNA strand having the same sequence as the RNA transcript and that is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand having the same sequence as the RNA transcript and that is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0115] The terms "recombinant antibody," "recombinant cobinder," or "recombinant polypeptide / protein" refer to an antibody, cobinder, polypeptide / protein that is prepared, expressed, created, or isolated by recombinant means. For example, a recombinant cobinder can be a cobinder expressed using a recombinant expression vector transfected into a host cell, or a cobinder isolated from a recombinant, combinatorial library, or a cobinder prepared, expressed, created, or isolated by any other means, including splicing immunoglobulin gene sequences to other DNA sequences. As a further example, a recombinant polypeptide / protein can be a polypeptide / protein expressed using a recombinant expression vector transfected into a host cell, or a polypeptide / protein isolated from a recombinant, combinatorial library, or a polypeptide / protein prepared, expressed, created, or isolated by any other means, including splicing immunoglobulin gene sequences to other DNA sequences. As another example, a recombinant antibody can be an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant, combinatorial antibody library, an antibody isolated from an animal (e.g., a mouse or a cow) transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor et al., 1992, Nucl. Acids Res. 20:6287-95), or an antibody prepared, expressed, created, or isolated by any other means, including splicing immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies can have variable and constant regions, including those derived from human germline immunoglobulin sequences (see, Kabat et al., supra).However, in certain embodiments, such recombinant antibodies may be subjected to in vitro mutagenesis (or, when transgenic animals of human Ig sequences are used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0116] As used herein, the term "therapeutic agent" refers to an agent that can be used to treat, manage, or ameliorate a disease and / or its associated symptoms. In certain embodiments, a therapeutic agent comprises a cobinding agent described herein.

[0117] As used herein, the term "diagnostic agent" refers to an agent that aids in the diagnosis of disease. Diagnostic agents can be used in vitro or in vivo. In some embodiments, diagnostic agents are used in in vitro assays. In some embodiments, diagnostic agents are administered to a subject. Such agents can be used to localize, pinpoint, and / or define disease-causing processes. In some embodiments, diagnostic agents aid in the diagnosis of cancer or tumorigenesis when administered to a subject or contacted with a sample from a subject. In certain embodiments, diagnostic agents comprise a co-binding agent described herein.

[0118] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, e.g., a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, the subject is a human.

[0119] "Substantially all" refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0120] The terms "detectable agent" or "detectable molecule" are used interchangeably herein and refer to a substance that can be used to confirm the existence or presence of a desired molecule, e.g., a co-binding agent described herein, in a sample or subject. A detectable agent can be a substance that can be visualized or otherwise identified and / or measured (e.g., by quantification).

[0121] The term "encoding nucleic acid" or grammatical equivalents, as used in reference to a nucleic acid molecule, refers to a nucleic acid molecule that, either naturally or when manipulated by methods well known to those of skill in the art, can be transcribed to produce mRNA that is subsequently translated into a polypeptide and / or fragment thereof. The antisense strand is the complementary strand of such a nucleic acid molecule, from which a coding sequence can be deduced.

[0122] The term "excipient" generally refers to an inert substance used as a diluent, vehicle, preservative, binder, or stabilizer, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkylsulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See also Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990), incorporated herein by reference in its entirety.

[0123] As used herein, the term "compound" encompasses small organic molecules and inorganic chemicals having a molecular weight of less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD, less than about 1 kD, or less than about 0.5 kD, including, but not limited to, all analogs, derivatives, salts, and solvates (e.g., hydrates) thereof. In some examples, the compounds may include nucleic acids, peptides, peptidomimetics, peptoids, other small organic compounds, or drugs. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened using any of the assays provided herein. Examples of methods for synthesizing compound libraries can be found in (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al., 1994).

[0124] As used herein, the term "fragment," in the context of a peptide or polypeptide, refers to a peptide or polypeptide that comprises less than the full-length amino acid sequence. Such a fragment may result, for example, from truncation at the amino terminus, truncation at the carboxy terminus, and / or internal deletion of a residue(s) from the amino acid sequence.

[0125] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0126] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., a cobinder described herein) that is external to the body into a patient, for example, by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0127] The term "composition" is intended to encompass a product containing specified ingredients (eg, antibodies provided herein), optionally in specified amounts.

[0128] As used herein, the term "and / or" when used in a phrase such as "A and / or B" is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or" when used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0129] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide sequence" includes a plurality of such sequences, and the like.

[0130] Numerical values ​​used herein are often expressed in range format throughout this specification. The use of range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure unless the context clearly dictates otherwise. Thus, the use of such ranges explicitly includes all possible subranges, all individual numerical values ​​within the range, and all numerical values ​​or numerical ranges, including integers within such ranges and fractions of values ​​or integers within such ranges, unless the context clearly dictates otherwise. This interpretation applies regardless of the breadth of the range and in all circumstances throughout this patent document.

[0131] For the sake of brevity, certain abbreviations are used herein. One example is the single-letter abbreviations that represent amino acid residues. The amino acids and their corresponding three-letter and single-letter abbreviations are as follows: [Table 3]

[0132] II. Binder molecules - their components and composition In some embodiments, provided herein are binder molecules comprising a second binding moiety that specifically recognizes a target site, e.g., a target polypeptide, where the second binding moiety is a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). As described herein, the second binding moiety of the binder molecules described herein enables a high-affinity binding platform that can include a variety of other components, providing numerous configurations useful for a variety of applications. It is understood that the term "second binding moiety" does not imply the presence of a separate first binding moiety. In other words, the binder molecule may include 1) a single binding moiety that is the second binding moiety; 2) a first moiety and a second binding moiety that is not a binding moiety, or may include a first binding moiety and a second binding moiety. Similar reasoning applies throughout other aspects of the description provided herein; for example, the description of a co-binder as including a second antibody moiety does not imply the presence of a separate first antibody moiety.

[0133] For example, in some embodiments, the binding agent molecule comprises a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"), and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain via a linker. In some embodiments, the first binding moiety is a first V H H domain, and the second binding moiety comprises a second V H domain having an N-terminal truncation. H H domain ("truncated V H the first V H domain) H The C-terminus of the H domain is the second V H It is connected to the N-terminus of the H domain via a linker.

[0134] In some embodiments, the binding agent molecule comprises a first moiety, e.g., an enzyme, drug, or toxin, which is connected to the second binding moiety via a linker through the N-terminus of the N-terminally truncated antibody variable domain.

[0135] In some embodiments, the binding agent molecule comprises a linker, and the second binding moiety is connected to the linker through the N-terminus of the N-terminal truncated antibody variable domain. In some embodiments, the binding agent molecule does not comprise a linker.

[0136] The following sections provide further description of various aspects of the binder molecule. Such modular description is not intended to limit the scope of the present disclosure, and based on the teachings provided herein, one of ordinary skill in the art will readily understand that certain modules may be at least partially integrated. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0137] In some embodiments, one or more features of a binding agent molecule, for example, one or more of FR1, CDR1, VH, or VL, are identified according to the IMGT numbering scheme or the Kabat numbering scheme.

[0138] A. Secondary Binding Moiety The binding agent molecules, e.g., co-binders, provided herein comprise a second binding moiety that is a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). The second antibody moieties provided herein can take many forms, and descriptions are included to identify the N-terminal truncation. In some embodiments, the second binding moiety further comprises another moiety, e.g., a conjugated label or drug.

[0139] 1. The antibody portion of the second binding moiety Provided herein is an antibody portion comprising an antibody variable domain with an N-terminal truncation (an "N-terminal truncated antibody variable domain"), wherein the antibody portion of the second binding moiety specifically recognizes a target site, e.g., a polypeptide epitope.

[0140] In some embodiments, the antibody portion of the second binding moiety is a variable region (in some embodiments, referred to herein as a VR, optionally with its numerical identification, e.g., VR2). In some embodiments, the antibody portion of the second binding moiety is a heavy chain variable region (in some embodiments, referred to herein as a VHAb or VH domain). In some embodiments, the heavy chain variable region is associated with a light chain variable region. In some embodiments, the heavy chain variable region associated with the light chain variable region is a single chain, e.g., an scFv. In some embodiments, the heavy chain variable region is associated with at least one constant domain, and / or the light chain variable region is associated with at least one constant domain, e.g., a Fab or scFab. In some embodiments, when the heavy chain variable region is associated with a light chain variable region, the heavy chain variable region and the light chain variable region are derived from the same antibody or antigen-binding fragment. In some embodiments, the heavy chain variable region associated with the light chain variable region forms a stable complex. In some embodiments, the heavy chain variable region and the light chain variable region associate with each other to form an antigen-binding domain.

[0141] In some embodiments, the antibody portion of the second binding moiety is a light chain variable region (in some embodiments, referred to herein as a VLAb or VL domain). In some embodiments, the light chain variable region is the light chain variable region of a human lambda (λ) light chain. In some embodiments, the light chain variable region is the light chain variable region of a human kappa (κ) light chain. In some embodiments, the light chain variable region is associated with a heavy chain variable region. In some embodiments, the light chain variable region associated with the heavy chain variable region is a single chain, e.g., an scFv. In some embodiments, the light chain variable region is linked to at least one constant domain, and / or the heavy chain variable region is linked to at least one constant domain, e.g., a Fab or scFab. In some embodiments, when the light chain variable region is associated with a heavy chain variable region, the light chain variable region and the heavy chain variable region are derived from the same antibody or antigen-binding fragment. In some embodiments, the light chain variable region associated with the heavy chain variable region forms a stable complex. In some embodiments, the light chain variable region and the heavy chain variable region associate with each other to form an antigen-binding domain.

[0142] In some embodiments, the antibody portion of the second binding moiety further comprises one or more constant domains, eg, any one or more of CH1, CH2, CH3, or CL.

[0143] In some embodiments, the antibody portion of the second binding moiety is V H In some embodiments, the antibody portion of the second binding moiety is selected from the group consisting of a Fab, an Fv, an scFv, a dsFv, an Fab', and an (Fab')2 fragment. In some embodiments, the antibody portion of the second binding moiety is a single domain antibody.

[0144] In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated variable region. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated heavy chain variable region. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated heavy chain variable region linked to a light chain variable region. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated light chain variable region. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated light chain variable region linked to a heavy chain variable region. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated V H In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety is a truncated single-domain antibody.

[0145] The second binding moieties provided herein, or at least a portion thereof, may be obtained or derived from a variety of sources. For example, in some embodiments, the second binding moiety, or at least a portion thereof, is obtained or derived from a camelid, such as a camelid single-chain V H It's H.

[0146] In some embodiments, the second binding moiety, or at least a portion thereof, is obtained or derived from an affibody, affilin, affimer, affitin, alphabody, anticalin, aptamer, avimer, DARPin, finomer, Kunitz domain peptide, monobody, nanobody (also called single domain antibody, sdAb), or nanoCLAMP. In some embodiments, the second binding moiety, or at least a portion thereof, is obtained or derived from an IgG, IgA, IgE, IgM, or IgD.

[0147] In some embodiments, the second binding moiety, or at least a portion thereof, is obtained or derived from a mammal, including a camelid, human, non-human primate (such as a monkey), livestock, farm animal, or zoo animal, such as a dog, horse, rabbit, cow, pig, hamster, gerbil, mouse, ferret, rat, or cat. In some embodiments, the second binding moiety, or at least a portion thereof, is obtained or derived from a synthetic source.

[0148] The antibody portion of the second binding moiety provided herein specifically recognizes a target site, which encompasses a wide variety of epitopes, including polypeptides, nucleic acids, and small molecules.

[0149] 2. Cutting and Identification In certain embodiments, the second binding moiety described herein is a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain").

[0150] In some embodiments, the truncation of the second binding moiety is a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the truncation of the N-terminus of the second binding moiety is a truncation in framework region 1 (FR1) of the second binding moiety. In some embodiments, the second binding moiety is a V comprising a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminus in framework region 1 (FR1) of the second binding moiety. H Contains H.

[0151] In some embodiments, the X3 amino acid of the polypeptide linker and the start of the complementarity determining region 1 (CDR1) of the second antibody moiety (or the amino acid at the N-terminus of the second antibody moiety), characterized by the first amino acid of the CDR1 amino acid to the N-terminus of the CDR1, are separated by no more than 25 amino acids, e.g., no more than 24 amino acids, no more than 23 amino acids, no more than 22 amino acids, no more than 21 amino acids, no more than 20 amino acids, no more than 19 amino acids, no more than 18 amino acids, no more than 17 amino acids, no more than 16 amino acids, no more than 15 amino acids, no more than 14 amino acids, no more than 13 amino acids, no more than 12 amino acids, no more than 11 amino acids, no more than 10 amino acids, no more than 9 amino acids, no more than 8 amino acids, no more than 7 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, or no more than 3 amino acids.

[0152] Truncation at or from the N-terminus of a polypeptide / protein refers to the N-terminal end of the polypeptide / protein, i.e., shortening of the polypeptide / protein sequence from the N-terminus. For antibody variable domains (e.g., the second antibody moiety) contained in a binding agent molecule, N-terminal truncations of the antibody variable domain are identified based on comparison with a full-length antibody variable domain. The FR1 region of the antibody variable domain is highly conserved, and whether a polypeptide contains an antibody variable domain with an N-terminal truncation can be easily determined by methods known in the art. For example, the corresponding positions of amino acids in a polypeptide comprising an antibody variable domain ("numbered amino acids") can be determined first by aligning the polypeptide sequence with a full-length antibody variable domain, or according to any of the established variable region residue numbering systems, such as Kabat, IMGT, EU numbering, AbM, Chothia, Contact, and AHo. Many computer algorithms have been developed and are available to those skilled in the art via the internet for inputting a sequence and obtaining a sequence numbered according to any one of the specific numbering schemes provided herein.Exemplary such tools include: Antigen Receptor Numbering And Receptor Classification (ANARCI, opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / , described in Dunbar et al., Bioinformatics. 2016 Jan 15;32(2):298-300, which is incorporated herein by reference in its entirety), abYsis online or the standalone tool developed by Prof. Andrew CMartin (bioinf.org.uk / abs / , abysis.org / ), AHo's Amazing Atlas of Antibody Anatomy (AAAAA, bioc.uzh.ch / antibody, described in A. Honegger & A. Pluckthun. J. Mol. Biol, 309(2001)657-670, which is incorporated herein by reference in its entirety). Second, each numbered amino acid of the cobinder (including the antibody variable domain sequence and, optionally, a portion of the linker sequence) is compared with naturally occurring amino acids at a certain frequency in the corresponding numbered position under the same numbering scheme. If the amino acid at position 1 of the cobinder occurs at a frequency of about 3% or less for naturally occurring antibody variable domains, the antibody variable domain in the cobinder is considered to have a first N-terminal amino acid truncation, and the amino acid at position 1 of the numbered amino acid sequence is considered to be part of the linker sequence. Similarly, if the amino acids at positions 1 and 2 of the cobinder occur at a frequency of about 3% or less for naturally occurring antibody variable domains, the antibody variable domain in the cobinder is considered to have a first and second N-terminal amino acid truncation (i.e., the N-terminal truncation of the N-terminal truncated antibody variable domain is two amino acids), and the amino acids at positions 1 and 2 of the numbered amino acid sequence are considered to be part of the linker sequence.If the amino acids at numbered amino acid positions 1, 2, and 3 of the cobinder occur at a frequency of about 3% or less for naturally occurring antibody variable domains, the antibody variable domain in the cobinder is considered to have a first, second, and third N-terminal amino acid truncation (i.e., the N-terminal truncation of the N-terminal truncated antibody variable domain is three amino acids), and the amino acids at numbered amino acid positions 1, 2, and 3 are considered to be part of the linker sequence. This comparison is repeated for the N-terminal N positions of the amino acids. If the amino acids at numbered amino acid positions 1, 2, 3, ..., and N of the cobinder occur at a frequency of about 3% or less for naturally occurring antibody variable domains, the antibody variable domain in the cobinder is considered to have a first, second, third, and N N-terminal amino acid truncation (i.e., the N-terminal truncation of the N-terminal truncated antibody variable domain is N amino acids), and the amino acids at numbered amino acid positions 1 through N are considered to be part of the linker sequence. In some embodiments, the N-terminal truncation is identified using the ANARCI program (see Dunbar et al., Nucleic Acids Res, 44, 2016). In some embodiments, the N-terminal truncation is identified using the abYsis program (e.g., version 3.4.1; see also Swindells et al., J Mol Biol, 429, 2017). In some embodiments, the N-terminal truncation is identified using the AAAAA program (see Honegger & Pluckthun, J Mol Biol, 309, 2001). Alternatively, or in addition, N-terminal truncations of an antibody variable domain (e.g., second antibody moiety) included in a binding agent molecule can be identified (or confirmed) by modeling the tertiary structure of the second binding moiety and, optionally, adjacent residues. A wild-type antibody moiety (e.g., V. H The shortened beta-sheet structure compared to the corresponding full-length FR1 region of (H) indicates the presence of an N-terminal truncation. Various computer programs for modeling the tertiary structure of antibodies, such as Alphafold (see Jumper et al., Nature, 596, 2021), are well known in the art.

