CD3 binding proteins and methods of use thereof

Abstract

The present disclosure includes target binding proteins having heavy and light chain variable domains with specific sequences that bind to CD3 epsilon, the heavy and light chain variable domains being arranged within one or more polypeptides. The present disclosure also includes compositions, nucleic acids, vectors, cells, and methods of making and using the proteins, compositions, and nucleic acids. In another aspect, the present disclosure provides a composition comprising the target binding protein herein and a carrier. In some embodiments, the composition is a pharmaceutical composition and the carrier is a pharma- ceutically acceptable carrier.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 326,668, filed April 1, 2022, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing The Sequence Listing submitted with this application by EFS, entitled "4862-124PCT.xml", was created on March 31, 2023, is 643,441 bytes in size, and is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to the field of biotechnology, and more specifically to target binding molecules, including molecules that bind to CD3. [Background technology]

[0004] Anti-CD3 epsilon (CD3ε) antibodies have been used for several years and are designed to function in a variety of roles and combat many diseases. Many of these anti-CD3ε antibodies are derived from a very small number of variants, including SP-34. Additional anti-CD3ε binding proteins are desired to further develop the efficacy, safety, and manufacturability of anti-CD3ε molecules. There is a need for anti-CD3ε binding proteins with different binding affinities, improved manufacturability, and improved stability compared to SP-34 antibodies to treat certain diseases, and related therapies with tailored CD3ε binding affinities. There is also a need for anti-CD3ε binding proteins that exhibit improved binding affinities, stability, and manufacturability in the context of molecular structures other than full-length antibodies, including in the context of both multivalent and monovalent binding proteins. Summary of the Invention

[0005] The present disclosure provides target binding proteins having a CD3ε binding domain, as well as related compositions and methods.

[0006] In one aspect, the disclosure provides a target binding protein comprising a heavy chain variable domain comprising: a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the sequence of TYAMN (SEQ ID NO:3), a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the sequence of HGNFGNSYVSWX1AY (SEQ ID NO:6), where X1 is W or F; a variable light chain complementarity determining region 1 (VL CDR1) comprising the sequence of X2SSTGAVTTSNYX3N (SEQ ID NO:10), where X2 is R or G and X3 is P or V; and a light chain variable domain comprising a heavy chain variable domain, a light chain variable domain comprising a heavy chain variable domain (HcCDR2), and a variable light chain complementarity determining region 3 (VL CDR3) comprising the sequence of X4LWYSNX5WV (SEQ ID NO: 15), where X4 is V or I and X5 is R or L, wherein the heavy chain variable domain and the light chain variable domain are arranged within one or more polypeptides, and wherein the target binding protein specifically binds to CD3 epsilon.

[0007] In some embodiments, the VH CDR1 comprises the sequence of TYAMN (SEQ ID NO:3), the VH CDR2 comprises the sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises the sequence of HGNFGNSYVSWWAY (SEQ ID NO:7) or HGNFGNSYVSWFAY (SEQ ID NO:8), the VL CDR1 comprises the sequence of RSSTGAVTTSNYPN (SEQ ID NO:11), RSSTGAVTTSNYVN (SEQ ID NO:12), or GSSTGAVTTSNYVN (SEQ ID NO:13), the VL CDR2 comprises the sequence of GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises the sequence of VLWYSNRWV (SEQ ID NO:16), VLWYSNLWV (SEQ ID NO:17), or ILWYSNRWV (SEQ ID NO:18).

[0008] In some embodiments, the VH CDR1 comprises TYAMN (SEQ ID NO:3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), the VL CDR1 comprises RSSTGAVTTSNYPN (SEQ ID NO:11), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16); or the VH CDR1 comprises TYAMN (SEQ ID NO:3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), the VL CDR1 comprises GSSTGAVTTSNYVN (SEQ ID NO:13), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16). and VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16), or VH CDR1 comprises TYAMN (SEQ ID NO:3), VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 comprises HGNFGNSYVSWFAY (SEQ ID NO:8), VL CDR1 comprises GSSTGAVTTSNYVN (SEQ ID NO:13), VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16), or VH CDR1 comprises TYAMN (SEQ ID NO:3), VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), VL CDR1 comprises RSSTGAVTTSNYVN (SEQ ID NO:12), VL CDR2 comprises GTNKRAP, or VH CDR1 comprises TYAMN (SEQ ID NO:3), VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), VL CDR1 comprises GSSTGAVTTSNYVN (SEQ ID NO:13), VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and VL CDR3 comprises VLWYSNLWV (SEQ ID NO:17);CDR1 comprises TYAMN (SEQ ID NO:3), VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), VL CDR1 comprises RSSTGAVTTSNYVN (SEQ ID NO:12), VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16).

[0009] In some embodiments, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO:11), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the VH CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises the VL CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 46, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 49.

[0010] In some embodiments, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the VH CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises the VL CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 64. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 64.

[0011] In some embodiments, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWFAY (SEQ ID NO:8), and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the VH CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 128, and the light chain variable domain comprises the VL CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 122. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 128, and the light chain variable domain comprises the sequence of SEQ ID NO: 122.

[0012] In some embodiments, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18). In some embodiments, the heavy chain variable domain comprises the VH CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises the VL CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 113. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 113.

[0013] In some embodiments, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO:17). In some embodiments, the heavy chain variable domain comprises the VH CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises the VL CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 98. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 98.

[0014] In some embodiments, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the VH CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises the VL CDRs above and comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 107. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 107.