[0153] Because the second binding moiety is usually preceded by another amino acid sequence (e.g., a linker sequence), the presence of an N-terminal truncation in the second binding moiety may not be easily detected by simply visually inspecting the amino acid sequence alignment. Under such circumstances, the truncation in the second binding moiety can be identified, for example, by the following exemplary process: First, the amino acid sequence of the binding agent molecule (or a portion thereof, including the second binding moiety and adjacent amino acid residues) is aligned with the amino acid sequence of an immunoglobulin protein (e.g., an isotype of the immunoglobulin (Ig) family to which the second binding moiety belongs). Second, each amino acid in the sequence of the second binding moiety is then numbered according to the position number of the amino acid in the Ig isotype to which the second binding moiety is aligned (Figure 1). Next, each numbered amino acid is compared with amino acids naturally occurring at that numbered position from the Ig family, or amino acids naturally occurring above a certain frequency. In some embodiments, such a comparison can be made using amino acids that naturally occur at a frequency of more than 1%, e.g., greater than any of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, at the same numbered position from the Ig family. This comparison is repeated for the N-terminal N positions of the amino acids in the second binding portion of the binding agent molecule (Figure 1). In some embodiments, N is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0154] Based on the comparison completed to identify a truncation, if the amino acid at a numbered position is different from the natural amino acid of the Ig family at the corresponding position, that numbered position is defined as a mismatch in the second binding portion of the binder molecule, and the mismatched amino acid is defined as the amino acid that is missing or missing in the second binding portion of the binder molecule (because the naturally occurring amino acid is missing at that position). The number of mismatches or deletions within the first N amino acids is calculated as M = the number of positions within the first N amino acids that do not match with naturally occurring residues. The mismatch percentage ("mismatch %") is calculated as (M / N) x 100%. This is a percentage calculated from the ratio of the number N to the number of positions within the first N amino acids that do not match with naturally occurring amino acids. If the mismatch % of the N-terminal N amino acids exceeds a certain threshold, the N-terminal N amino acids are truncated according to the disclosure provided herein. In some embodiments, a certain threshold of the mismatch percentage is at least about 50%, for example, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, when the mismatch percentage of the N-terminal N amino acids is at least 50%, for example, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, the present disclosure defines the N-terminal N amino acids as being truncated. In some embodiments, when the mismatch percentage of the N-terminal N amino acids is 100%, the present disclosure defines the N-terminal N amino acids as being truncated. In some embodiments, when the mismatch percentage of the N-terminal N amino acids is 50% or more, the N-terminal N amino acids are truncated. Without being bound by theory, the present inventors believe that a mismatch of 50% or more of the N-terminal N amino acids correlates with disruption of the beta-sheet structure in the N-terminal N amino acids of the second binding portion. The presence of an N-terminal truncation can therefore be further confirmed by a shortened beta-sheet compared to the corresponding full-length FR1 region of the wild-type antibody portion through structural analysis.

[0155] The flowchart in Figure 1 illustrates the iterative process of identifying the total number of amino acids missing, deleted, and / or truncated from the second binding portion of the binding agent molecule. In some embodiments, to identify amino acid truncations, an alignment is performed between the sequence of the binding agent molecule or a portion thereof, e.g., the sequence of the second binding portion, and one or more of the sequences of the framework 1 region (FR1, framework region 1) of isotype Ig listed in Tables 3, 4, and 5 (which can be found in the section entitled "Particular Table"). In some embodiments, to identify amino acid truncations, an alignment is performed between the sequence of the binding agent molecule or a portion thereof, e.g., the sequence of the second binding portion, and one or more of the sequences of isotype Ig. The sequences of isotype Ig are disclosed in the databases according to the database identifiers listed in the left column of Tables 3, 4, and 5, which sequences are incorporated herein by reference. In some embodiments, to identify amino acid truncations, an alignment is performed based on the isotype of the binding agent molecule or portion thereof, e.g., the sequence of the second binding moiety, with one or more of the sequences of the framework 1 region (FR1, framework region 1) of isotype Ig listed in Tables 3, 4, and 5.

[0156] The N-terminal truncation of the second binding moiety can also be identified, for example, by the following further exemplary process: First, the sequence of the second binding moiety is numbered according to any one of known antibody numbering schemes, including, for example, Kabat, Chothia, AbM, Contact, IMGT, or AHo numbering, known to those skilled in the art and provided herein (FIG. 2). Many computer algorithms have been developed and are available to those skilled in the art via the Internet for inputting a sequence and obtaining a sequence numbered according to any one of the specific numbering schemes provided herein. Exemplary such tools include: Antigen Receptor Numbering And Receptor Classification (ANARCI, opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / , described in Dunbar et al., Bioinformatics. 2016 Jan 15;32(2):298-300, which is incorporated herein by reference in its entirety), abYsis online or the standalone tool developed by Prof. Andrew CMartin (bioinf.org.uk / abs / , abysis.org / ), AHo's Amazing Atlas of Antibody Anatomy (AAAAA, bioc.uzh.ch / antibody, described in A. Honegger & A. Pluckthun. J. Mol. Biol, 309(2001)657-670, which is incorporated herein by reference in its entirety). Second, each numbered amino acid in the sequence of the second binding moiety is compared to naturally occurring amino acids or amino acids that occur naturally above a certain frequency at that numbered position (under the same numbering scheme) from the same Ig family to which the second binding moiety belongs.In some embodiments, such a comparison can be made using amino acids that naturally occur at greater than any of the following frequencies at the same numbered position from the same Ig family to which the second binding moiety belongs: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. This comparison is repeated for the N-terminal N-position of the amino acid in the second binding moiety (Figure 1). In some embodiments, N is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0157] Based on the comparison described herein, if the second binding moiety at a numbered position differs from a natural amino acid in the Ig family at the corresponding position, the numbered position is defined as a mismatch in the second binding moiety, and the mismatched amino acid is defined as the amino acid that is missing or missing in the second binding moiety (because the naturally occurring amino acid is missing at that position). The number of mismatches or deletions within the first N amino acids is calculated as M = the number of positions within the first N aa that do not match naturally occurring residues. The mismatch percentage ("mismatch %") is calculated as (M / N) x 100%, which is a percentage calculated from the ratio of the number N to the number of positions within the first N amino acids that do not match naturally occurring amino acids. If the mismatch % of the N-terminal N amino acids exceeds a certain threshold, the present disclosure defines the N-terminal N amino acids as truncated. In one embodiment, the present disclosure defines the N-terminal N amino acids as truncated if the mismatch percentage of the N-terminal N amino acids is at least 20%, for example, at least any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0158] Thus, the total number of missing, deleted, and / or truncated amino acids may be determined as described herein. The flowchart in Figure 2 shows the iterative process of determining the total number of missing, deleted, and / or truncated amino acids from a second binding moiety in order to classify the second binding moiety as having an "N-terminally truncated antibody variable domain."

[0159] In some embodiments, the naturally occurring frequencies of amino acids in the second binding moiety, etc. are identified based on any one or more of the sequences shown in Table 3, Table 4, and Table 5. In some embodiments, the naturally occurring frequencies of amino acids in the second binding moiety, etc. are identified based on Table 7, Table 9, and / or Table 11.

[0160] In some embodiments, amino acids naturally occurring in variable heavy chains at greater than 1% frequency at each position according to an antibody numbering scheme, for example according to the IMGT numbering scheme, are set forth in Table 6. [Table 4]

[0161] In some embodiments, the naturally occurring amino acids in the variable heavy chain at each position according to an antibody numbering scheme, for example, according to the IMGT numbering scheme, and their frequency of occurrence are set forth in Table 7. [Table 5]

[0162] In some embodiments, amino acids naturally occurring in variable kappa light chains at greater than 2% frequency at each position according to an antibody numbering scheme, e.g., according to the IMGT numbering scheme, are set forth in Table 8. [Table 6]

[0163] In some embodiments, the naturally occurring amino acids in variable kappa light chains at each position according to an antibody numbering scheme, for example, according to the IMGT numbering scheme, and their frequency of occurrence are set forth in Table 9. [Table 7]

[0164] In some embodiments, amino acids naturally occurring in variable lambda light chains at greater than 2% frequency at each position according to an antibody numbering scheme, for example according to the IMGT numbering scheme, are set forth in Table 10. [Table 8]

[0165] In some embodiments, the naturally occurring amino acids in variable lambda light chains at each position according to an antibody numbering scheme, for example according to the IMGT numbering scheme, and their frequency of occurrence are set forth in Table 11. [Table 9]

[0166] In some embodiments, the second binding moiety may be considered to contain an "internal" deletion and / or insertion. Such internal deletion and / or insertion may also be considered to be an N-terminal truncation based on the N-terminal truncation identification process described herein. In this situation, the N-terminal sequence of the "internal" deletion and / or truncation is considered to be part of the linker sequence instead of being part of the second binding moiety. The presence of an N-terminal truncation in the second binding moiety may be further confirmed by modeling the tertiary structure of the binding agent molecule.

[0167] In some embodiments, the first amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not E or Q. In some embodiments, the first amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not E, Q, or R. In some embodiments, the second amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not I, L, M, or V. In some embodiments, the third amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not Q or T. In some embodiments, the third amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not Q, T, H, or R. In some embodiments, the fourth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not L or V. In some embodiments, the fourth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not L, V, or R. In some embodiments, the fifth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not K, L, Q, R, or V. In some embodiments, the sixth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not E or Q. In some embodiments, the sixth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not E, K, Q, or D. In some embodiments, the seventh amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not P, S, or W. In some embodiments, the seventh amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not P, S, W, L, or T. In some embodiments, the eighth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not G. In some embodiments, the eighth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not G, A, or V. In some embodiments, the ninth amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not A, E, G, P, or S. In some embodiments, the eleventh amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not A, E, G, T, or V.In some embodiments, the 12th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not L or V. In some embodiments, the 13th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not I, K, L, R, or V. In some embodiments, the 14th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not K, Q, or R. In some embodiments, the 14th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not K, Q, R, or N. In some embodiments, the 15th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not A or P. In some embodiments, the 15th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not A, P, D, L, or T. In some embodiments, the 16th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not G, P, S, or T. In some embodiments, the 17th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not A, D, E, G, Q, R, or S. In some embodiments, the 17th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not A, D, E, G, Q, R, S, P, T, or V. In some embodiments, the 18th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not S or T. In some embodiments, the 18th amino acid at the N-terminus of the truncated second binding moiety, e.g., VHAb2, is not S, T, A, L, or M.

[0168] In some embodiments, the N-terminal truncated antibody variable domain of the second binding moiety further comprises 1 to 18 amino acid substitutions, for example in the framework 1 (FR1) region.

[0169] In some embodiments, a binder molecule comprising the second binding moiety comprises N-terminal amino acid A1, where A1 is any amino acid other than E or Q. In some embodiments, a binder molecule comprising the second binding moiety comprises N-terminal amino acids A1-A2, where A1 is any amino acid other than E or Q, and A2 is any amino acid other than I, L, M, or V. In some embodiments, a binder molecule comprising the second binding moiety comprises N-terminal amino acids A1-A2-A3, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, and A3 is any amino acid other than Q or T. In some embodiments, a binder molecule comprising the second binding moiety comprises N-terminal amino acids A1-A2-A3-A4, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, and A4 is any amino acid other than L or V. In some embodiments, a binder molecule comprising the second binding moiety comprises N-terminal amino acids A1-A2-A3-A4-A5, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, A4 is any amino acid other than L or V, and A5 is any amino acid other than K, L, Q, R, or V.

[0170] 3. Other features related to the second binding moiety In some embodiments, the second binding moiety is associated with another feature useful in the description provided herein. In some embodiments, the second binding moiety is associated with a drug, e.g., the second binding moiety is covalently bound to a drug. In some embodiments, the second binding moiety is associated with a label, e.g., the second binding moiety is covalently bound to an affinity label (e.g., biotin) or a visual label (e.g., a fluorescent label). In some embodiments, the second binding moiety is associated with an enzyme, e.g., the second binding moiety is covalently bound to an enzyme. In some embodiments, the second binding moiety is associated with a toxin, e.g., the second binding moiety is covalently bound to a toxin. In some embodiments, the second binding moiety is associated with a nucleic acid, e.g., the second binding moiety is covalently bound to a nucleic acid. In some embodiments, the second binding moiety is associated with albumin, e.g., human serum albumin.

[0171] B. Co-binder In certain aspects, provided herein are co-binding agents comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, optionally wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"), and wherein the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain, optionally via a linker. In some embodiments, the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"). In some embodiments, the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain via a linker, e.g., a peptide linker. In some embodiments, the co-binding agent comprises a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"), and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain via a linker. In some embodiments, the co-binding agent is a single amino acid chain.

[0172] In some embodiments, the co-binding agent specifically recognizes two target sites (epitopes) on a single target antigen, e.g., a polypeptide. As described herein, the co-binding agent is configured to increase affinity and specificity for the target antigen by specifically recognizing two target sites (epitopes). In some embodiments, the co-binding agent is a multispecific co-binding agent, e.g., a bispecific co-binding agent. In some embodiments, the bispecific co-binding agent recognizes two target antigens in spatial proximity, e.g., in a complex. In some embodiments, the bispecific co-binding agent recognizes two of the same target antigens, e.g., comprised in a homodimer.

[0173] In some embodiments, provided are co-binders comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V H H domain ("N-terminally truncated V H H domain), and the first binding portion comprises a first V H In some embodiments, the N-terminal truncated antibody variable domain comprises an H domain, and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain via a linker. H The H domain is the V H In some embodiments, the N-terminal truncated V domain comprises a truncation in the FR1 region of the H domain. H In some embodiments, the N-terminal truncated V domain comprises a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. H The H domain comprises an N-terminal amino acid A1, where A1 is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2, where A1 is any amino acid other than E or Q, and A2 is any amino acid other than I, L, M, or V. In some embodiments, the N-terminal truncated V H The H domain comprises N-terminal amino acids A1-A2-A3, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, and A3 is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, and A4 is any amino acid other than L or V. In some embodiments, the N-terminally truncated V HThe H domain comprises N-terminal amino acids A1-A2-A3-A4-A5, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, A4 is any amino acid other than L or V, and A5 is any amino acid other than K, L, Q, R, or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises, from the N-terminus to the C-terminus, a contiguous series of three amino acids X1-X2-X3 that form the C-terminus of the polypeptide linker, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0174] In some embodiments, provided is a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety specifically recognizes the V H A second V containing an N-terminal truncation in the FR1 region of the H domain H H domain ("N-terminally truncated V H H domain), and the first binding portion comprises a first V H In some embodiments, the N-terminal truncated antibody variable domain comprises an H domain, and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain via a linker. H In some embodiments, the N-terminal truncated V domain comprises a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. H The H domain comprises an N-terminal amino acid A1, where A1 is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2, where A1 is any amino acid other than E or Q, and A2 is any amino acid other than I, L, M, or V. In some embodiments, the N-terminal truncated VH The H domain comprises N-terminal amino acids A1-A2-A3, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, and A3 is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, and A4 is any amino acid other than L or V. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4-A5, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, A4 is any amino acid other than L or V, and A5 is any amino acid other than K, L, Q, R, or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises, from the N-terminus to the C-terminus, a contiguous series of three amino acids X1-X2-X3 that form the C-terminus of the polypeptide linker, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0175] In some embodiments, provided is a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety specifically recognizes the V H a second V containing a truncation of any of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the FR1 region of the H domain; H H domain ("N-terminally truncated V H H domain), and the first binding portion comprises a first V HIn some embodiments, the N-terminal truncated antibody variable domain comprises an H domain, and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain via a linker. H The H domain comprises an N-terminal amino acid A1, where A1 is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2, where A1 is any amino acid other than E or Q, and A2 is any amino acid other than I, L, M, or V. In some embodiments, the N-terminal truncated V H The H domain comprises N-terminal amino acids A1-A2-A3, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, and A3 is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, and A4 is any amino acid other than L or V. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4-A5, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, A4 is any amino acid other than L or V, and A5 is any amino acid other than K, L, Q, R, or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises, from the N-terminus to the C-terminus, a contiguous series of three amino acids X1-X2-X3 that form the C-terminus of the polypeptide linker, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0176] In some embodiments, provided is a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety specifically recognizes the V H a second V containing a truncation of any of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the FR1 region of the H domain; H H domain ("N-terminally truncated V H H domain), and the first binding portion comprises a first V H H domain, and the N-terminal truncated V H The H domain comprises an N-terminal amino acid A1, where A1 is any amino acid other than E or Q, and the first binding moiety is connected to the second binding moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain. In some embodiments, the N-terminal truncated V H The H domain comprises N-terminal amino acids A1-A2, where A1 is any amino acid other than E or Q, and A2 is any amino acid other than I, L, M, or V. In some embodiments, the N-terminal truncated V H The H domain comprises N-terminal amino acids A1-A2-A3, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, and A3 is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, and A4 is any amino acid other than L or V. In some embodiments, the N-terminally truncated V HThe H domain comprises N-terminal amino acids A1-A2-A3-A4-A5, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, A4 is any amino acid other than L or V, and A5 is any amino acid other than K, L, Q, R, or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises, from the N-terminus to the C-terminus, a contiguous series of three amino acids X1-X2-X3 that form the C-terminus of the polypeptide linker, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0177] In some embodiments, provided is a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety specifically recognizes the V H a second V containing a truncation of any of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the FR1 region of the H domain; H H domain ("N-terminally truncated V H H domain), and the first binding portion comprises a first V H H domain, and the N-terminal truncated V H The H domain comprises an N-terminal amino acid A1, where A1 is any amino acid other than E or Q, and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminal truncated antibody variable domain via a linker, which linker comprises, from N- to C-terminal, a contiguous series of three amino acids X1-X2-X3 that form the C-terminus of the polypeptide linker, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G. In some embodiments, the N-terminally truncated V HThe H domain comprises N-terminal amino acids A1-A2, where A1 is any amino acid other than E or Q, and A2 is any amino acid other than I, L, M, or V. In some embodiments, the N-terminal truncated V H The H domain comprises N-terminal amino acids A1-A2-A3, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, and A3 is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, and A4 is any amino acid other than L or V. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4-A5, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, A4 is any amino acid other than L or V, and A5 is any amino acid other than K, L, Q, R, or V. In some embodiments, the linker is a polypeptide linker.

[0178] In some embodiments, provided are co-binding agents comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"), and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain, optionally via a linker. In some embodiments, the co-binding agent binds to the second target site with an affinity that is at least about three times greater than that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the co-binding agent binds to the target molecule with an affinity that is at least about three times greater than that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first antibody portion is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the N-terminal truncated antibody variable domain is a truncated VH or a truncated VL domain. In some embodiments, the second antibody portion is a single-domain antibody. In some embodiments, the N-terminal truncated antibody variable domain has an N-terminal truncation of about 1 to about 25 amino acids. In some embodiments, the N-terminal truncated antibody variable domain has an N-terminal truncation of 1 amino acid.

[0179] In some embodiments, provided are co-binding agents comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"); the first binding moiety is a first antibody moiety; and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain, optionally via a linker. In some embodiments, the co-binding agent binds to the second target site with an affinity that is at least about three times greater than that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the co-binding agent binds to the target molecule with an affinity that is at least about three times greater than that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first antibody portion is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the N-terminal truncated antibody variable domain is a truncated VH or a truncated VL domain. In some embodiments, the second antibody portion is a single-domain antibody. In some embodiments, the N-terminal truncated antibody variable domain has an N-terminal truncation of about 1 to about 25 amino acids. In some embodiments, the N-terminal truncated antibody variable domain has an N-terminal truncation of 1 amino acid.

[0180] In some embodiments, provided are co-binders comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V having an N-terminal truncation. H H domain ("N-terminally truncated V H H domain), and the first binding portion comprises a first V H H domain, H The C-terminus of the H domain is the second V HThe co-binding agent is connected to the N-terminus of the H domain via a linker. In some embodiments, the co-binding agent binds to the second target site with an affinity that is at least about three times greater than that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the co-binding agent binds to the target molecule with an affinity that is at least about three times greater than that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first antibody portion is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the N-terminal truncation of the N-terminal truncated antibody variable domain is from about 1 to about 25 amino acids.

[0181] In some embodiments, provided are co-binders comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V having an N-terminal truncation. H H domain ("N-terminally truncated V H H domain), and H The N-terminal truncation of H is one amino acid, and the first binding moiety is a first V H H domain, H The C-terminus of the H domain is the second V HThe co-binding agent is connected to the N-terminus of the H domain via a linker. In some embodiments, the co-binding agent binds to the second target site with an affinity at least about three times that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the co-binding agent binds to the target molecule with an affinity at least about three times that of a control co-binding agent comprising an antibody variable domain without the N-terminal truncation. In some embodiments, the first antibody portion is selected from the group consisting of Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragments. In some embodiments, the C-terminal amino acid of the peptide linker directly connecting the N-terminal truncated antibody variable domain is G. In some embodiments, the C-terminal three amino acids of the peptide linker directly connected to the N-terminal truncated antibody variable domain are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0182] In some embodiments, provided is a cobinder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, and the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain via a peptide linker, and the C-terminal three amino acids of the peptide linker directly connecting the antibody variable domain of the second binding moiety are X1-X2-X3, where X1 is any amino acid, X2 is K, R, Y, M, G, or N, and X3 is R, G, Y, or P. In some embodiments, the cobinder binds to the second target site with an affinity that is at least about three times greater than that of a linker-control cobinder. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the cobinder binds to the target molecule with an affinity that is at least about three times that of a linker control cobinder.

[0183] In some embodiments, provided is a cobinder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety is a second antibody moiety comprising an antibody variable domain, the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain via a peptide linker, and the C-terminal three amino acids of the peptide linker directly connecting the antibody variable domain of the second binding moiety are X1-X2-X3, where X1 is any amino acid, X2 is K, R, Y, M, G, or N, and X3 is R, G, Y, or P, and the first binding moiety is a first antibody moiety. In some embodiments, the cobinder binds to the second target site with an affinity that is at least about three times greater than that of a linker-control cobinder. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the cobinder binds to the target molecule with an affinity at least about three times that of a linker control cobinder. In some embodiments, the antibody variable domain is a VH or VL domain. In some embodiments, the second antibody moiety is a single domain antibody.

[0184] In some embodiments, provided is a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the first binding moiety is a first V H H domain, and the second binding moiety comprises a second V H H domain, H The C-terminus of the H domain is the second V HThe C-terminal three amino acids of the peptide linker connected via the peptide linker to the N-terminus of the H domain and directly connected to the antibody variable domain of the second binding moiety are X1-X2-X3, where X1 is any amino acid, X2 is K, R, Y, M, G, or N, and X3 is R, G, Y, or P. In some embodiments, the cobinder binds to the second target site with an affinity at least about three times that of the linker-control cobinder. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the cobinder binds to the target molecule with an affinity at least about three times that of the linker-control cobinder.

[0185] In some embodiments, provided are co-binders comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V that does not include an N-terminal truncation. H H domain, and the first binding moiety comprises a first V H and an H domain, wherein the first binding moiety is connected to the second binding moiety via a linker through the N-terminus of the N-terminal truncated antibody variable domain, and the three N-terminal amino acids of the second binding moiety are selected from the group consisting of HKR, FKR, MKR, CKR, QKR, VKR, RKR, LKR, KKR, WKR, SKR, KRG, EKR, YKR, IKR, TKR, NKR, FRR, YRR, AKR, ZLE, ZHQ, MZL, AMV, EHY, TYP, WAP, YMY, IYK, YTY, YYP, QNY, DKR, and SGY.

[0186] In some embodiments, the N-terminally truncated V H The H domain is the V H In some embodiments, the N-terminal truncated V domain comprises a truncation in the FR1 region of the H domain. H In some embodiments, the N-terminal truncated V domain comprises a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. HThe H domain comprises an N-terminal amino acid A1, where A1 is any amino acid other than E or Q. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2, where A1 is any amino acid other than E or Q, and A2 is any amino acid other than I, L, M, or V. In some embodiments, the N-terminal truncated V H The H domain comprises N-terminal amino acids A1-A2-A3, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, and A3 is any amino acid other than Q or T. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, and A4 is any amino acid other than L or V. In some embodiments, the N-terminally truncated V H The H domain comprises N-terminal amino acids A1-A2-A3-A4-A5, where A1 is any amino acid other than E or Q, A2 is any amino acid other than I, L, M, or V, A3 is any amino acid other than Q or T, A4 is any amino acid other than L or V, and A5 is any amino acid other than K, L, Q, R, or V. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises, from the N-terminus to the C-terminus, a contiguous series of three amino acids X1-X2-X3 that form the C-terminus of the polypeptide linker, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0187] In some embodiments, the co-binding agent binds to the second target site with an affinity that is at least about 3-fold, e.g., at least about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, or 50-fold greater than that of a control co-binding agent comprising an antibody variable domain without N-terminal truncation. In some embodiments, the first target site and the second target site are non-overlapping binding sites on a target molecule. In some embodiments, the first antibody portion and the second antibody portion specifically bind to different targets, e.g., the first antibody portion specifically binds to a first polypeptide target and the second antibody portion specifically binds to a second polypeptide target that is different from the first polypeptide target. In some embodiments, the first target site and the second target site are on different target molecules, including homo- and hetero-target complexes. In some embodiments, the co-binder binds to the target molecule with an affinity that is at least about 3-fold, e.g., at least about any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, or 50-fold greater than that of a control co-binder comprising an antibody variable domain without the N-terminal truncation.

[0188] In some aspects, provided herein are co-binding agents that specifically bind to a target (e.g., high affinity and / or high specificity co-binding agents), and complexes thereof with the target. In some embodiments, the co-binding agent has a first binding moiety, a second binding moiety, and a linker connecting the first binding moiety and the second binding moiety. In some embodiments, the complex comprises a co-binding agent and a target, e.g., a target molecule, and the co-binding agent comprises a first binding moiety, a second binding moiety, and a linker connecting the first binding moiety and the second binding moiety. In some embodiments, the first binding moiety and the second binding moiety bind to non-overlapping epitopes on the target, e.g., a polypeptide or polypeptide complex. In some embodiments, the first and second binding moieties simultaneously bind to non-overlapping epitopes on the target, e.g., a polypeptide or polypeptide complex. In some embodiments, the co-binder has an affinity for the target that is at least 50 times, e.g., at least 100 times, 200 times, 500 times, 1,000 times, 2,000 times, 5,000 times, or 10,000 times greater than that of the first binding moiety and / or the second binding moiety. In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker is a nucleic acid linker. In some embodiments, the linker is a chemical linker.

[0189] 1. First binding portion The co-binding agents provided herein comprise a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, details of which are provided in the section above.

[0190] In some embodiments, the first binding moiety is a first antibody moiety. In some embodiments, the first binding moiety is a non-cleavable antibody moiety, e.g., a non-cleavable form of a second binding moiety with an N-terminal truncation as described herein. In some embodiments, the first binding moiety is a first antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). In some embodiments, the first binding moiety is a first antibody moiety comprising an antibody variable domain with a C-terminal truncation. In some embodiments, the first binding moiety is another molecule that confers affinity to a target site. For example, in some embodiments, the first binding moiety is a ligand that recognizes a receptor or a portion thereof. In some embodiments, the first binding moiety is a receptor or a portion thereof, e.g., the extracellular domain of a receptor, that recognizes a ligand. In some embodiments, the first binding moiety is an aptamer. In some embodiments, the first binding moiety is a non-protein binding moiety, e.g., biotin or a nucleic acid. In some embodiments, the first binding moiety is a non-immunoglobulin binding agent.

[0191] In some embodiments, the antibody portion of the first binding moiety comprises a variable region (in some embodiments, referred to herein as a VR, optionally with its numerical identification, e.g., VR1 or VR2). In some embodiments, the antibody portion of the first binding moiety comprises a heavy chain variable region (in some embodiments, referred to herein as a VHAb or VH domain). In some embodiments, the heavy chain variable region is associated with a light chain variable region. In some embodiments, when the heavy chain variable region is associated with a light chain variable region, the heavy chain variable region and the light chain variable region are derived from the same antibody or antigen-binding fragment. In some embodiments, the heavy chain variable region associated with the light chain variable region forms a stable complex.

[0192] In some embodiments, the antibody portion of the first binding moiety comprises a light chain variable region (in some embodiments, referred to herein as a VLAb or VL domain). In some embodiments, the light chain variable region is a light chain variable region of a human lambda (λ) light chain. In some embodiments, the light chain variable region is a light chain variable region of a human kappa (κ) light chain. In some embodiments, the light chain variable region is associated with a heavy chain variable region. In some embodiments, when the light chain variable region is associated with a heavy chain variable region, the light chain variable region and the heavy chain variable region are derived from the same antibody or antigen-binding fragment. In some embodiments, the light chain variable region associated with the heavy chain variable region forms a stable complex.

[0193] In some embodiments, the antibody portion of the first binding moiety further comprises one or more constant domains, eg, any one or more of CH1, CH2, CH3, or CL.

[0194] In some embodiments, the antibody portion of the first binding moiety is V H In some embodiments, the antibody portion of the first binding moiety is selected from the group consisting of a Fab, an Fv, an scFv, a dsFv, an Fab', and an (Fab')2 fragment. In some embodiments, the antibody portion of the first binding moiety is a single domain antibody.

[0195] In some embodiments, the first binding moiety is a truncated first binding moiety, e.g., comprising an N-terminal and / or C-terminal truncation. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated variable region. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated heavy chain variable region. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated heavy chain variable region joined to a light chain variable region. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated light chain variable region. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated light chain variable region joined to a heavy chain variable region. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated V H In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragment. In some embodiments, the truncated antibody variable domain of the first binding moiety is a truncated single-domain antibody.

[0196] The first binding moieties provided herein, or at least a portion thereof, may be obtained or derived from a variety of sources. For example, in some embodiments, the first binding moiety, or at least a portion thereof, is obtained or derived from a camelid, such as a camelid single-chain V H It's H.

[0197] In some embodiments, the first binding moiety, or at least a portion thereof, is obtained or derived from an affibody, affilin, affimer, affitin, alphabody, anticalin, aptamer, avimer, DARPin, finomer, Kunitz domain peptide, monobody, nanobody (also called single domain antibody, sdAb), or nanoCLAMP. In some embodiments, the first binding moiety, or at least a portion thereof, is obtained or derived from an IgG, IgA, IgE, IgM, or IgD.

[0198] In some embodiments, the truncation, e.g., the N-terminal truncation of the first binding moiety, is a truncation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the N-terminal truncation of the first binding moiety is a truncation in framework region 1 (FR1) of the second binding moiety. In some embodiments, the first binding moiety is a V that includes an N-terminal truncation in framework region 1 (FR1) of the first binding moiety of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. H Contains H.

[0199] In some embodiments, the first binding moiety, or at least a portion thereof, is obtained or derived from a mammal, including a camelid, human, non-human primate (such as a monkey), livestock, farm animal, or zoo animal, such as a dog, horse, rabbit, cow, pig, hamster, gerbil, mouse, ferret, rat, cat, etc. In some embodiments, the first binding moiety, or at least a portion thereof, is obtained or derived from a synthetic source.