[0015] In another aspect, the disclosure provides a target binding protein, the target binding protein comprising a heavy chain variable domain comprising a sequence of: EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWX1AYWGX6GTLVTVSS (SEQ ID NO: 20); and a light chain variable domain comprising the sequence of: GX9QPEDEAEYYCX4LWYSNX5WVFGGGTKLTVL (SEQ ID NO: 21), wherein X1 is a) W or F, X2 is R or G, X3 is P or V, X4 is V or I, and X5 is R or L, b) X6 is Q, X7 is Q, or X6 is C and X7 is C, c) J is L if X9 is V, or J is I if X9 is A, and d) the target binding protein specifically binds CD3 epsilon.

[0016] In some embodiments, X6 is Q and X7 is Q. In some embodiments, X6 is C and X7 is C.

[0017] In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO:46 and the light chain variable domain comprises the sequence of SEQ ID NO:49, or the heavy chain variable domain comprises the sequence of SEQ ID NO:46 and the light chain variable domain comprises the sequence of SEQ ID NO:64, or the heavy chain variable domain comprises the sequence of SEQ ID NO:46 and the light chain variable domain comprises the sequence of SEQ ID NO:113, or the heavy chain variable domain comprises the sequence of SEQ ID NO:46 and the light chain variable domain comprises the sequence of SEQ ID NO:98, or the heavy chain variable domain comprises the sequence of SEQ ID NO:46 and the light chain variable domain comprises the sequence of SEQ ID NO:107, or the heavy chain variable domain comprises the sequence of SEQ ID NO:128 and the light chain variable domain comprises the sequence of SEQ ID NO:122.

[0018] In some embodiments, the heavy chain variable domain and the light chain variable domain are located within the same polypeptide. In some embodiments, the heavy chain variable domain and the light chain variable domain are linked via a linker. In some embodiments, the linker has a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids.

[0019] In some embodiments, the target binding protein comprises the sequence of SEQ ID NO: 50, 28, 114, 99, 108, or 32. In some embodiments, the target binding protein comprises a single chain variable fragment (scFv). In some embodiments, the target binding protein is selected from the group consisting of BiTE, (scFv)2, NANOBODY®, nanobody-HSA VHH-scAb, VHH-Fab, dual scFab, F(ab')2, diabody, CROSSMAB®, DAF(2-in-1), DAE(4-in-1), DUTAMAB®, DT-IgG, knobs-in-hole common light chain, knobs-in-hole assembly, charge pair, Fab-arm exchange, SEED body, LUZ-Y, FcAb, kl-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, ZYBODY™, DVI-IgG, diabody-CH3, triple body, mini antibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2,

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[0020] In some embodiments, the target binding protein is or comprises an IgG, IgM, IgA, IgE, or IgD antibody, or a fragment thereof. In some embodiments, the target binding protein is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the target binding protein is humanized.

[0021] In some embodiments, the target binding protein further comprises a masking moiety that inhibits the target binding protein from binding to CD3 in an inactive state. In some embodiments, the masking moiety is attached to the target binding protein via a cleavable moiety (directly or indirectly, e.g., via one or more linkers), and the cleavable moiety is a substrate for a protease. In some embodiments, the protease is ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartic protease, BACE, renin, aspartic cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, Caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, cysteine ​​cathepsin, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteinase, cruzipain, legumain, otubain-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, metalloproteinase, meprin, nep Relysin, PSMA, BMP-1, MMP, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine protease, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXII a, elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane protein, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, or TMPRSS4.

[0022] In some embodiments, the target binding protein further comprises a second target binding domain that specifically binds to a second target.

[0023] In some embodiments, the heavy chain variable domain and / or the light chain variable domain are conjugated to a toxin, radioisotope, small molecule, diagnostic agent, therapeutic macromolecule, targeting moiety, or detectable moiety via a conjugate moiety. In some embodiments, the conjugate moiety is cleavable by a protease. In some embodiments, the conjugate moiety is not cleavable by a protease.

[0024] In another aspect, the disclosure provides a composition comprising a target binding protein herein and a carrier, in some embodiments, the composition is a pharmaceutical composition and the carrier is a pharma- ceutically acceptable carrier.

[0025] In another aspect, the disclosure provides a container, vial, syringe, injector pen, or kit comprising at least one dose of a composition herein.

[0026] In another aspect, the disclosure provides a nucleic acid comprising a sequence encoding a target binding protein herein.

[0027] In another aspect, the disclosure provides a vector comprising a nucleic acid herein.

[0028] In another aspect, the disclosure provides a cell comprising a nucleic acid herein or a vector herein.

[0029] In another aspect, the disclosure provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a target binding protein herein, or a composition herein, hi some embodiments, the subject has been identified or diagnosed as having cancer.

[0030] In another aspect, the disclosure provides a method of producing a target binding protein, comprising culturing a cell herein in a culture medium under conditions sufficient to produce the target binding protein, and recovering the target binding protein from the cell or culture medium. In some embodiments, the method further comprises isolating the recovered target binding protein from the cell or culture medium. In some embodiments, the method further comprises formulating the target binding protein into a pharmaceutical composition.

[0031] The features and advantages of the present invention will be understood by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention may be utilized and the accompanying drawings of which: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Provided herein are target binding proteins that specifically bind to cluster of differentiation 3 (CD3) (e.g., CD3 epsilon). In one aspect, the target binding proteins can include a heavy chain variable domain and a light chain variable domain that form a target binding domain that specifically binds to CD3 (e.g., CD3 epsilon). In some embodiments, compared to known anti-CD3 proteins produced from non-human species (e.g., sp34 mouse anti-human CD3 antibody), the CD3 binding molecules herein include one or more mutations in their amino acid sequence that render them more human, e.g., increase their similarity to CD3 binding molecules naturally produced in humans.