[0200] The antibody portion of the first binding moiety provided herein specifically recognizes a target site, which encompasses a wide variety of epitopes, including polypeptides, nucleic acids, and small molecules.

[0201] 2. Specific co-binder configuration In certain aspects, the co-binding agents described herein comprise a first antibody portion of a first binding moiety and a second antibody portion of a second binding moiety, wherein the first antibody portion and the second antibody portion independently comprise one of the following: a variable region (VR), a heavy chain variable region (VH or VHAb), or a light chain variable region (VL or VLAb). One of skill in the art will readily recognize that many combinations of pairings of first binding moieties and second antibody portions are possible, including, but not limited to, any of the following pairs of first antibodies and second antibody portions: (i) VR1 and VR2, (ii) VHAb1 and VHAb2, (iii) VHAb1 and VLAb2, (iv) VLAb1 and VHAb2, (v) VLAb1 and VLAb2, (vi) VR1 and VHAb2, (vii) VHAb1 and VR2, (viii) VR1 and VLAb2, and (vii) VLAb1 and VR2. In some embodiments, the heavy chain variable region (e.g., VHAb1 or VHAb2) is associated with a light chain variable region. In some embodiments, the light chain variable region (e.g., VLAb1 or VLAb2) is associated with a heavy chain variable region.

[0202] In one embodiment, provided herein is a co-binding agent that specifically binds to a target, the co-binding agent comprising: (i) a first binding moiety comprising a first variable region (VR1) of a first antibody, (ii) a second binding moiety comprising a second variable region (VR2) of a second antibody comprising an N-terminal truncation, and (iii) a polypeptide linker connecting the C-terminal amino acid of the VR1 and the N-terminal amino acid of the truncated VR2.

[0203] In one embodiment, provided herein is a co-binding agent that specifically binds to a target, the co-binding agent comprising: (i) a first binding moiety comprising a first variable region (VR1) of a first antibody; (ii) a second binding moiety comprising a second variable region (VR2) of a second antibody comprising a truncation of 1 to 18 amino acids at its N-terminus; and (iii) a polypeptide linker connecting the C-terminal amino acid of the VR1 and the N-terminal amino acid of the truncated VR2, wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

[0204] In one embodiment, provided herein is a co-binding agent that specifically binds to a target, the co-binding agent comprising: (i) a first binding moiety comprising a first variable region (VR1) of a first antibody; (ii) a second binding moiety comprising a second variable region (VR2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acid of the VR1 and the N-terminal amino acid of the truncated VR2, wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

[0205] In some embodiments, provided herein are co-binding agents that specifically bind to a target, the co-binding agent comprising: (i) a first binding moiety comprising a first variable region (VR1) of a first antibody; (ii) a second binding moiety comprising a second variable region (VR2) of a second antibody comprising an N-terminal truncation in the framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acid of the VR1 and the N-terminal amino acid of the truncated VR2, wherein VR1 and VR2 bind to non-overlapping epitopes on the target.

[0206] In some embodiments, provided herein is a co-binding agent comprising: (i) a first binding moiety comprising a first antibody moiety that specifically recognizes a first target site; (ii) a second binding moiety comprising a second antibody moiety that specifically recognizes a second target site, wherein the second antibody moiety comprises an antibody variable domain having an N-terminal truncation of 1 to 18 amino acids; and (iii) a polypeptide linker connecting the C-terminal amino acid of the first antibody moiety and the N-terminal amino acid of the second antibody moiety.

[0207] In some embodiments, the truncation of 1 to 18 amino acids N-terminal to the second antibody portion is in the framework 1 (FR1) region of the second antibody portion. In some embodiments, the X3 amino acid of the polypeptide linker and the start of complementarity determining region 1 (CDR1) of the second antibody portion, characterized by the first amino acid of CDR1 at the N-terminal amino acid side of the CDR1, are separated by 5 to 25 amino acids. In some embodiments, the X3 amino acid of the polypeptide linker and the start of complementarity determining region 1 (CDR1) of the second antibody portion are separated by 25 or fewer amino acids.

[0208] Linkers are described in more detail herein in the section entitled "Linkers." In some embodiments, the polypeptide linker comprises, from N- to C-terminal, a contiguous series of three amino acids, X1-X2-X3, that form the C-terminus of the polypeptide linker, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0209] In one aspect, provided herein is a co-binding agent that specifically binds to a target, the co-binding agent comprising: (i) a first variable region (VR1) of a first antibody, (ii) a second variable region (VR2) of a second antibody, and (iii) a polypeptide linker connecting the C-terminal amino acid of VR1 and the N-terminal amino acid of VR2. In some embodiments, the C-terminal three amino acids of the polypeptide linker are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G. In some embodiments, VR1 and VR2 bind to non-overlapping epitopes on the target. In some embodiments, VR2 comprises an N-terminal truncation of 1 to 18 amino acids.

[0210] In one aspect, provided herein is a co-binding agent that specifically binds to a target, the co-binding agent comprising: (i) a first variable region (VR1) of a first antibody, (ii) a second variable region (VR2) of a second antibody comprising a truncation of 1 to 18 amino acids at its N-terminus, and (iii) a polypeptide linker connecting the C-terminal amino acids of VR1 and the N-terminal amino acids of the truncated VR2. In some embodiments, the C-terminal three amino acids of the polypeptide linker are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G. In some embodiments, VR1 and VR2 bind to non-overlapping epitopes on the target.

[0211] In some embodiments, provided herein are co-binders that specifically bind to a target, the co-binders comprising: (i) a first variable region (VR1) of a first antibody; (ii) a second variable region (VR2) of a second antibody comprising a truncation of 1 to 18 amino acids at its N-terminus in the framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acids of the VR1 and the N-terminal amino acids of the truncated VR2, wherein the C-terminal three amino acids of the polypeptide linker are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R; X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E; and X3 is G; and VR1 and VR2 bind to non-overlapping epitopes on the target.

[0212] In some embodiments, provided herein are co-binding agents that specifically bind to a target, the co-binding agent comprising: (i) a first variable region (VR1) of a first antibody; (ii) a second variable region (VR2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acids of the VR1 and the N-terminal amino acids of the truncated VR2, wherein the C-terminal three amino acids of the polypeptide linker are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G; and wherein the VR1 and VR2 bind to non-overlapping epitopes on the target.

[0213] In another aspect, provided herein is a co-binder that specifically binds to a target, the co-binder comprising: (i) a first variable region (VR1) of a first antibody; (ii) a second variable region (VR2) of a second antibody comprising a truncation in its framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acids of VR1 and the N-terminal amino acid of the truncated VR2, wherein the C-terminal three amino acids of the polypeptide linker are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R; X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E; and X3 is G; the X3 amino acid of the polypeptide linker and the complementarity determining region 1 (CDR1) of VR2 are separated by 5 to 25 amino acids; and VR1 and VR2 bind to non-overlapping epitopes on the target.

[0214] In another aspect, provided herein is a co-binder that specifically binds to a target, the co-binder comprising: (i) a first variable region (VR1) of a first antibody; (ii) a second variable region (VR2) of a second antibody comprising a truncation in its framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acids of VR1 and the N-terminal amino acid of the truncated VR2, wherein the C-terminal three amino acids of the polypeptide linker are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R; X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E; and X3 is G; the X3 amino acid of the polypeptide linker and the complementarity determining region 1 (CDR1) of VR2 are separated by 25 or fewer amino acids; and VR1 and VR2 bind to non-overlapping epitopes on the target.

[0215] In another aspect, provided herein is a co-binder that specifically binds to a target, the co-binder comprising: (i) a first variable region (VR1) of a first antibody; (ii) a second variable region (VR2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acid of the VR1 and the N-terminal amino acid of the truncated VR2, wherein the polypeptide linker the three C-terminal amino acids of VR1 are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G; the X3 amino acid of the polypeptide linker and the complementarity-determining region 1 (CDR1) of VR2 are separated by 5 to 25 amino acids; and VR1 and VR2 bind to non-overlapping epitopes on the target.

[0216] In another aspect, provided herein is a co-binder that specifically binds to a target, the co-binder comprising: (i) a first variable region (VR1) of a first antibody; (ii) a second variable region (VR2) of a second antibody comprising a truncation of 1 to 18 amino acids in the framework 1 (FR1) region; and (iii) a polypeptide linker connecting the C-terminal amino acid of the VR1 and the N-terminal amino acid of the truncated VR2, wherein the polypeptide linker the three C-terminal amino acids of VR1 are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G; the X3 amino acid of the polypeptide linker and the complementarity-determining region 1 (CDR1) of VR2 are separated by 25 or fewer amino acids; and VR1 and VR2 bind to non-overlapping epitopes on the target.

[0217] In some embodiments, the VR1 is a light chain variable region. In some embodiments, the VR1 is a heavy chain variable region. In some embodiments, the VR2 is a light chain variable region. In some embodiments, the VR2 is a heavy chain variable region. In some embodiments, the VR1 is a light chain variable region and the VR2 is a light chain variable region. In some embodiments, the VR1 is a light chain variable region and the VR2 is a heavy chain variable region. In some embodiments, the VR1 is a heavy chain variable region and the VR2 is a light chain variable region. In some embodiments, the VR1 is a heavy chain variable region and the VR2 is a heavy chain variable region. In some embodiments, the VR1 is a V H In some embodiments, VR2 is V H H. In some embodiments, VR is V H H, and VR2 is V H It's H.

[0218] In some embodiments, the truncation of the N-terminus of the second binding moiety is by any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the X3 amino acid of the polypeptide linker and the CDR1 of the second binding moiety are separated by no more than 25 amino acids, e.g., no more than 24 amino acids, no more than 23 amino acids, no more than 22 amino acids, no more than 21 amino acids, no more than 20 amino acids, no more than 19 amino acids, no more than 18 amino acids, no more than 17 amino acids, no more than 16 amino acids, no more than 15 amino acids, no more than 14 amino acids, no more than 13 amino acids, no more than 12 amino acids, no more than 11 amino acids, no more than 10 amino acids, no more than 9 amino acids, no more than 8 amino acids, no more than 7 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, or no more than 3 amino acids. In some embodiments, the X3 amino acid of the polypeptide linker and the CDR1 of the second binding moiety are separated by any of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acids. In some embodiments, the truncation at the N-terminus of the second binding moiety is a truncation in framework region 1 (FR1) of the second binding moiety.

[0219] In some embodiments, the variable region, e.g., an N-terminal truncated antibody variable domain of the second binding moiety, comprises V H In some embodiments, the first binding moiety is a first V H H domain, and the second binding moiety comprises a second V H domain having an N-terminal truncation. H H domain ("truncated V H the first V H domain) H The C-terminus of the H domain is the second V HIt is connected to the N-terminus of the H domain via a linker.

[0220] Thus, in some embodiments, provided herein is a co-binder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, wherein the second binding moiety comprises a second V having an N-terminal truncation. H H domain ("truncated V H H domain), and the first binding moiety is a first V H In some embodiments, the truncated VH domain of the second binding moiety is connected to the first binding moiety through the N-terminus of the N-terminal truncated antibody variable domain via a linker. HThe H domain is truncated by any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the X3 amino acid of the polypeptide linker and the CDR1 of the second binding moiety are separated by 25 or fewer amino acids, e.g., 24 or fewer amino acids, 23 or fewer amino acids, 22 or fewer amino acids, 21 or fewer amino acids, 20 or fewer amino acids, 19 or fewer amino acids, 18 or fewer amino acids, 17 or fewer amino acids, 16 or fewer amino acids, 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, or 3 or fewer amino acids. In some embodiments, the X3 amino acid of the polypeptide linker and the CDR1 of the second binding moiety are separated by any of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acids. In some embodiments, the truncation at the N-terminus of the second binding moiety is a truncation in framework region 1 (FR1) of the second binding moiety. In some embodiments, the C-terminal three amino acids of the linker are X1-X2-X3, where X1 is V, L, W, P, S, G, K, D, F, M, T, N, or R, X2 is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 is G.

[0221] In some embodiments, the co-binding agent has only the first and second binding moieties that bind to distinct, non-overlapping epitopes on the target molecule. In some embodiments, the co-binding agent can also have a third binding moiety that binds to a third, non-overlapping, distinct epitope on the target molecule. In some embodiments, the co-binding agent can also have a third binding moiety and a fourth binding moiety that bind to a third and fourth, non-overlapping, distinct epitope on the target molecule, respectively. These third and / or fourth binding moieties may or may not be N-terminally truncated, as described for the second binding moiety.

[0222] The co-binding agent may be a monomeric molecule or a multimeric complex. In some embodiments, the co-binding agent is a monomeric molecule having a pair of the binding moieties. In some embodiments, the co-binding agent is a monomeric molecule having a pair of the first and second binding moieties. In some embodiments, the co-binding agent is a monomeric molecule having a pair of the first, second, and third binding moieties. In some embodiments, the co-binding agent is a monomeric molecule having a pair of the first, second, third, and fourth binding moieties.

[0223] In some embodiments, the co-binding agent is a multimeric complex having at least two pairs of the binding moieties. In some embodiments, the co-binding agent is a multimeric complex having at least three pairs of the binding moieties. In some embodiments, the co-binding agent is a multimeric complex having at least four pairs of the binding moieties. In some embodiments, the co-binding agent is a multimeric complex having two pairs of the binding moieties. In some embodiments, the co-binding agent is a multimeric complex having two pairs of the first and second binding moieties. In some embodiments, the co-binding agent is a multimeric complex having two pairs of the first, second, and third binding moieties. In some embodiments, the co-binding agent is a multimeric complex having two pairs of the first, second, third, and fourth binding moieties. In some embodiments, the co-binding agent is a multimeric complex having three pairs of the binding moieties. In some embodiments, the co-binding agent is a multimeric complex having three pairs of the first and second binding moieties. In some embodiments, the co-binding agent is a multimeric complex having three pairs of the first, second, and third binding moieties. In some embodiments, the co-binding agent is a multimeric complex having three pairs of the first, second, third, and fourth binding moieties. In some embodiments, the co-binding agent is a multimeric complex having four pairs of the binding moieties. In some embodiments, the co-binding agent is a multimeric complex having four pairs of the first and second binding moieties. In some embodiments, the co-binding agent is a multimeric complex having four pairs of the first, second, and third binding moieties. In some embodiments, the co-binding agent is a multimeric complex having four pairs of the first, second, third, and fourth binding moieties.

[0224] In some embodiments, the multimeric complex co-binder may have the sets of binding moieties in different orientations. In some embodiments, the sets of binding moieties are arranged consecutively. For example, the two sets of the first binding moiety (comprising paratope P1) and the second binding moiety (comprising paratope P2) may be arranged as P1-P2-P1-P2. As another example, the two sets of the first binding moiety (comprising paratope P1), the second binding moiety (comprising paratope P2), and their binding moieties (comprising paratope P3) may be arranged as P1-P2-P3-P1-P2-P3. In some embodiments, the sets of binding moieties are arranged in reverse. For example, the two sets of the first binding moiety (comprising paratope P1) and the second binding moiety (comprising paratope P2) may be arranged as P1-P2-P2-P1. As another example, the two sets of the first binding moiety (comprising paratope P1), the second binding moiety (comprising paratope P2), and their binding moieties (comprising paratope P3) can be arranged as P1-P2-P3-P3-P2-P1. In some embodiments, the sets of binding moieties are arranged alternately. For example, the two sets of the first binding moiety (comprising paratope P1) and the second binding moiety (comprising paratope P2) can be arranged as P1-P1-P2-P2. As another example, the two sets of the first binding moiety (comprising paratope P1), the second binding moiety (comprising paratope P2), and their binding moieties (comprising paratope P3) can be arranged as P1-P1-P2-P2-P3-P3. As will be appreciated by those skilled in the art, the binding moieties of the multimeric cobinders described herein can be arranged in any order. In some embodiments, the order of arrangement of the binding moieties of the multimeric cobinder is optimized to maximize binding affinity to the target molecule and / or minimize any non-specific binding.