[0033] In some embodiments, the target binding protein can be a single chain protein, such as a single chain antibody. For example, the single chain antibody can be a single chain fragment variable (scFv) antibody. In some embodiments, the target binding protein can be a multi-chain protein (e.g., a multi-chain antibody) that includes a protein complex formed by multiple polypeptides.

[0034] In some embodiments, the target binding protein can be an activatable molecule, e.g., an activatable CD3 binding molecule, that includes a masking moiety linked to the CD3 binding domain via a cleavable moiety (directly or indirectly, e.g., via one or more linkers). The cleavable moiety can be cleaved under certain conditions (e.g., when exposed to proteases in the tumor microenvironment), thereby releasing the masking moiety from the CD3 binding domain.

[0035] In some embodiments, the target binding protein can be a multispecific (e.g., bispecific) binding protein that binds to one or more additional targets other than CD3. For example, a multispecific protein can specifically bind to CD3 and a tumor-associated antigen, such as HER2, Jagged, EGFR, etc.

[0036] Also provided herein are related compositions, kits, nucleic acids, vectors, and recombinant cells, as well as related methods, including methods of using and producing any of the target binding proteins described herein.

[0037] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in this disclosure are described herein, and other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0038] The terms "a" and "an" refer to one or more (i.e., to at least one) of the grammatical object of the article. As an example, "a cell" includes one or more cells.

[0039] As used herein, the terms "about" and "approximately," when used to modify an amount specified in a numerical value or range, indicate reasonable deviations from that numerical value as well as values ​​known to one of ordinary skill in the art, for example, ±20%, ±10%, or ±5%, as appropriate, within the intended meaning of the recited value.

[0040] Concentrations, amounts, and other numerical data may be expressed or presented in a range format herein. It should be understood that such range formats are used for convenience and brevity only, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limitations, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. As an illustration, a numerical range of "about 0.01 to 2.0" should be interpreted to include not only the explicitly recited values ​​of about 0.01 to about 2.0, but also the individual values ​​and subranges within the stated range. Thus, this numerical range includes individual values ​​such as 0.5, 0.7, and 1.5, as well as subranges such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5. Moreover, such interpretation should be applied regardless of the breadth of the range or the properties being described. Furthermore, it should be noted that all percentages are by weight unless otherwise specified.

[0041] In understanding the scope of the present disclosure, the terms "including" or "comprising" and their derivatives, as used herein, are intended to be open-ended terms specifying the presence of the stated features, elements, components, groups, integers, and / or steps, but not excluding the presence of other unstated features, elements, components, groups, integers, and / or steps. The above also applies to words of similar meaning, such as "including" and "having" and their derivatives. As used herein, the term "consisting" and its derivatives, are intended to be limiting terms specifying the presence of the stated features, elements, components, groups, integers, and / or steps, but excluding the presence of other unstated features, elements, components, groups, integers, and / or steps. As used herein, the term "consisting essentially of" is intended to specify the presence of the described features, elements, components, groups, integers, and / or steps, as well as those that do not substantially affect the basic and novel characteristic(s) of the features, elements, components, groups, integers, and / or steps. Reference to any one of these transition terms (i.e., "comprises," "consists of," or "consists essentially of") is understood to provide direct support for substituting any of the other transition terms not specifically used. For example, the modification of the term "comprises" to "consistes essentially of" or "consists of" finds direct support for any element disclosed throughout this disclosure to be so defined. Based on this definition, any element disclosed herein or incorporated by reference may be included or excluded from the claimed invention.

[0042] As used herein, for convenience, a plurality of compounds, elements or steps may be presented in common lists. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, the individual members of such lists should not be construed as being effectively equivalent to other members of the same list solely based on their presentation in a common group, absent any indication to the contrary.

[0043] The term "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more specific manner.

[0044] Additionally, certain molecules, constructs, compositions, elements, moieties, excipients, diseases, conditions, properties, steps, etc. may be discussed in the context of a particular embodiment or aspect of this disclosure, or in a separate paragraph or section. It should be understood that this is merely for convenience and brevity, and that any such disclosure is intended to be equally applicable to and combined with any other embodiment or aspect found in this disclosure and claims that form the invention in this application and claims as of the filing date. For example, a listing of a construct, molecule, method step, kit, or composition described with respect to a construct, composition, or method is intended to find direct support for the relevant embodiment of the construct, composition, formulation, and method described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0045] Target-binding proteins The target binding proteins herein may comprise a heavy chain variable domain and a light chain variable domain that form a target binding domain that specifically binds to CD3 (e.g., CD3 epsilon). An exemplary CD3 epsilon molecule that may be bound by the proteins herein is wild-type human CD3 epsilon (SEQ ID NO: 1).

[0046] In some embodiments, the heavy chain variable domain and the light chain variable domain are located in the same polypeptide. In certain embodiments, the heavy chain variable domain and the light chain variable domain are linked together by one or more linkers. The linkers can be peptide linkers as described in the linker section below. In some examples, the target binding domain in the single chain polypeptide can be an scFv. Alternatively, the heavy chain variable domain and the light chain variable domain are located in two different polypeptides.

[0047] In some embodiments, the target binding protein can be a protein complex that includes multiple polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides). In some examples, some or all of the multiple polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides) can be identical. In some examples, each of the multiple polypeptides in the target binding complex is different from the other polypeptides.

[0048] The target binding domain may be an antibody or fragment thereof, a VH domain, a VHH domain, a VNAR domain, and a single chain fragment variable (scFv), a BiTE or a component thereof, (scFv)2, NANOBODY®, nanobody-HSA, VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, CROSSMAB®, DAF(2-in-1), DAE(4-in-1), DUTAM AB®, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab-arm exchange, SEED body, LUZ-Y, FcAb, kl-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, ZYBODY™, DVI-IgG, diabody-CH3, triple body, mini antibody, mini body, TriBi mini body, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2,

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number

[0049] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more target binding domains that specifically bind to an antigen or epitope. Examples of antibodies include intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multispecific antibodies. An example of a target binding domain is the V H -V L It is formed by a dimer. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0050] The "light chain" contains one variable domain (VL) and one constant domain (CL). There are two different light chains, called kappa or lambda. The "heavy chain" consists of one variable domain (VH) and three constant region domains (CH1, CH2, CH3). There are five main classes or isotypes of heavy chains, some of which have multiple subtypes, which determine the functional activity of the antibody molecule. The five major classes of immunoglobulins are Immunoglobulin M (IgM), Immunoglobulin D (IgD), Immunoglobulin G (IgG), Immunoglobulin A (IgA), and Immunoglobulin E (IgE). IgG is by far the most abundant immunoglobulin and has several subclasses (IgG1, IgG2, IgG3, and IgG4 in humans).

[0051] The "fragment antigen binding" (Fab) comprises an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment is formed when an antibody is cleaved by pepsin (or otherwise cleaved) below the hinge region, in which case the two fragment target binding domains (Fab) of the antibody molecule remain linked. The F(ab')2 fragment comprises two intact light chains paired with the two VH and CH1 domains of the heavy chain joined together by the hinge region. The "fragment crystallizable" (Fc) fragment (also referred to herein as the Fc domain) corresponds to the paired CH2 and CH3 domains and is the part of the antibody molecule that interacts with effector molecules and cells. The functional difference between the heavy chain isotypes is mainly in the Fc fragment. The "single chain fragment variable" (scFv) comprises only the variable domain of the light chain (VL) linked by a peptide stretch to the variable domain of the heavy chain (VH). The name single chain Fv comes from the fragment variable. A "hinge region" or "interdomain" is a flexible stretch of amino acids that joins or links the Fab fragment to the Fc domain. A "synthetic hinge region" is an amino acid sequence that joins or links the Fab fragment to the Fc domain.

[0052] An "Fv" fragment comprises a non-covalent dimer of one heavy chain variable domain and one light chain variable domain. "Dual variable domain immunoglobulin G" or "DVD-IgG" refers to a multivalent and multispecific target binding protein as described, for example, in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Patent Nos. 7,612,181, 8,258,268, 8,586,714, 8,716,450, 8,722,855, 8,735,546, and 8,822,645, each of which is incorporated by reference in its entirety. Examples of DART are described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0053] In some embodiments, the target binding protein may be a mouse, rat, rabbit, goat, camel, donkey, primate, chimeric, human, or humanized protein. In one example, the target binding protein may be a human protein. In one example, the target binding protein may be a humanized (e.g., fully human) protein.

[0054] The term "humanized" refers to target binding proteins having amino acid sequences that include VH and VL region sequences derived from a reference protein produced in a non-human species (e.g., mouse), but also include modifications in those sequences intended to make them more "human-like", i.e., more similar to human germline variable sequences, as compared to the reference protein. In some embodiments, a "humanized" target binding protein is a protein that immunospecifically binds to an antigen of interest, has framework (FR) regions having amino acid sequences substantially as those of a human protein, and complementarity determining regions (CDRs) having amino acid sequences substantially as those of a non-human protein, and comprises humanized VH and VL regions.

[0055] The term "human protein" is intended to include target binding proteins having variable and constant regions generated, assembled, or derived from human immunoglobulin sequences. In some embodiments, a target binding protein may be considered to be "human" even if its amino acid sequence includes residues or elements, e.g., in one or more CDRs, that are not encoded by human germline immunoglobulin sequences (including sequence variations that may have been (originally) introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0056] In some embodiments, in the target binding protein, the heavy chain variable domain may comprise three complementarity determining regions (CDRs) (VH CDR1, VH CDR2, and VH CDR3) and the light chain variable domain may comprise three CDRs (VL CDR1, VL CDR2, and VL CDR3). VH CDR1 may comprise the sequence of SEQ ID NO:3. VH CDR2 may comprise the sequence of SEQ ID NO:5. VH CDR3 may comprise the sequence of SEQ ID NO:6. For example, VH CDR3 may comprise the sequence of SEQ ID NO:7 or 8. VL CDR1 may comprise the sequence of SEQ ID NO:10. For example, VL CDR1 may comprise the sequence of SEQ ID NO:11, 12, or 13. VL CDR2 may comprise the sequence of SEQ ID NO:14. VL CDR3 may comprise the sequence of SEQ ID NO:15. For example, VL CDR3 may comprise the sequence of SEQ ID NO:16, 17, or 18.