[0225] In some embodiments, the co-binding agents disclosed herein have a first binding moiety and a second binding moiety that bind to two distinct, non-overlapping epitopes on a target molecule. The two distinct, non-overlapping epitopes on the target molecule can be relatively close to each other. In some embodiments, the two epitopes recognized by the co-binding agent are located close to each other but still separated by a sufficient distance to accommodate the co-binding agent's linker. In some embodiments, the first and second epitopes are separated by 150 angstroms or less, e.g., about 120 angstroms or less, 100 angstroms or less, 80 angstroms or less, 50 angstroms or less, 40 angstroms or less, 30 angstroms or less, 15 angstroms or less, 10 angstroms or less, or 5 angstroms or less.

[0226] In the case of linear epitopes on a target peptide or protein, the two epitopes can be spaced within 200 amino acids of each other, for example, within approximately 150 amino acids of each other, 120 amino acids of each other, 100 amino acids of each other, 80 amino acids of each other, 50 amino acids of each other, 40 amino acids of each other, 30 amino acids of each other, 20 amino acids of each other, 15 amino acids of each other, 10 amino acids of each other, or 5 amino acids of each other. In some embodiments, the two epitopes recognized by the co-binders are selected so that the two binding interactions are cooperative and synergistic and do not interfere with each other.

[0227] In some embodiments, the co-binding agent comprises a first antibody portion that is a variable region (VR1) and a second antibody portion that is a variable region (VR2). In some embodiments, VR1 binds to an epitope of the target to produce a desired biological effect on the target, but with insufficient affinity for therapeutic or diagnostic use by itself, and VR2 binds to a different epitope of the target with sufficient affinity, such that the resulting co-binding agent binds to the target with sufficient affinity to produce the desired biological effect. In some embodiments of the co-binding agents provided herein, VR2 binds to an epitope of the target to produce a desired biological effect on the target, but with insufficient affinity for therapeutic or diagnostic use by itself, and VR1 binds to a different epitope of the target with sufficient affinity, such that the resulting co-binding agent binds to the target with sufficient affinity to produce the desired biological effect. In some embodiments of the cobinding agents provided herein, the first binding moiety binds to an epitope of the target to produce a desired biological effect on the target but with insufficient affinity for therapeutic or diagnostic use by itself, and the second binding moiety binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect. In some embodiments of the cobinding agents provided herein, the second binding moiety binds to an epitope of the target to produce a desired biological effect on the target but with insufficient affinity for therapeutic or diagnostic use by itself, and the first binding moiety binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect.

[0228] In some embodiments, the cobinding agent comprises a first antibody portion that is a heavy chain variable region of a first antibody (VHAb1) and a second antibody portion that is a heavy chain variable region of a second antibody (VHAb2). In some embodiments, VHAb1 binds to an epitope of the target to produce a desired biological effect on the target, but with insufficient affinity for therapeutic or diagnostic use by itself, and VHAb2 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect. In some embodiments of the cobinding agents provided herein, VHAb2 binds to an epitope of the target to produce a desired biological effect on the target, but with insufficient affinity for therapeutic or diagnostic use by itself, and VHAb1 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect.

[0229] In some embodiments, the cobinding agent comprises a first antibody portion that is a light chain variable region of a first antibody (VLAb1) and a second antibody portion that is a heavy chain variable region of a second antibody (VHAb2). In some embodiments, VLAb1 binds to an epitope of the target to produce a desired biological effect on the target, but with an affinity that is insufficient for therapeutic or diagnostic use by itself, and VHAb2 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect. In some embodiments of the cobinding agents provided herein, VHAb2 binds to an epitope of the target to produce a desired biological effect on the target, but with an affinity that is insufficient for therapeutic or diagnostic use by itself, and VLAb1 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect.

[0230] In some embodiments, the cobinding agent comprises a first antibody portion that is a heavy chain variable region of a first antibody (VHAb1) and a second antibody portion that is a light chain variable region of a second antibody (VLAb2). In some, VHAb1 binds to an epitope of the target to produce a desired biological effect on the target, but with insufficient affinity for therapeutic or diagnostic use by itself, and VLAb2 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect. In some embodiments of the cobinding agents provided herein, VLAb2 binds to an epitope of the target to produce a desired biological effect on the target, but with insufficient affinity for therapeutic or diagnostic use by itself, and VHAb1 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect.

[0231] In some embodiments, the cobinding agent comprises a first antibody portion that is a light chain variable region of a first antibody (VLAb1) and a second antibody portion that is a light chain variable region of a second antibody (VLAb2). In some embodiments of the cobinding agents provided herein, VLAb1 binds to an epitope of the target to produce a desired biological effect on the target, but with an affinity that is insufficient for therapeutic or diagnostic use by itself, and VLAb2 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect. In some embodiments of the cobinding agents provided herein, VLAb2 binds to an epitope of the target to produce a desired biological effect on the target, but with an affinity that is insufficient for therapeutic or diagnostic use by itself, and VLAb1 binds to a different epitope of the target with sufficient affinity, such that the resulting cobinding agent binds to the target with sufficient affinity to produce the desired biological effect.

[0232] In some embodiments of the cobinders provided herein, VR1 binds to an epitope on the target to produce a desired biological effect on the target but also binds to an epitope on a non-target that causes undesired side effects, and VR2 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinder that binds to the target with sufficient affinity to produce the desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesired side effects. In some embodiments of the cobinders provided herein, VR2 binds to an epitope on the target to produce a desired biological effect on the target but also binds to an epitope on the non-target that causes undesired side effects, and VR1 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinder that binds to the target with sufficient affinity to produce the desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesired side effects.

[0233] In some embodiments of the cobinders provided herein, the first binding moiety binds to an epitope of the target to cause a desired biological effect on the target but also binds to an epitope of a non-target that causes undesired side effects, and the second binding moiety binds to a different epitope of the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinder that binds to the target with sufficient affinity to cause the desired biological effect but is unable to bind to the non-target with sufficient affinity to cause the undesired side effects. In some embodiments of the cobinders provided herein, the second binding moiety binds to an epitope of the target to cause a desired biological effect on the target but also binds to an epitope of a non-target that causes undesired side effects, and the first binding moiety binds to a different epitope of the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinder that binds to the target with sufficient affinity to cause the desired biological effect but is unable to bind to the non-target with sufficient affinity to cause the undesired side effects.

[0234] In some embodiments of the cobinding agents provided herein, VHAb1 binds to an epitope on the target to produce a desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VHAb2 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce a desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects. In some embodiments of the cobinding agents provided herein, VHAb2 binds to an epitope on the target to produce a desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VHAb1 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce a desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects.

[0235] In some embodiments of the cobinding agents provided herein, VLAb1 binds to an epitope on the target to produce a desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VHAb2 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce a desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects. In some embodiments of the cobinding agents provided herein, VHAb2 binds to an epitope on the target to produce a desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VLAb1 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce a desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects.

[0236] In some embodiments of the cobinding agents provided herein, VHAb1 binds to an epitope on the target to produce a desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VLAb2 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce a desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects. In some embodiments of the cobinding agents provided herein, VLAb2 binds to an epitope on the target to produce a desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VHAb1 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce a desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects.

[0237] In some embodiments of the cobinding agents provided herein, VLAb1 binds to an epitope on the target to produce the desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VLAb2 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce the desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects. In some embodiments of the cobinding agents provided herein, VLAb2 binds to an epitope on the target to produce the desired biological effect on the target but also binds to a non-target epitope that causes undesirable side effects, and VLAb1 binds to a different epitope on the target with high affinity but does not bind to the non-target with sufficient affinity, resulting in a cobinding agent that binds to the target with sufficient affinity to produce the desired biological effect but is unable to bind to the non-target with sufficient affinity to produce the undesirable side effects.

[0238] The present disclosure allows for the construction of cobinders provided herein using a variety of VHs or VLs, resulting in improved affinity and / or specificity for such cobinders. In one embodiment, the antigen-binding fragment (e.g., VH and / or VL) included in the second binding moiety used in the cobinders provided herein can be selected based on the proximity of the paratope of the antigen-binding fragment to the N-terminus of the antigen-binding fragment. In certain embodiments, cobinders can be constructed using a VH, which can result in improved affinity and / or specificity for cobinders of the present disclosure, and the paratope of the VH is in close proximity to the N-terminus of the VH. In some embodiments, cobinders can be constructed using a VH as part of the second binding moiety, which can result in improved affinity and / or specificity for cobinders of the present disclosure, and the paratope of the VH is in close proximity to the N-terminus of the VH. In other embodiments, cobinders can be constructed using VHAb2 and can result in improved affinity and / or specificity for cobinders of the present disclosure, with the paratope of VHAb2 in close proximity to the N-terminus of the VHAb2. In certain embodiments, cobinders can be constructed using VL and can result in improved affinity and / or specificity for cobinders of the present disclosure, with the paratope of VL in close proximity to the N-terminus of the VL. In some embodiments, cobinders can be constructed using VL as part of the second binding moiety and can result in improved affinity and / or specificity for cobinders of the present disclosure, with the paratope of VL in close proximity to the N-terminus of the VL. In further embodiments, cobinders can be constructed using VLAb2 and can result in improved affinity and / or specificity for cobinders of the present disclosure, with the paratope of VLAb2 in close proximity to the N-terminus of the VLAb2. In some embodiments, cobinders can be constructed with variable regions (VRs) and the paratope of the VR is in close proximity to the N-terminus of the VR, which can result in improved affinity and / or specificity conferred to the cobinders of the present disclosure.In certain embodiments, a cobinder may be constructed with a variable region (VR2) as part of the second binding moiety, which may result in improved affinity and / or specificity for the cobinders of the present disclosure, and the paratope of the VR2 is in close proximity to the N-terminus of the VR2. The VH, VH, VHAb, VHAb2, and VL as part of the second binding moiety, VL, VLAb, VLAb2, VR, and VR2 of this paragraph may be any of the corresponding embodiments described herein.

[0239] The proximity between the paratope of the antigen-binding fragment (e.g., VH, VL, VHAb, VHAb2, VLAb, VLAb2, VR, and VR2 described herein) and the N-terminus of the antigen-binding fragment can be determined based on the structure of the antigen-binding fragment and / or the structure of a complex of the antigen-binding fragment with its target antigen. In some embodiments, the nearest non-hydrogen atom on the antigen surface in the structure of the complex of the antigen-binding fragment with its target antigen can be used as a surrogate for the paratope to determine the proximity of the paratope to the N-terminus of the antigen-binding fragment. Thus, The proximity can be determined based on the spacing between the first Cα atom (N-terminus) of the antigen-binding fragment and the nearest non-hydrogen atom on the surface of the antigen in the structure of a complex of such antigen-binding fragment with its target antigen. In one embodiment, antigen-binding fragments (e.g., VH, VL, VHAb, VHAb2, VLAb, VLAb2, VR, and VR2 described herein) are suitable for use in constructing cobinders provided herein, resulting in improved affinity and / or specificity for the cobinders of the present disclosure, and the proximity can be determined based on the spacing between the first Cα atom (N-terminus) of the antigen-binding fragment and the nearest non-hydrogen atom on the surface of the antigen in the structure of a complex of such antigen-binding fragment with its target antigen. A threshold value, as determined by the spacing of nearest non-hydrogen atoms on the antigen surface, below or about a certain threshold is required to provide sufficient spacing for the two binding moieties of the cobinder to link without interfering with binding to the target antigen. In another embodiment, antigen-binding fragments (e.g., VH, VL, VHAb, VHAb2, VLAb, VLAb2, VR, and VR2 described herein) are suitable for use as the second binding moiety or part of the second binding moiety to construct the cobinders provided herein, and may be used in conjunction with the cobinders of the present disclosure. A proximity threshold is required to provide sufficient spacing for the two binding moieties of the cobinder to be linked without interfering with binding to the target antigen, where the proximity is less than or equal to a certain threshold, as determined by the spacing between the first Cα atom (N-terminus) of the antigen-binding fragment and the nearest non-hydrogen atom on the antigen surface, resulting in improved affinity and / or specificity. In one particular embodiment, the proximity referred to herein is less than or equal to about 15 Å, e.g., less than or equal to about 14 Å, 13 Å, 12 Å, 11 Å, 10 Å, 9 Å, 8 Å, 7 Å, 6 Å, or 5 Å.

[0240] The present disclosure further allows the proximity between the N-terminus of the antigen-binding fragment and the paratope of the antigen-binding fragment (e.g., using the nearest non-hydrogen atom on the surface of the bound antigen) to be determined by examining existing structures contained in a database (e.g., PDB). Such proximity can also be determined by homology structure modeling using a large number of structures available in a structural database, as practiced by those skilled in the art. Furthermore, such proximity can be determined through other structures identified by other structure simulation software or methods, such as molecular dynamics or molecular mechanics (e.g., CHARMM, AMBER, and NAMD) and ab initio protein modeling (e.g., Rosetta), as practiced by those skilled in the art. Thus, the present disclosure allows the structure of the antigen bound to the antigen-binding fragment and the proximity between the N-terminus of the antigen-binding fragment and the paratope (e.g., using the nearest non-hydrogen atom on the surface of the bound antigen as a proxy) to be determined without the need to experimentally identify any structure, as will be understood by those skilled in the art.

[0241] Alternatively, the proximity between the N-terminus of the antigen-binding fragment and the paratope can be determined using the functional effect of placing a linkage at the N-terminus of the antigen-binding fragment as a reporter of such proximity (e.g., using the nearest non-hydrogen atom on the surface of the bound antigen as a surrogate). As described above, the proximity between the N-terminus of the antigen-binding fragment and the paratope is useful for determining whether there is sufficient space for the two binding moieties of the cobinder to be linked without interfering with binding to the target antigen. The present disclosure further provides that the affinity of the antigen-binding fragment is adversely affected after inserting or linking a linker to the N-terminus of the antigen-binding fragment if the proximity between the N-terminus of the antigen-binding fragment and the paratope is below a certain threshold necessary to provide sufficient space for the two binding moieties of the cobinder to be linked without interfering with binding to the target antigen. Thus, the present disclosure provides that the change in affinity after inserting or linking a linker to the N-terminus of the antigen-binding fragment can be correlated with determining whether the proximity between the N-terminus of the antigen-binding fragment and the paratope is below a certain threshold that is sufficient to link the two binding moieties of the cobinder without interfering with binding to the target antigen.

[0242] Thus, the present disclosure provides that antigen-binding fragments (e.g., VH, VL, VHAb, VHAb2, VLAb, VLAb2, VR, and VR2 described herein) are suitable for use in constructing cobinders provided herein, and that if the affinity of the antigen-binding fragment for the antigen changes beyond a certain threshold after inserting or linking a linker to the N-terminus of the antigen-binding fragment, improved affinity and / or specificity is achieved in the cobinders of the present disclosure. Briefly, in some embodiments, an antigen-binding fragment ("ABF") against a target is fused to a reference immunoglobulin domain (refIg) that does not specifically bind to the target via a (GGGS)4 linker. The fusion construct refIg-GS-ABF can be fused to create a refIg-GS-ABF construct. Such ABFs are suitable for use in constructing the cobinders provided herein and for generating improved affinity and / or specificity resulting in cobinders of the present disclosure when the affinity of the fusion construct refIg-GS-ABF for the target is weaker than the affinity of the ABF for the target by a certain threshold. In one specific embodiment, the affinity of the fusion construct refIg-GS-ABF for the target is at least 2-fold weaker than the affinity of the ABF for the target, e.g., at least any of the following: 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, or 20-fold weaker.

[0243] Similarly, affinity is expressed as the dissociation equilibrium constant (K D ), the present disclosure provides that antigen-binding fragments (e.g., VH, VL, VHAb, VHAb2, VLAb, VLAb2, VR, and VR2 described herein are suitable for use in constructing the cobinders provided herein, and that the K DIt is defined that if the K changes beyond a certain threshold after inserting or linking a linker to the N-terminus of the antigen-binding fragment, it will result in improved affinity and / or specificity resulting in a cobinder of the present disclosure. Briefly, in some embodiments, an antigen-binding fragment ("ABF") against a target can be fused to a reference immunoglobulin domain (refIg) that does not specifically bind to the target via a (GGGS)4 linker to create a refIg-GS-ABF construct. Such ABFs can be used to construct the cobinders provided herein, as well as to determine the K of the fusion construct refIg-GS-ABF against the target. D is the K D In one particular embodiment, the K of the fusion construct refIg-GS-ABF for the target is suitable for use in generating improved affinity and / or specificity conferred to the cobinder of the present disclosure when the K is greater than a certain threshold value. D is the K of ABF for the target D at least two times greater than, for example, at least any of the following: 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times greater.