[0057] The target binding protein can comprise a heavy chain variable domain comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable domain comprising a VL CDR1, a VL CDR2, and a VL CDR3. In some examples, in the target binding protein, the VH CDR1 may comprise the sequence of TYAMN (SEQ ID NO:3), the VH CDR2 may comprise the sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 may comprise the sequence of HGNFGNSYVSWWAY (SEQ ID NO:7) or HGNFGNSYVSWFAY (SEQ ID NO:8), the VL CDR1 may comprise the sequence of RSSTGAVTTSNYPN (SEQ ID NO:11), RSSTGAVTTSNYVN (SEQ ID NO:12), or GSSTGAVTTSNYVN (SEQ ID NO:13), the VL CDR2 may comprise the sequence of GTNKRAP (SEQ ID NO:14), and the VL CDR3 may comprise the sequence of VLWYSNRWV (SEQ ID NO:16), VLWYSNLWV (SEQ ID NO:17), or ILWYSNRWV (SEQ ID NO:18).

[0058] In one example, in a target binding protein, VH CDR1 may comprise TYAMN (SEQ ID NO:3), VH CDR2 may comprise RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 may comprise HGNFGNSYVSWWAY (SEQ ID NO:7), VL CDR1 may comprise RSSTGAVTTSNYPN (SEQ ID NO:11), VL CDR2 may comprise GTNKRAP (SEQ ID NO:14), and VL CDR3 may comprise VLWYSNRWV (SEQ ID NO:16). In another example, VH CDR1 may comprise TYAMN (SEQ ID NO:3), VH CDR2 may comprise RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 may comprise HGNFGNSYVSWWAY (SEQ ID NO:7), VL CDR1 may comprise GSSTGAVTTSNYVN (SEQ ID NO:13), VL CDR2 may comprise GTNKRAP (SEQ ID NO:14), and VL CDR3 may comprise VLWYSNRWV (SEQ ID NO:16). In another example, VH CDR1 may comprise TYAMN (SEQ ID NO:3), VH CDR2 may comprise RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 may comprise HGNFGNSYVSWFAY (SEQ ID NO:8), VL CDR1 may comprise GSSTGAVTTSNYVN (SEQ ID NO:13), VL CDR2 may comprise GTNKRAP (SEQ ID NO:14), and VL CDR3 may comprise VLWYSNRWV (SEQ ID NO:16). In another example, the VH CDR1 may comprise TYAMN (SEQ ID NO:3), the VH CDR2 may comprise RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 may comprise HGNFGNSYVSWWAY (SEQ ID NO:7), the VL CDR1 may comprise RSSTGAVTTSNYVN (SEQ ID NO:12), the VL CDR2 may comprise GTNKRAP (SEQ ID NO:14), and the VL CDR3 may comprise ILWYSNRWV (SEQ ID NO:18).In another example, VH CDR1 may comprise TYAMN (SEQ ID NO:3), VH CDR2 may comprise RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 may comprise HGNFGNSYVSWWAY (SEQ ID NO:7), VL CDR1 may comprise GSSTGAVTTSNYVN (SEQ ID NO:13), VL CDR2 may comprise GTNKRAP (SEQ ID NO:14), and VL CDR3 may comprise VLWYSNLWV (SEQ ID NO:17). In another example, VH CDR1 may comprise TYAMN (SEQ ID NO:3), VH CDR2 may comprise RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 may comprise HGNFGNSYVSWWAY (SEQ ID NO:7), VL CDR1 may comprise RSSTGAVTTSNYVN (SEQ ID NO:12), VL CDR2 may comprise GTNKRAP (SEQ ID NO:14), and VL CDR3 may comprise VLWYSNRWV (SEQ ID NO:16).

[0059] Additional examples of VH CDR1 include the sequence of amino acids at positions 31-35 of the heavy chain variable domain of Table 9B. Additional examples of VH CDR2 include the sequence of amino acids at positions 50-68 of the heavy chain variable domain of Table 9B. Additional examples of VH CDR3 include the sequence of amino acids at positions 101-114 of the heavy chain variable domain of Table 9B. Additional examples of VL CDR1 include the sequence of amino acids at positions 23-36 of the light chain variable domain of Table 9B. Additional examples of VH CDR2 include the sequence of amino acids at positions 52-58 of the light chain variable domain of Table 9B. Additional examples of VH CDR3 include the sequence of amino acids at positions 91-99 of the light chain variable domain of Table 9B.

[0060] In some embodiments, in the target binding protein, the heavy chain variable domain may comprise the sequence of SEQ ID NO:20 and the light chain variable domain may comprise the sequence of SEQ ID NO:21.

[0061] In some examples, the heavy chain variable domain and the light chain variable domain may contain one or more cysteines that form a disulfide bond. In some examples, the disulfide bond does not interfere with the target binding of the molecule, but may improve stability. For example, the heavy chain domain may include the sequence of SEQ ID NO: 24, and the light chain domain may include the sequence of SEQ ID NO: 25. In some examples, the heavy and light chain variable domains do not have such cysteine ​​mutations. For example, the heavy chain domain may include the sequence of SEQ ID NO: 22, and the light chain domain may include the sequence of SEQ ID NO: 23.

[0062] For example, the heavy chain variable domain may comprise the sequence of SEQ ID NO: 46, or 128, and the light chain variable domain within the light chain variable domain may comprise the sequence of SEQ ID NO: 49, 64, 113, 98, 107, or 122. In some examples, the heavy chain variable domain comprises a VH CDR1 comprising the sequence of SEQ ID NO:3, a VH CDR2 comprising the sequence of SEQ ID NO:5, and a VH CDR3 comprising the sequence of SEQ ID NO:6, 7, or 8; the heavy chain variable domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NOs:46 or 128; and the light chain variable domain comprises an LH CDR1 comprising the sequence of SEQ ID NO:10, 11, 12, or 13, an LH CDR2 comprising the sequence of SEQ ID NO:14, and an LH CDR3 comprising the sequence of SEQ ID NO:15, 16, 17, or 18. The light chain variable domain, including the CDR3, comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NOs: 49, 64, 113, 98, 107, or 122. In one example, the heavy chain variable domain comprises a VH CDR1 comprising the sequence of SEQ ID NO:3, a VH CDR2 comprising the sequence of SEQ ID NO:5, and a VH CDR3 comprising the sequence of SEQ ID NO:6, 7, or 8, the heavy chain variable domain comprises a sequence at least 95% identical to any one of SEQ ID NO:46 or 128, the light chain variable domain comprises an LH CDR1 comprising the sequence of SEQ ID NO:10, 11, 12, or 13, an LH CDR2 comprising the sequence of SEQ ID NO:14, and an LH CDR3 comprising the sequence of SEQ ID NO:15, 16, 17, or 18, and the light chain variable domain comprises a sequence at least 95% identical to any one of SEQ ID NO:49, 64, 113, 98, 107, or 122. In one example, the heavy chain variable domain comprises the sequence of SEQ ID NO:46 and the light chain variable domain comprises the sequence of SEQ ID NO:49.In some examples, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), wherein the heavy chain variable domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:46, and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO:11), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). The light chain variable domain, including the CDR3, comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:49.

[0063] In one example, the heavy chain variable domain in the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), wherein the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO: 46, and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO: 11), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16), wherein the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO: 49.

[0064] In another example, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 64. In some examples, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), wherein the heavy chain variable domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 46, and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16). The light chain variable domain, including CDR3, comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:64. In one example, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), wherein the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46, and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:64.

[0065] In another example, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 113. In some examples, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), wherein the heavy chain variable domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 46, and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO: 12), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO: 18). The light chain variable domain, including CDR3, comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:113. In one example, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), wherein the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46, and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18), wherein the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:113.

[0066] In another example, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 98. In some examples, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), wherein the heavy chain variable domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 46, and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO: 17). The light chain variable domain, including the CDR3, comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:98. In one example, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), wherein the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46, and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO:17), wherein the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:98.

[0067] In another example, the heavy chain variable domain comprises the sequence of SEQ ID NO: 46, and the light chain variable domain comprises the sequence of SEQ ID NO: 107. In some examples, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), wherein the heavy chain variable domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 46, and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO: 12), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16). The light chain variable domain, including the CDR3, comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:107. In one example, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), wherein the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46, and the light chain variable domain comprises a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:107.

[0068] In another example, the heavy chain variable domain in the heavy chain variable domain can comprise the sequence of SEQ ID NO: 128, and the light chain variable domain in the light chain variable domain can comprise the sequence of SEQ ID NO: 122. In some examples, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), and a VH CDR3 comprising HGNFGNSYVSWFAY (SEQ ID NO: 8), the heavy chain variable domain comprises a sequence at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 128, and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16). The light chain variable domain, including CDR3, comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%, or 100% identical) to SEQ ID NO:122. In one example, the heavy chain variable domain comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWFAY (SEQ ID NO:8), wherein the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:128, and the light chain variable domain comprises a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:122.

[0069] Further examples of heavy and light chain variable domains include those in Table 9B.

[0070] Where the target binding protein is a single chain protein (eg, scFv), the single chain target binding protein may comprise the sequence of SEQ ID NO:50, 28, 114, 99, 108, or 32. In some examples, the single chain target binding protein comprises a VH CDR1 comprising the sequence of SEQ ID NO:3, a VH CDR2 comprising the sequence of SEQ ID NO:5, a VH CDR3 comprising the sequence of SEQ ID NO:6, 7, or 8, an LH CDR1 comprising the sequence of SEQ ID NO:10, 11, 12, or 13, an LH CDR2 comprising the sequence of SEQ ID NO:14, and an LH CDR3 comprising the sequence of SEQ ID NO:15, 16, 17, or 18, wherein the single chain target binding protein comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NOs:50, 28, 114, 99, 188, or 32. For example, the single chain target binding protein comprises a VH CDR1 comprising the sequence of SEQ ID NO:3, a VH CDR2 comprising the sequence of SEQ ID NO:5, a VH CDR3 comprising the sequence of SEQ ID NO:6, 7, or 8, an LH CDR1 comprising the sequence of SEQ ID NO:10, 11, 12, or 13, an LH CDR2 comprising the sequence of SEQ ID NO:14, and an LH CDR3 comprising the sequence of SEQ ID NO:15, 16, 17, or 18, wherein the single chain target binding protein comprises a sequence that is at least 95% identical to any one of SEQ ID NOs:50, 28, 114, 99, 188, or 32.

[0071] In some examples, the single chain target binding protein may comprise the sequence of SEQ ID NO: 50. In some examples, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), a VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO: 11), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16), wherein the single chain target binding protein comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical) to SEQ ID NO: 50. For example, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO:11), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the single chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:50.

[0072] In some examples, the single chain target binding protein may comprise the sequence of SEQ ID NO: 28. In some examples, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), a VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16), and the single chain target binding protein comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical) to SEQ ID NO: 28. For example, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the single chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:28.

[0073] In some examples, the single chain target binding protein may comprise the sequence of SEQ ID NO: 114. In some examples, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18), wherein the single chain target binding protein comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical) to SEQ ID NO:114. For example, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18), wherein the single chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:114.

[0074] In some examples, the single chain target binding protein may comprise the sequence of SEQ ID NO: 99. In some examples, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO:17), wherein the single chain target binding protein comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical) to SEQ ID NO:99. For example, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), and a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO:17), wherein the single chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:99.