[0244] Furthermore, in some specific embodiments provided herein, an antigen-binding fragment ("ABF") against a target can be fused to a reference immunoglobulin domain (refIg) that does not specifically bind to the target via a (GGGS)x linker to create a refIg-GS-ABF construct, where x can be 1, 2, 3, 4, 5, 6, 7, or 8. In one embodiment, an ABF against the target can be fused to a refIg that does not specifically bind to the target via a GGGS linker to create a refIg-GS-ABF construct described herein. In another embodiment, an ABF against the target can be fused to a refIg that does not specifically bind to the target via a (GGGS)2 linker to create a refIg-GS-ABF construct described herein. In a further embodiment, an ABF against the target can be fused to a refIg that does not specifically bind to the target via a (GGGS)3 linker to create a refIg-GS-ABF construct described herein. In yet another embodiment, an ABF for the target can be fused to a refIg that does not specifically bind to the target via a (GGGS) linker to create the refIg-GS-ABF construct described herein. In one embodiment, an ABF for the target can be fused to a refIg that does not specifically bind to the target via a (GGGS) linker to create the refIg-GS-ABF construct described herein. In another embodiment, an ABF for the target can be fused to a refIg that does not specifically bind to the target via a (GGGS) linker to create the refIg-GS-ABF construct described herein. In a further embodiment, an ABF for the target can be fused to a refIg that does not specifically bind to the target via a (GGGS) linker to create the refIg-GS-ABF construct described herein. In yet another embodiment, an ABF for the target can be fused to a refIg that does not specifically bind to the target via a (GGGS) linker to create the refIg-GS-ABF construct described herein.

[0245] The present disclosure provides that the refIg described herein can be any immunoglobulin domain (e.g., VH, VL, scFv, VHH) as long as the refIg does not specifically bind to the same antigen as the antigen the co-binder is constructed to specifically bind. For example, as demonstrated herein, when the co-binder is constructed to bind to EGFR, the anti-human lysozyme VHH, HuL6, can be used as such a refIg. Similarly, in some embodiments, the refIg can be an antigen-binding domain or fragment (e.g., VH, VL, scFv, or VHH) of an isotype immunoglobulin. In certain embodiments, the refIg can be an antigen-binding domain or fragment (e.g., VH, VL, scFv, or VHH) that binds to an antigen different from the antigen the co-binder is constructed to specifically bind.

[0246] C. Linker In certain aspects of the binding agent molecules described herein, the binding agent molecule comprises a linker. Generally, the linkers described herein are linked to one or more components of the binding agent molecules described herein, for example, by a covalent bond. For example, in some embodiments, the cobinder comprises a second binding moiety and a linker, and the linker is attached to the second binding moiety via the N-terminus of the second binding moiety. In some embodiments, the cobinder comprises a linker connecting the first binding moiety and the second binding moiety, and the linker is attached to the second binding moiety via the N-terminus of the second binding moiety, and the linker is attached to the first binding moiety via the C-terminus of the first binding moiety. In some embodiments, the second binding moiety is an N-terminally truncated antibody variable domain. In some embodiments, the linker connects the first binding moiety and the second binding moiety via a covalent bond. In some embodiments, the linker connects the first binding moiety and the second binding moiety via a combination of covalent and non-covalent bonds, e.g., the linker is covalently attached to the second binding moiety or the first binding moiety and non-covalently attached to the other binding moiety. In some embodiments, the linker of the co-binding agent facilitates the co-binding agent achieving cooperative and / or synergistic binding interactions with its target molecule. As described herein, linkers may take many forms and may be selected based on a variety of characteristics.

[0247] In some embodiments, the linker comprises a polypeptide. In some embodiments, the linker is a polypeptide. In some embodiments, the linker comprises a polypeptide complex, e.g., comprising two or more polypeptide subunits. In some embodiments, the linker comprises a polynucleotide. In some embodiments, the linker is a polynucleotide. In some embodiments, the linker is a polynucleotide complex, e.g., a first polynucleotide strand and a second polynucleotide strand having a complementary region. In some embodiments, the linker is a chemical linker or a synthetic linker, e.g., a polymer-based linker.

[0248] In some embodiments, the linker is covalently attached to the binding moiety of the binding agent molecule. For example, in some embodiments, the binding agent molecule comprises a second binding moiety and a linker, wherein the second binding moiety and the linker are a single polypeptide. In some embodiments, the linker is non-covalently attached to the binding moiety of the binding agent molecule.

[0249] In some embodiments, the linker is attached, e.g., covalently attached, to the N-terminus of the second binding moiety. In some embodiments, the terminal portion of the linker attached, e.g., covalently attached, to the N-terminus of the second binding moiety comprises three amino acids, i.e., X1-X2-X3. For example, in some embodiments, the linker comprises a polypeptide, and the C-terminal portion of the linker attached, e.g., covalently attached, to the N-terminus of the second binding moiety comprises, from N-terminus to C-terminus, three amino acids, i.e., X1-X2-X3. In some embodiments, X3 is covalently attached, e.g., via a peptide bond, to the N-terminal residue of the second binding moiety.

[0250] In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G, R, or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or R. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is Y.

[0251] In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is V, L, W, P, S, G, K, D, F, M, T, N, or R. In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is V, L, W, P, S, G, K, D, F, M, T, N, or R and X3 of X1-X2-X3 at the C-terminal portion of the linker is G, R, or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or R. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is Y.

[0252] In certain embodiments, X2 of X1-X2-X3 at the C-terminal portion of the linker is V, A, L, S, G, R, K, M, C, F, T, P, or E, and X3 of X1-X2-X3 at the C-terminal portion of the linker is G, R, or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or R. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R. In some embodiments, X3 of X1-X2-X3 of the C-terminal portion of the linker is Y.

[0253] In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is V, L, W, P, S, G, K, D, F, M, T, N, or R; X2 of X1-X2-X3 at the C-terminal portion of the linker is V, A, L, S, G, R, K, M, C, F, T, P, or E; and X3 of X1-X2-X3 at the C-terminal portion of the linker is G, R, or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or R. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R or Y. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is G. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is R. In some embodiments, X3 of X1-X2-X3 at the C-terminal portion of the linker is Y.

[0254] In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, X2 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, and X3 of X1-X2-X3 at the C-terminal portion of the linker is G, R, or Y. In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, X2 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, and X3 of X1-X2-X3 at the C-terminal portion of the linker is G or R. In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, X2 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, and X3 of X1-X2-X3 at the C-terminal portion of the linker is G or Y. In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, X2 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, and X3 of X1-X2-X3 at the C-terminal portion of the linker is R or Y. In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, X2 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, and X3 of X1-X2-X3 at the C-terminal portion of the linker is G. In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, X2 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, and X3 of X1-X2-X3 at the C-terminal portion of the linker is R. In some embodiments, X1 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, X2 of X1-X2-X3 at the C-terminal portion of the linker is any amino acid, and X3 of X1-X2-X3 at the C-terminal portion of the linker is Y.

[0255] In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is VVG, VAG, VLG, VSG, VGG, VRG, VKG, VMG, VCG, VFG, VTG, VPG, or VEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is LVG, LAG, LLG, LSG, LGG, LRG, LKG, LMG, LCG, LFG, LTG, LPG, or LEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is WVG, WAG, WLG, WSG, WGG, WRG, WKG, WMG, WCG, WFG, WTG, WPG, or WEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is PVG, PAG, PLG, PSG, PGG, PRG, PKG, PMG, PCG, PFG, PTG, PPG, or PEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is SVG, SAG, SLG, SSG, SGG, SRG, SKG, SMG, SCG, SFG, STG, SPG, or SEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is GVG, GAG, GLG, GSG, GGG, GRG, GKG, GMG, GCG, GFG, GTG, GPG, or GEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is KVG, KAG, KLG, KSG, KGG, KRG, KKG, KMG, KCG, KFG, KTG, KPG, or KEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is DVG, DAG, DLG, DSG, DGG, DRG, DKG, DMG, DCG, DFG, DTG, DPG, or DEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is FVG, FAG, FLG, FSG, FGG, FRG, FKG, FMG, FCG, FFG, FTG, FPG, or FEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is MVG, MAG, MLG, MSG, MGG, MRG, MKG, MMG, MCG, MFG, MTG, MPG, or MEG.In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is TVG, TAG, TLG, TSG, TGG, TRG, TKG, TMG, TCG, TFG, TTG, TPG, or TEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is NVG, NAG, NLG, NSG, NGG, NRG, NKG, NMG, NCG, NFG, NTG, NPG, or NEG. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is RVG, RAG, RLG, RSG, RGG, RRG, RKG, RMG, RCG, RFG, RTG, RPG, or REG.

[0256] In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is VVR, VAR, VLR, VSR, VGR, VRR, VKR, VMR, VCR, VFR, VTR, VPR, or VER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is LVR, LAR, LLR, LSR, LGR, LRR, LKR, LMR, LCR, LFR, LTR, LPR, or LER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is WVR, WAR, WLR, WSR, WGR, WRR, WKR, WMR, WCR, WFR, WTR, WPR, or WER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is PVR, PAR, PLR, PSR, PGR, PRR, PKR, PMR, PCR, PFR, PTR, PPR, or PER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is SVR, SAR, SLR, SSR, SGR, SRR, SKR, SMR, SCR, SFR, STR, SPR, or SER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is GVR, GAR, GLR, GSR, GGR, GRR, GKR, GMR, GCR, GFR, GTR, GPR, or GER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is KVR, KAR, KLR, KSR, KGR, KRR, KKR, KMR, KCR, KFR, KTR, KPR, or KER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is DVR, DAR, DLR, DSR, DGR, DRR, DKR, DMR, DCR, DFR, DTR, DPR, or DER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is FVR, FAR, FLR, FSR, FGR, FRR, FKR, FMR, FCR, FFR, FTR, FPR, or FER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is MVR, MAR, MLR, MSR, MGR, MRR, MKR, MMR, MCR, MFR, MTR, MPR, or MER.In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is TVR, TAR, TLR, TSR, TGR, TRR, TKR, TMR, TCR, TFR, TTR, TPR, or TER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is NVR, NAR, NLR, NSR, NGR, NRR, NKR, NMR, NCR, NFR, NTR, NPR, or NER. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is RVR, RAR, RLR, RSR, RGR, RRR, RKR, RMR, RCR, RFR, RTR, RPR, or RER.

[0257] In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is VVY, VAY, VLY, VSY, VGY, VRY, VKY, VMY, VCY, VFY, VTY, VPY, or VEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is LVY, LAY, LLY, LSY, LGY, LRY, LKY, LMY, LCY, LFY, LTY, LPY, or LEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is WVY, WAY, WLY, WSY, WGY, WRY, WKY, WMY, WCY, WFY, WTY, WPY, or WEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is PVY, PAY, PLY, PSY, PGY, PRY, PKY, PMY, PCY, PFY, PTY, PPY, or PEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is SVY, SAY, SLY, SSY, SGY, SRY, SKY, SMY, SCY, SFY, STY, SPY, or SEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is GVY, GAY, GLY, GSY, GGY, GRY, GKY, GMY, GCY, GFY, GTY, GPY, or GEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is KVY, KAY, KLY, KSY, KGY, KRY, KKY, KMY, KCY, KFY, KTY, KPY, or KEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is DVY, DAY, DLY, DSY, DGY, DRY, DKY, DMY, DCY, DFY, DTY, DPY, or DEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is FVY, FAY, FLY, FSY, FGY, FRY, FKY, FMY, FCY, FFY, FTY, FPY, or FEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is MVY, MAY, MLY, MSY, MGY, MRY, MKY, MMY, MCY, MFY, MTY, MPY, or MEY.In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is TVY, TAY, TLY, TSY, TGY, TRY, TKY, TMY, TCY, TFY, TTY, TPY, or TEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is NVY, NAY, NLY, NSY, NGY, NRY, NKY, NMY, NCY, NFY, NTY, NPY, or NEY. In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is RVY, RAY, RLY, RSY, RGY, RRY, RKY, RMY, RCY, RFY, RTY, RPY, or REY.

[0258] In some embodiments, the C-terminal portion of the linker (X1-X2-X3) is any one selected from Table 2. [Table 10]

[0259] In some embodiments, the linker is (EAAAK) n wherein n is an integer between 1 and 25 (e.g., between 1 and 20 or between 1 and 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker is a peptide sequence comprising: (XP) n , (XPP) n , or (XPPP) n wherein X is any amino acid and n is an integer between 1 and 25 (e.g., between 1 and 20 or between 1 and 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker is a peptide sequence comprising: (X n , (XPP) n , or (XPPP) nwherein each X is G, A, P, or S, and n is an integer between 1 and 25 (e.g., between 1 and 20 or between 1 and 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker is a peptide sequence comprising: (AP) n or (APAP) n wherein n is an integer between 1 and 25 (e.g., between 1 and 20 or between 1 and 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker is a peptide sequence comprising: (EEEEKKKK) n wherein n is an integer between 1 and 25 (e.g., between 1 and 20 or between 1 and 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker is a peptide sequence comprising: (G x S y ) n wherein x is 1 to 5, y is 1 to 5, and n is an integer between 1 and 25 (e.g., 1 to 20 or 1 to 10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker is a peptide sequence comprising: (GGGGS) n where n is an integer between 1 and 25 (e.g., 1-20 or 1-10), including any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker comprises a peptide sequence disclosed herein and X1-X2-X3 of the C-terminal portion of a linker disclosed herein.

[0260] In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0261] In some embodiments, the rigidity of the rigid linker is maximized to increase the affinity of the covalent binding moiety. In some embodiments, the rigid linker can bend or flex no more than 90 degrees, for example, no more than 75 degrees, no more than 60 degrees, no more than 45 degrees, no more than 30 degrees, no more than 15 degrees, or no more than 5 degrees. In some embodiments, the rigid linker can bend or flex no more than 45 degrees and can twist no more than 30 degrees. In some embodiments, the linker can twist no more than 360 degrees, for example, no more than 300 degrees, no more than 240 degrees, no more than 180 degrees, no more than 150 degrees, no more than 120 degrees, no more than 90 degrees, no more than 60 degrees, no more than 30 degrees, no more than 15 degrees, or no more than 5 degrees. In some embodiments, the linker can twist no more than 5 degrees. In some embodiments, the rigid linker can bend or flex no more than 90 degrees, e.g., no more than 75 degrees, no more than 60 degrees, no more than 45 degrees, no more than 30 degrees, no more than 15 degrees, or no more than 5 degrees, and can twist no more than 360 degrees, no more than 300 degrees, no more than 240 degrees, no more than 180 degrees, no more than 150 degrees, no more than 120 degrees, no more than 90 degrees, no more than 60 degrees, no more than 30 degrees, no more than 15 degrees, or no more than 5 degrees.

[0262] In some embodiments, the rigid linker has a rigid middle portion and a less rigid tip on one or more ends that connect to the binding moieties. In some embodiments, the rigid linker has a rigid middle portion and a less rigid tip on one or more ends that connect to the binding moieties. For example, such a rigid linker can facilitate simultaneous binding of binding moieties to non-overlapping epitopes on a target molecule.

[0263] In some embodiments, the linker connects the first binding moiety and the second binding moiety via a non-covalent interaction. In some embodiments, the first binding moiety and / or the second binding moiety comprise a moiety that participates in the non-covalent interaction. For example, in some embodiments, the linker comprises a leucine zipper, the first binding moiety comprises a first portion of the leucine zipper, and the second binding moiety comprises a second portion of the leucine zipper. In some embodiments, the linker comprises a double-stranded nucleic acid comprising two strands having complementary regions, the first binding moiety comprises a nucleic acid strand, and the second binding moiety comprises a second nucleic acid strand.

[0264] In some embodiments, the nucleic acid linker comprises a polynucleotide, such as an oligonucleotide, double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA. In some embodiments, the nucleic acid linker comprises 200 nucleotides or less, such as 180 nucleotides or less, 160 nucleotides or less, 140 nucleotides or less, 120 nucleotides or less, 100 nucleotides or less, 80 nucleotides or less, 60 nucleotides or less, 40 nucleotides or less, 20 nucleotides or less, or 10 nucleotides or less.