[0075] In some examples, the single chain target binding is the sequence of SEQ ID NO: 108. In some examples, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the single chain target binding protein comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical) to SEQ ID NO:108. For example, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO:7), a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the single chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:108.

[0076] In some examples, the single chain target binding protein may comprise the sequence of SEQ ID NO: 32. In some examples, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO: 3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), a VH CDR3 comprising HGNFGNSYVSWFAY (SEQ ID NO: 8), a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), a VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16), wherein the single chain target binding protein comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical) to SEQ ID NO: 32. For example, the single chain target binding protein comprises a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO:5), a VH CDR3 comprising HGNFGNSYVSWFAY (SEQ ID NO:8), a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNKRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), wherein the single chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:32.

[0077] Further examples of single chain target binding proteins include those disclosed in Table 9B.

[0078] The target binding protein may specifically bind to CD3 (e.g., CD3 epsilon). As used herein, the terms "specific binding" and "specifically binds" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and the antigen for which the immunoglobulin is specific. The strength or affinity of a binding interaction is determined by the dissociation constant (K d or K D ), where the smaller K drepresents a higher affinity. The strength or affinity of a binding interaction can be expressed in terms of the association rate (Kon) or dissociation rate (Koff). The strength or affinity of a binding interaction refers to the strength of the sum of non-covalent interactions between a target binding domain and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of a target binding domain and an antigen or epitope. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity of a target binding domain to its corresponding antigen or epitope are known in the art.