[0265] In some embodiments, the linker has a length based on the primary structure of the linker, e.g., a linear chain of amino acids. In some embodiments, the length of the linker is estimated based on the primary structure, e.g., a linear chain of amino acids. In some embodiments, the linker has a length of 250 angstroms or less, e.g., 240 angstroms or less, 230 angstroms or less, 220 angstroms or less, 210 angstroms or less, 200 angstroms or less, 190 angstroms or less, 180 angstroms or less, 170 angstroms or less, 160 angstroms or less, 150 angstroms or less, 140 angstroms or less, 130 angstroms or less, 120 angstroms or less, 110 angstroms or less, 100 angstroms or less, 90 angstroms or less, 80 angstroms or less, 70 angstroms or less, 60 angstroms or less, 50 angstroms or less, 40 angstroms or less, 30 angstroms or less, 20 angstroms or less, 15 angstroms or less, 10 angstroms or less, or 5 angstroms or less. In some embodiments, the linker has a length of approximately any of 250 angstroms, 240 angstroms, 230 angstroms, 220 angstroms, 210 angstroms, 200 angstroms, 190 angstroms, 180 angstroms, 170 angstroms, 160 angstroms, 150 angstroms, 140 angstroms, 130 angstroms, 120 angstroms, 110 angstroms, 100 angstroms, 90 angstroms, 80 angstroms, 70 angstroms, 60 angstroms, 50 angstroms, 40 angstroms, 30 angstroms, 20 angstroms, 15 angstroms, 10 angstroms, or 5 angstroms. In some embodiments, the length of the linker is reduced to the minimum length necessary to provide at least a link between the first binding moiety and the second binding moiety without interfering with the binding of each binding molecule to the target molecule.In some embodiments, the length of the linker is configured to achieve both minimal entropy loss and minimal interference with binding of the binding molecule to the target molecule.

[0266] In some embodiments, the linker has a length of 120 amino acids or less, e.g., 115 amino acids or less, 110 amino acids or less, 105 amino acids or less, 100 amino acids or less, 95 amino acids or less, 90 amino acids or less, 85 amino acids or less, 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, 35 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less. In some embodiments, the linker has a length of approximately any of 120 amino acids, 115 amino acids, 110 amino acids, 105 amino acids, 100 amino acids, 95 amino acids, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, 10 amino acids, or 5 amino acids.

[0267] In some embodiments, the linker is a chemical linker, e.g., a synthetic chemical structure or a polymer. In some embodiments, the linker comprises multiple polyethylene glycol subunits. In some embodiments, the linker is a non-peptidyl polymer.

[0268] D. Other Configurations, Alternatives, and Variants of Binder Molecules Provided herein are various configurations of binding agent molecules comprising a second binding moiety that specifically recognizes a target site, the second binding moiety being a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"). In some embodiments, the binding agent molecule comprises a linker. In some embodiments, the binding agent molecule does not comprise a linker. In some embodiments, the binding agent molecule comprises a first moiety that is not a binding moiety, e.g., an enzyme, a drug, or a toxin. In some embodiments, the binding agent molecule is a multispecific binding agent molecule, e.g., a bispecific cobinder. In some embodiments, the binding agent molecule, e.g., a cobinder, is a multimeric binding agent molecule comprising at least a third binding moiety. In some embodiments, the binding agent molecule is a multispecific CAR, e.g., a CAR, including a bispecific CAR. In some embodiments, the binding agent molecule is a conjugate, including a bispecific conjugate, e.g., a cobinder conjugated to a drug or label.

[0269] In some embodiments, the binder molecule comprises a first portion that is a non-immunoglobulin binder molecule that specifically binds to a target. These alternative binder molecules may include, for example, any of the engineered protein scaffolds known in the art. Such scaffolds may comprise one or more CDRs of an antibody against the target. Such scaffolds include, for example, anticalins, which are based on the lipocalin scaffold, a protein structure characterized by a rigid beta-barrel supporting four hypervariable loops that form the ligand-binding site. Novel binding specificities can be engineered by targeted random mutagenesis in the loop region in combination with functional display and guided selection (see, e.g., Skerra, 2008, FEBS J. 275:2677-83).Other suitable scaffolds include, for example, adnectins or monobodies based on the tenth extracellular domain of human fibronectin III (see, e.g., Koide and Koide, 2007, Methods Mol. Biol. 352:95-109), affibodies based on the Z domain of staphylococcal protein A (see, e.g., Nygren et al., 2008, FEBS J. 275:2668-76), DARPins based on ankyrin repeat proteins (see, e.g., Stumpp et al., 2008, Drug. Discov. Today 13:695-701), fynomers based on the SH3 domain of human Fyn protein kinase (see, e.g., Grabulovski et al., 2007, J. Biol. Chem. 282:3196-204), affitins based on Sac7d from Sulfolobus acidolarius (see, e.g., Krehenbrink et al., 2007, J. Biol. Chem. 282:3196-204), and affibodies based on the Z domain of staphylococcal protein A (see, e.g., Krehenbrink et al., 2007, J. Biol. Chem. 282:3196-204). al., 2008, J. Mol. Biol. 383:1058-68), affilins based on human yB-crystallin (see, e.g., Ebersbach et al., 2007, J. Mol. Biol. 372:172-85), avimers based on the A domain of membrane receptor proteins (see, e.g., Silverman et al., 2005, Biotechnol. 23:1556-61), cysteine-rich knottin peptides (see, e.g., Kolmar, 2008, FEBS J. 275:2684-90), and engineered Kunitz-type inhibitors (see, e.g., Nixon and Wood, 2006, Curr. Opin. Drug. Discov. Dev. 9:261-68). For a review, see, e.g., Gebauer and Skerra, 2009, Curr. Opin. Chem. Biol. 13:245-55.

[0270] In some embodiments, the present disclosure encompasses modification(s) of the amino acid sequence of the binding agent molecule, e.g., a cobinder. In some embodiments, the antibody or antigen-binding fragment thereof comprises modification(s) of the amino acid sequence. For example, it may be desirable to improve other biological properties of the antibody, including, but not limited to, binding affinity and / or specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Accordingly, it is contemplated that variants of the binding agent molecule, e.g., a cobinder, can be prepared. For example, variants of the cobinder can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art will understand that amino acid changes can alter post-translational processing of the cobinder, or the antibody or antigen-binding fragment thereof that is part of the cobinder, for example, changing the number or position of glycosylation sites or altering membrane anchoring properties.

[0271] In some embodiments, the binding agent molecules, e.g., cobinders, provided herein are chemically modified, e.g., by covalently attaching any type of molecule to the binding agent molecule, e.g., cobinder, or the antibody or antigen-binding fragment thereof. Such derivatives may include cobinders chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.

[0272] The alterations may be substitutions, deletions, or insertions of one or more codons encoding the polypeptide (of the cobinding agent, or of the antibody or antigen-binding fragment thereof that is part of the cobinding agent), resulting in a change in the amino acid sequence compared to the native sequence of the polypeptide. Amino acid substitutions may result in the replacement of one amino acid with another having similar structural and / or chemical properties, e.g., replacing leucine with serine, e.g., conservative amino acid substitutions. The insertions or deletions may optionally span from about 1 to 5 amino acids. In certain embodiments, the substitutions, deletions, or insertions include no more than 25 amino acid substitutions, e.g., no more than 20 amino acid substitutions, no more than 18 amino acid substitutions, no more than 15 amino acid substitutions, no more than 10 amino acid substitutions, no more than 5 amino acids, no more than 4 amino acid substitutions, no more than 3 amino acid substitutions, or no more than 2 amino acid substitutions relative to the original molecule. In certain embodiments, the substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Permissible changes can be identified by systematically inserting, deleting, or substituting amino acids within the sequence and testing the resulting variants for the activity exhibited by the full-length or mature native sequence.

[0273] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from a single residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which extends the serum half-life of the antibody.

[0274] Substantial alteration of the biological properties of the binding agent molecule, e.g., cobinder, or antibody or antigen-binding fragment thereof, is achieved by selecting substitutions that differ significantly in their impact on (a) the structure of the polypeptide backbone, e.g., sheet or helix structure, in the area of ​​substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Alternatively, conservative (e.g., within amino acid groups with similar properties and / or side chains) substitutions can be made to maintain or not significantly alter the properties. Amino acids can be classified based on similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[0275] Alternatively, naturally occurring residues may be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0276] Non-conservative substitutions involve exchanging a member of one of these classes for another. Such substituted residues may be introduced into conservative substitution sites or into remaining (non-conserved) sites. The changes can be made using methods known in the art, such as oligonucleotide-directed (site-specific) mutagenesis, alanine scanning, and PCR mutagenesis. Site-specific mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7, and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315-23), or other known techniques can be performed on cloned DNA to produce variant DNAs of the co-binding agent.

[0277] Any cysteine ​​residue not involved in maintaining the proper conformation of the binding agent molecule, e.g., a cobinder, or the antibody or antigen-binding fragment thereof, can also be substituted, for example, with another amino acid, e.g., alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, for example, cysteine ​​bond(s) can be added to a cobinder, or an antibody or antigen-binding fragment thereof that is part of the cobinder, to improve the stability of the cobinder, or the antibody or antigen-binding fragment thereof that is part of the cobinder (e.g., where the antibody is an antibody fragment such as an Fv fragment).

[0278] In some embodiments, the binding agent molecule, e.g., a cobinder, or the antibody or antigen-binding fragment thereof, is "deimmunized." In some embodiments, a "deimmunized" binding agent molecule, e.g., a cobinder, comprises a humanized or chimeric antibody, having one or more alterations in its amino acid sequence that reduce the immunogenicity of the antibody compared to the respective original, non-deimmunized antibody. One procedure for generating such antibody variants involves identifying and removing T cell epitopes from the antibody molecule. In a first step, the immunogenicity of the antibody molecule can be determined by several methods known in the art, such as in vitro measurement of T cell epitopes or in silico prediction of such epitopes. After residues important for T cell epitope function are identified, they can be mutated to remove immunogenicity and retain antibody activity. For a review, see, e.g., Jones et al., 2009, Methods in Molecular Biology 525:405-23.

[0279] In certain embodiments, covalent modifications of binder molecules, e.g., cobinders, are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of binder molecules, e.g., cobinders, with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues of the binder molecule. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0280] Other types of covalent modifications of binder molecules, e.g., co-binders, within the scope of the present disclosure include altering the native glycosylation pattern (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501, and Walsh, 2010, Drug Discov. Today 15:773-80), as well as linking the binder molecule to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, e.g., by methods described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0281] In some embodiments, binding agent molecules, e.g., co-binders, of the present disclosure may be modified to form chimeric molecules comprising the co-binder fused to another heterologous polypeptide or amino acid sequence, e.g., an epitope tag (see, e.g., Terpe, 2003, Appl. Microbiol. Biotechnol. 60:523-33) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[0282] Also provided herein in some embodiments is a fusion protein comprising a binding agent molecule provided herein, e.g., a co-binding agent, and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the binding agent molecule, e.g., a co-binding agent, is fused is useful for targeting the binding agent molecule to a particular cell.

[0283] The present disclosure also provides conjugates that include a binding agent molecule, eg, a co-binder, of the present disclosure covalently attached, eg, by a linker (eg, a synthetic linker), to one or more agents.

[0284] In some embodiments, a binding agent molecule provided herein, eg, a co-binder, is conjugated or recombinantly fused, eg, to a detectable molecule.

[0285] Such detection may be achieved, for example, by coupling the co-binding agent to a detectable substance, including various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent materials, such as, but not limited to, acridinium-based compounds or HALOTAG; radioactive materials, such as, but not limited to, iodine ( 131 I, 125 I, 123 I, and 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In, and 111 In), technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga and 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F). 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La,175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, or 117 These include, but are not limited to, Sn, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions.

[0286] Also provided herein are binding agent molecules, e.g., cobinders, recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to a heterologous protein or polypeptide (or fragment thereof, e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to generate a fusion protein, and uses thereof. In particular, provided herein are fusion proteins comprising a cobinder provided herein and a heterologous protein, polypeptide, or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide to which the antibody is fused is useful for targeting the cobinder to a particular cell type. For example, a cobinder that binds to a cell surface receptor expressed by a particular cell type can be fused or conjugated to a cytotoxic antibody or peptide.

[0287] Furthermore, the binder molecules provided herein, e.g., cobinders, can be fused to a marker or "tag" sequence, e.g., a peptide, to facilitate purification. In certain embodiments, the amino acid sequence of the marker or tag is a hexahistidine peptide, such as the tag provided in, among others, pQE vectors (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the "FLAG" tag.

[0288] Methods for fusing or conjugating moieties (including polypeptides) to binding agent molecules, e.g., co-binding agents, are known (e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); Analysis, Results, and Future Prospect of Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (Baldwin et al. eds., 1985), Thorpe et al. al., 1982, Immunol.Rev.62:119-58, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,0 No. 53, No. 5,447,851, No. 5,723,125, No. 5,783,181, No. 5,908,626, No. 5,844,095, and No. 5,112,946, EP 307,434, EP 367,166, EP 394,827, PCT Publication No. WO 91 / 06570, No. WO 96 / 04388, No. WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813, Ashkenazi et al., 1991, Proc. Natl.Acad. Sci. USA, 88:10535-39; Traunecker et al., 1988, Nature, 331:84-86; Zheng et al., 1995, J. Immunol. 154:5590-600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-41).

[0289] Fusion proteins can be generated, for example, through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be used to alter the activity of the cobinders provided herein, including, for example, cobinders with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). Co-binding agents, or antibodies provided herein to the co-binding agents, may be altered by subjecting them to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding the antibodies provided herein may be recombined with one or more components, motifs, sections, portions, domains, fragments, etc., of one or more heterologous molecules.

[0290] The binding agent molecules provided herein, eg, co-binders, can also be conjugated to a second antibody to form an antibody heteroconjugate, eg, as described in US Pat. No. 4,676,980.

[0291] The binding agent molecules provided herein, e.g., co-binders, can also be attached to solid supports, particularly useful for immunoassays or purification of the target antigen, including, but not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0292] The antibody-agent conjugates can be made using a variety of bifunctional protein coupling agents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further contemplates that co-binding agent-agent conjugates can be prepared using any suitable method disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0293] Traditional conjugation strategies for binding agent molecules, e.g., cobinders and agents, have been based on random conjugation chemistry involving the ε-amino group of Lys residues or the thiol group of Cys residues, resulting in heterogeneous conjugates. Recently developed techniques enable site-specific conjugation to polypeptides, resulting in uniform loading and avoiding subpopulations of conjugates with altered antigen binding or pharmacokinetics. These include engineering "thiomabs," which involve cysteine ​​substitutions at positions in the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332:41-52, and Junutula et al., 2008, Nature Biotechnol. 26:925-32). Alternatively, selenocysteine ​​can be cotranslationally inserted into the antibody sequence by recoding the stop codon UGA to a stop-to-selenocysteine ​​insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56, and Hofer et al., 2009, Biochemistry 48(50):12047-57).

[0294] 1. Chimeric antigen receptor (CAR) In some aspects, the present disclosure provides a chimeric antigen receptor (CAR) comprising a binder molecule, e.g., a cobinder, provided herein. In some aspects, the present disclosure provides a cell, e.g., a CAR effector cell, expressing a CAR provided herein. In some embodiments, the cell is an immune cell, e.g., a T cell. The CAR provided herein comprises (a) an extracellular domain comprising a binder molecule described herein, and (b) an intracellular signaling domain. In some embodiments, the CAR comprises a transmembrane domain located between the extracellular domain and the intracellular domain.

[0295] In some embodiments, there may be a spacer domain between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain may be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or intracellular domain of the polypeptide chain. The spacer domain may contain up to about 300 amino acids, e.g., about 10 to about 100, or about 25 to about 50 amino acids.

[0296] The transmembrane domain can be derived from either natural or synthetic sources. If the origin is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions particularly useful in the present invention can be derived from (i.e., include at least the transmembrane region(s) thereof) the α, β, δ, or γ chain of the T cell receptor, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain can be synthetic and, if so, can contain primarily hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine can be found at each end of a synthetic transmembrane domain. In some embodiments, for example, a short oligopeptide or polypeptide linker having a length of about 2 to about 10 amino acids (e.g., about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10) can form the link between the transmembrane domain and the intracellular signaling domain of the CAR. In some embodiments, the linker is a glycine-serine doublet.

[0297] In some embodiments, a transmembrane domain that is naturally associated with one of the sequences of the intracellular domain of the CAR is used. In some embodiments, the transmembrane domain can be selected or modified by amino acid substitutions that prevent the domain from binding to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interaction with other members of the receptor complex.

[0298] The intracellular signaling domain of the CAR is involved in the activation of at least one of the normal effector functions of the immune cell to which the CAR is placed. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits a signal for the effector function and instructs the cell to perform a specific function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the signal for the effector function. Therefore, the term "intracellular signaling sequence" is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transmit the signal for the effector function.

[0299] Examples of intracellular signaling domains for use in the CARs of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act together to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same function.

[0300] It is known that signals generated solely through the TCR are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (costimulatory signaling sequences).

[0301] The primary signaling sequence regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. In some embodiments, the CAR construct contains one or more ITAMs.

[0302] Examples of ITAMs containing primary signaling sequences that are particularly useful in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0303] In some embodiments, the CAR comprises a primary signaling sequence derived from CD3ζ. For example, the intracellular signaling domain of the CAR can comprise the intracellular signaling sequence of CD3ζ by itself or in combination with any other desired intracellular signaling sequence(s) useful in the context of the CAR described herein. For example, the intracellular domain of the CAR can comprise the intracellular signaling sequence of CD3ζ and a costimulatory signaling sequence. The costimulatory signaling sequence can be part of the intracellular domain of a costimulatory molecule, such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like.

[0304] In some embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling sequence of CD3ζ and the intracellular signaling sequence of CD28. In some embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling sequence of CD3ζ and the intracellular signaling sequence of 4-1BB. In some embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling sequence of CD3ζ and the intracellular signaling sequences of CD28 and 4-1BB.

[0305] Also provided herein are effector cells (such as lymphocytes, e.g., T cells) that express a CAR described herein.

[0306] Also provided is a method for producing an effector cell that expresses a CAR described herein, the method comprising introducing a vector comprising a nucleic acid encoding the CAR into the effector cell. In some embodiments, introducing the vector into the effector cell comprises transducing the effector cell with the vector. In some embodiments, introducing the vector into the effector cell comprises transfecting the effector cell with the vector. Transduction or transfection of the vector into the effector cell can be performed using any method known in the art.

[0307] 2. Immunoconjugates The binding agent molecule, in some embodiments, comprises a binding agent molecule attached to an effector molecule, e.g., an immunoconjugate (also referred to herein as an "immunoconjugate") comprising a co-binding agent. In some embodiments, the effector molecule is a therapeutic agent, e.g., a cancer therapeutic agent, which is either cytotoxic, cytostatic, or provides some therapeutic effect. In some embodiments, the effector molecule is a label capable of generating a detectable signal directly or indirectly.

[0308] In some embodiments, provided are immunoconjugates (also referred to herein as "antibody drug conjugates" or "ADCs") comprising a binding agent molecule and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic, cytostatic, or toxin that disrupts or reduces the ability of a target cell to divide. The use of ADCs for the local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Research 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278), allows for targeted delivery of the drug moiety to target cells and their intracellular accumulation therein, where systemic administration of these unconjugated therapeutic agents can result in unacceptable levels of toxicity not only to the target cells being eliminated but also to normal cells (Baldwin et al., Lancet (Mar. 15, 1986):603-605 (1986); Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal (Antibodies '84: Biological and Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506) Therefore, maximum efficacy with minimum toxicity is required.

[0309] Therapeutic agents used in immunoconjugates include, for example, daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21:183-187 (1986)). Toxins used in immunoconjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581(2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028(2000); Mandler et al., Bioconjugate Chem. 13:786-791(2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623(1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928(1998); Hinman et al., Cancer Res. 53:3336-3342 (1993). The toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to become inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0310] Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, e.g., WO 93 / 21232, published October 28, 1993.

[0311] Immunoconjugates of a binding agent molecule and one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a dolastatin, an aurostatin, a trichothecene, and CC1065, and derivatives of these toxins that have toxin activity, are also contemplated herein.

[0312] In some embodiments, an immunoconjugate is provided that includes a therapeutic agent having intracellular activity. In some embodiments, the immunoconjugate is internalized, and the therapeutic agent is a cytotoxin that blocks protein synthesis in the cell, resulting in cell death. In some embodiments, the therapeutic agent is a cytotoxin that includes a polypeptide with ribosome-inactivating activity, including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, Pseudomonas exotoxin A, and variants thereof. In some embodiments, when the therapeutic agent is a cytotoxin that includes a polypeptide with ribosome-inactivating activity, the anti-AMC immunoconjugate must be internalized after binding to the target cell in order for the protein to be cytotoxic to the cell.

[0313] In some embodiments, immunoconjugates are provided that include a therapeutic agent that acts to disrupt DNA. In some embodiments, the therapeutic agent that acts to disrupt DNA is selected from the group consisting of, for example, enediynes (e.g., calicheamicin and esperamicin) and non-enediyne small molecule agents (e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)). Other cancer therapeutic agents useful in accordance with the present application include, but are not limited to, daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidin, duocarmycin / CC-1065, and bleomycin / pepleomycin.

[0314] The present invention further contemplates immunoconjugates formed between a binding agent molecule and a compound with nucleolytic activity (eg, a ribonuclease or DNA endonuclease, eg, a deoxyribonuclease, DNase).

[0315] In some embodiments, the immunoconjugate comprises an agent that acts to disrupt tubulin. Such agents may include, for example, rhizoxin / maytansine, paclitaxel, vincristine and vinblastine, colchicine, auristatin, dolastatin 10 MMAE, and peloruside A.

[0316] In some embodiments, the immunoconjugate comprises an alkylating agent, including, for example, Asaley NSC 167780, AZQ NSC 182986, BCNU NSC 409962, busulfan NSC 750, carboxyphthalatoplatinum NSC 271674, CBDCA NSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248, cisplatin NSC 119875, clomesone NSC 338947, cyanomorpholinodoxorubicin NSC 357704, cyclodizone NSC 348948, dianhydrogalactitol NSC 132313, fluorodopan NSC 132314, fluorodopan NSC 132315, fluorodopan NSC 132316, fluorodopan NSC 132317, fluorodopan NSC 132318, fluorodopan NSC 132319 ... 73754, hepsulfam NSC 329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU NSC 95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustard NSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC 135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoin mustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812, thiotepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogen mustard NSC 34462, and Yoshi-864 NSC 102627.

[0317] In some embodiments, the cancer therapeutic agent portion of the immunoconjugate of the present application may comprise an antimitotic agent, including, but not limited to, allocolchicine NSC 406042, halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (derived from NG-auristatin), maytansine NSC 153858, rhizoxin NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine ​​NSC 83265, vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574.

[0318] In some embodiments, the immunoconjugate comprises a topoisomerase I inhibitor, including, but not limited to, camptothecin NSC 94600, camptothecin Na salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971, camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501, camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610459, camptothecin derivative NSC 606499, camptothecin derivative NSC 610456, camptothecin derivative NSC 364830, camptothecin derivative NSC 606497, and morpholinodoxorubicin NSC 354646.

[0319] In some embodiments, the immunoconjugate comprises a topoisomerase II inhibitor, including, but not limited to, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivatives NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCl NSC 337766, daunorubicin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC 269148, N,N-dibenzyldaunomycin NSC 268242, oxantrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC 122819, and VP-16 NSC 141540.

[0320] In some embodiments, the immunoconjugate comprises an RNA or DNA antimetabolite, including, but not limited to, L-alanosine NSC 153353, 5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC 163501, aminopterin derivative NSC 132483, aminopterin derivative NSC 184692, aminopterin derivative NSC 134033, antifol NSC 633713, antifol NSC 623017, Baker's soluble antifol NSC 139105, dichloroallyl lawsone NSC 126771, brequinar NSC 368390, ftorafur (prodrug) NSC 148958, 5,6-dihydro-5-azacytidine NSC 264880, methotrexate NSC 740, methotrexate derivative NSC 174121, N-(phosphonoacetyl)-L-aspartate (PALA) NSC 224131, pyrazofurin NSC 143095, trimetrexate NSC 352122, 3-HP NSC 95678, 2'-deoxy-5-fluorouridine NSC 27640, 5-HP NSC 107392, α-TGDR NSC 71851, aphidicolin glycinate NSC 303812, ara-C NSC 63878, 5-aza-2'-deoxycytidine NSC 127716, β-TGDR NSC 71261, cyclocytidine NSC 145668, guanazole NSC 1895, hydroxyurea NSC 32065, inosine glycodialdehyde NSC 118994, macbecin II NSC 330500, pyrazoloimidazole NSC 51143, thioguanine NSC 752, and thiopurine NSC 755.

[0321] In some embodiments, the immunoconjugate comprises a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm,212 Bi, 32 P, 212 These include radioactive isotopes of Pb and Lu.

[0322] In some embodiments, the binding agent molecule can be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pre-targeting, where the binding agent molecule-receptor conjugate is administered to a patient, after which unbound conjugate is removed from the circulation using a clearing agent, and then a "ligand" (e.g., avidin) conjugated to a cytotoxic drug (e.g., a radionucleotide) is administered.

[0323] In some embodiments, the immunoconjugate may comprise a binding agent molecule conjugated to a prodrug-activating enzyme. In some such embodiments, the prodrug-activating enzyme converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) to an active drug, such as an anti-cancer drug. Enzymes that can be conjugated to an antibody include alkaline phosphatase, useful for converting phosphate-containing prodrugs to the free drug; arylsulfatases, useful for converting sulfate-containing prodrugs to the free drug; cytosine deaminases, useful for converting the non-toxic 5-fluorocytosine to the anti-cancer drug, 5-fluorouracil; proteases useful for converting peptide-containing prodrugs to the free drug, such as Serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (e.g., cathepsins B and L); and enzymes containing D-amino acid substituents. Examples of enzymes useful for converting prodrugs containing hydroxybenzoates to free drugs include, but are not limited to, D-alanylcarboxypeptidases, useful for converting prodrugs containing hydroxybenzoates to free drugs; carbohydrate-cleaving enzymes, such as β-galactosidase a...

Claims

1. A co-binding agent comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, the second binding moiety is a second antibody moiety comprising an antibody variable domain with an N-terminal truncation (an "N-terminally truncated antibody variable domain"); The co-binding agent, wherein the first binding moiety is connected to the second binding moiety through the N-terminus of the N-terminally truncated antibody variable domain, optionally via a linker.

2. binds to said second target site with an affinity at least three times that of a control cobinder comprising an antibody variable domain without said N-terminal truncation; and / or 10. The cobinder of claim 1, which binds to a target molecule with an affinity at least three times that of a control cobinder comprising an antibody variable domain without said N-terminal truncation.

3. the first binding moiety is a first antibody moiety; the first antibody portion is selected from the group consisting of a Fab, Fv, scFv, dsFv, Fab', or a (Fab')2 fragment; or the first antibody portion is a single domain antibody; and / or the second antibody portion comprising: selected from the group consisting of Fab, Fv, scFv, dsFv, Fab', or (Fab')2 fragments; or The cobinding agent of claim 1 which is a single domain antibody.

4. The cobinding agent of claim 1 , wherein the N-terminal truncated antibody variable domain is a truncated VH or a truncated VL domain.

5. The N-terminal truncated antibody variable domain is a truncated V Ｈ The cobinding agent of claim 1 which is an H domain.

6. The first binding moiety is a first V Ｈ H domain, and the second binding moiety comprises a second V H domain having an N-terminal truncation. Ｈ H domain ("truncated V Ｈ 6. The cobinding agent according to claim 1 , comprising a nucleotide sequence ("H domain"), The first V Ｈ The C-terminus of the H domain is the second V Ｈ The co-binder is connected to the N-terminus of the H domain via a linker.

7. The cobinding agent of any one of claims 1 to 5, wherein the N-terminal truncation of the N-terminal truncated antibody variable domain is from about 1 to about 25 amino acids.

8. The cobinding agent according to any one of claims 1 to 5, wherein the linker is a peptide linker.

9. The C-terminal three amino acids of the peptide linker directly connected to the N-terminal truncated antibody variable domain are １ -X ２ -X ３ and X １ is V, L, W, P, S, G, K, D, F, M, T, N, or R; X ２ is V, A, L, S, G, R, K, M, C, F, T, P, or E; X ３ is G, or the C-terminal three amino acids of the peptide linker directly connecting the antibody variable domain of the second binding moiety are X 1 -X 2 -X 3 ; X 1 is any amino acid; X2 is K, R, Y, M, G, or N; X 3 is R, G, Y, or P; or the C-terminal amino acid of the peptide linker directly connecting said N-terminal truncated antibody variable domain is R, G, Y, or P; or the C-terminal amino acid of the peptide linker directly connected to the N-terminal truncated antibody variable domain is G; The cobinder of claim 8.

10. The linker is ｘ S ｙ ) ｎ where x is 1 to 5, y is 0 to 5, and n is 1 or greater; or the linker comprises [EAAAK] n or [EEEEKKKK] n , where n is 1 or greater; or The linker comprises [AP] n , where n is 1 or greater; The cobinder of claim 8.

11. The cobinding agent of any one of claims 1 to 5, further comprising a third binding moiety that specifically recognizes a third target site.

12. The cobinding agent of any one of claims 1 to 5, which is an antibody comprising an Fc region.

13. 6. The cobinding agent of any one of claims 1 to 5, which is a chimeric antigen receptor ("CAR").

14. A co-binding agent comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, the second binding moiety is a second antibody moiety comprising an antibody variable domain; the first binding moiety is connected to the second binding moiety through the N-terminus of the antibody variable domain via a peptide linker; The C-terminal three amino acids of the peptide linker that directly connects the antibody variable domain of the second binding moiety are X １ -X ２ -X ３ and X １ is any amino acid, X ２ is K, R, Y, M, G, or N; X ３ is R, G, Y, or P.

15. 1. A library comprising a plurality of co-binding agents or a plurality of polynucleotides encoding a plurality of co-binding agents, each co-binding agent comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second target site, said second binding moiety being a second antibody moiety comprising an antibody variable domain having an N-terminal truncation (an "N-terminally truncated antibody variable domain"), said first binding moiety being connected to said second binding moiety through the N-terminus of said antibody variable domain via a peptide linker, and at least two co-binding agents comprised in said library differ from each other in terms of said peptide linker sequence.

16. The library of claim 15, wherein the first binding moiety comprises a first VHH domain and / or the second binding moiety comprises a second VHH domain.

17. The library of claim 15, wherein the antibody variable domain of the second binding moiety has an N-terminal truncation (an "N-terminally truncated antibody variable domain").

18. 1. A method of screening for a co-binder that specifically binds to a second target site with a desired affinity, comprising: (1) contacting the library according to claim 15 with a target molecule comprising the second target site to form a complex between the target molecule and a co-binding agent that specifically binds to the target molecule; and (2) identifying a co-binder that binds to the second target site with the desired affinity.

19. 1. A method for screening for a co-binder that specifically binds to a target molecule with a desired affinity, comprising: (1) contacting the library according to claim 15 with the target molecule to form a complex between the target molecule and a co-binding agent that specifically binds to the target molecule; and (2) identifying a co-binder that binds to the target molecule with the desired affinity.

20. 1. A method for increasing the binding affinity of a control cobinder that specifically binds to a target molecule, the control cobinder comprising a first binding moiety that specifically recognizes a first target site and a second binding moiety that specifically recognizes a second binding target site, the second binding moiety being a second antibody moiety comprising an antibody variable domain, the first binding moiety being connected to the second binding moiety through an N-terminus of the antibody variable domain via a linker, the control cobinder comprising a full-length antibody variable domain, the binding affinity of the control cobinder to the second target site being lower than that of the second antibody moiety in the free state, the method comprising obtaining a cobinder having an N-terminal truncation in the antibody variable domain of the second antibody moiety compared to the control cobinder.

Images