[0079] As used herein, a CD3 binding protein "specifically binds" to CD3 refers to a protein that binds to CD3 with a dissociation constant (Kd) of less than 100 μM (e.g., less than 5 μM or less than 10 μM). The target binding protein may be present at a concentration of from about 0.01 nM to about 500 nM (e.g., from about 0.01 nM to about 450 nM, from about 0.01 nM to about 400 nM, from about 0.01 nM to about 350 nM, from about 0.01 nM to about 300, from about 0.01 nM to about 250 nM, from about 0.01 nM to about 200 nM, from about 0.01 nM to about 150 nM, from about 0.01 nM to about 100 nM, from about 0.01 nM to about 80 nM, from about 0.01 nM to about 60 nM, from about 0.01 nM to about 50 nM, from about 0.01 nM to about 40 nM, from about 0.01 nM to about 25 nM, from about 0.01 nM to about 20nM, about 0.01nM to about 15nM, about 0.01nM to about 10nM, about 0.01nM to about 8nM, about 0.01nM to about 6nM, about 0.01nM to about 5nM, about 0.01nM to about 4nM, about 0.01nM to about 3nM, about 0.01nM to about 2 nM, about 0.01nM to about 1nM, about 0.01nM to about 0.8nM, about 0.01nM to about 0.6nM, about 0.01nM to about 0.4nM, about 0.01nM to about 0.2nM, about 0.01nM to about 0.1nM, about 0.01nM to about 0.05nM, about 0.05 nM to about 500nM, about 0.05nM to about 450nM, about 0.05nM to about 400nM, about 0.05nM to about 350nM, about 0.05nM to about 300, about 0.05nM to about 250nM, about 0.05nM to about 200nM, about 0.05nM to about 150nM , about 0.05nM to about 100nM, about 0.05nM to about 80nM, about 0.05nM to about 60nM, about 0.05nM to about 50nM, about 0.05nM to about 40nM, about 0.05nM to about 25nM, about 0.05nM to about 20nM, about 0.05nM to about 15n M, about 0.05nM to about 10nM, about 0.05nM to about 8nM, about 0.05nM to about 6nM, about 0.05nM to about 5nM, about 0.05nM to about 4nM, about 0.05nM to about 3nM, about 0.05nM to about 2nM, about 0.05nM to about 1nM, about 0.05 nM to about 0.8nM, about 0.05nM to about 0.6nM, about 0.05nM to about 0.4nM, about 0.05nM to about 0.2nM, about 0.05nM to about 0.1nM, about 0.1nM to about 500nM, about 0.1nM to about 450nM, about 0.1nM to about 400nM,from about 0.1 nM to about 350 nM, from about 0.1 nM to about 300, from about 0.1 nM to about 250 nM, from about 0.1 nM to about 200 nM, from about 0.1 nM to about 150 nM, from about 0.1 nM to about 100 nM, from about 0.1 nM to about 80 nM, from about 0.1 nM to about 60 nM, from about 0.1 nM to about 50 nM, from about 0.1 nM to about 40 nM, from about 0.1 nM to about 25 nm, from about 0.1 nM to about 20 nM, from about 0.1 nM to about 15 nM, from about 0.1 nM to about 10 nM, from about 0.1 nM to about 8 nM, from about 0.1 nM to about 6 nM, from about 0.1 nM to about 5 nM, from about 0.1 nM to about 4 nM, from about 0.1 nM to about 3 nM, from about 0.1 nM to about 2 nM, from about 0.1 nM to about 1 nM, from about 0.1 nM to about 0.8 nM, from about 0.1 nM to about 0.6 nM, from about 0.1 nM to about 0.4 nM, from about 0.1 nM to about 0.2 nM, from about 0.2 nM to about 500 nM, from about 0.2 nM to about 450 nM, from about 0.2 nM to about 400 nM, from about 0.2 nM to about 350 nM, from about 0.2 nM to about 300, from about 0.2 nM to about 250 nM, from about 0.2 nM to about 200 nM, from about 0.2 nM to about 150 nM, from about 0.2 nM to about 100 nM, from about 0.2 nM to about 80 nM, from about 0.2 nM to about 60 nM, from about 0.2 nM to about 50 nM, from about 0.2 nM to about 40 nM, from about 0.2 nM to about 25 nm, from about 0.2 nM to about 20 nM, from about 0.2 nM to about 15 nM, from about 0.2 nM to about 10 nM, from about 0.2 nM to about 8 nM, from about 0.2 nM to about 6 nM, from about 0.2 nM to about 5 nM, from about 0.2 nM to about 4 nM, from about 0.2 nM to about 3 nM, from about 0.2 nM to about 2 nM, from about 0.2 nM to about 1 nM, from about 0.2 nM to about 0.8 nM, from about 0.2 nM to about 0.6 nM, from about 0.2 nM to about 0.4 nM, from about 0.4 nM to about 500 nM, from about 0.4 nM to about 450 nM, from about 0.4 nM to about 400 nM, from about 0.4 nM to about 350 nM, from about 0.4 nM to about 300, from about 0.4 nM to about 250 nM, from about 0.4 nM to about 200 nM, from about 0.4 nM to about 150 nM, from about 0.4 nM to about 100 nM, from about 0.4 nM to about 80 nM, from about 0.4 nM to about 60 nM, from about 0.4 nM to about 50 nM, from about 0.4 nM to about 40 nM, from about 0.4 nM to about 25 nm, from about 0.4 nM to about 20 nM, from about 0.4 nM to about 15 nM, from about 0.4 nM to about 10 nM, from about 0.4 nM to about 8 nM, from about 0.4 nM to about 6 nM, from about 0.4 nM to about 5 nM, from about 0.4 nM to about 4 nM, from about 0.4 nM to about 3 nM, from about 0.4 nM to about 2 nM, from about 0.4 nM to about 1 nMfrom about 0.4 nM to about 0.8 nM, from about 0.4 nM to about 0.6 nM, from about 0.5 nM to about 500 nM, from about 0.5 nM to about 450 nM, from about 0.5 nM to about 400 nM, from about 0.5 nM to about 350 nM, from about 0.5 nM to about 300, from about 0.5 nM to about 250 nM, from about 0.5 nM to about 200 nM, from about 0.5 nM to about 150 nM, from about 0.5 nM to about 100 nM, from about 0.5 nM to about 80 nM, from about 0.5 nM to about 60 nM, from about 0.5 nM to about 50 nM, from about 0.5 nM to about 40 nM, from about 0.5 nM to about 25 nm, from about 0.5 nM to about 20 nM, from about 0.5 nM to about 15 nM, from about 0.5 nM to about 10 nM, from about 0.5 nM to about 8 nM, from about 0.5 nM to about 6 nM, from about 0.5 nM to about 5 nM, from about 0.5 nM to about 4 nM, from about 0.5 nM to about 3 nM, from about 0.5 nM to about 2 nM, from about 0.5 nM to about 1 nM, from about 0.5 nM to about 0.8 nM, from about 0.5 nM to about 0.6 nM, from about 0.6 nM to about 500 nM, from about 0.6 nM to about 450 nM, from about 0.6 nM to about 400 nM, from about 0.6 nM to about 350 nM, from about 0.6 nM to about 300, from about 0.6 nM to about 250 nM, from about 0.6 nM to about 200 nM, from about 0.6 nM to about 150 nM, from about 0.6 nM to about 100 nM, from about 0.6 nM to about 80 nM, from about 0.6 nM to about 60 nM, from about 0.6 nM to about 50 nM, from about 0.6 nM to about 40 nM, from about 0.6 nM to about 25 nm, from about 0.6 nM to about 20 nM, from about 0.6 nM to about 15 nM, from about 0.6 nM to about 10 nM, from about 0.6 nM to about 8 nM, from about 0.6 nM to about 6 nM, from about 0.6 nM to about 5 nM, from about 0.6 nM to about 4 nM, from about 0.6 nM to about 3 nM, from about 0.6 nM to about 2 nM, from about 0.6 nM to about 1 nM, from about 0.6 nM to about 0.8 nM, from about 1 nM to about 500 nM, from about 1 nM to about 450 nM, from about 1 nM to about 400 nM, from about 1 nM to about 350 nM, from about 1 nM to about 300, from about 1 nM to about 250 nM, from about 1 nM to about 200 nM, from about 1 nM to about 150 nM, from about 1 nM to about 100 nM, from about 1 nM to about 80 nM, from about 1 nM to about 60 nM, from about 1 nM to about 50 nM, from about 1 nM to about 40 nM, from about 1 nM to about 25 nm, from about 1 nM to about 20 nM, from about 1 nM to about 15 nM, from about 1 nM to about 10 nM, from about 1 nM to about 8 nM, from about 1 nM to about 6 nM, from about 1 nM to about 5 nM, from about 1 nM to about 4 nM, from about 1 nM to about 3 nM, from about 1 nM to about 2 nM, from about 2 nM to about 500 nM, from about 2 nM to about 450 nM, from about 2 nM to about 400 nM, from about 2 nM to about 350 nMfrom about 2 nM to about 300, from about 2 nM to about 250 nM, from about 2 nM to about 200 nM, from about 2 nM to about 150 nM, from about 2 nM to about 100 nM, from about 2 nM to about 80 nM, from about 2 nM to about 60 nM, from about 2 nM to about 50 nM, from about 2 nM to about 40 nM, from about 2 nM to about 25 nM, from about 2 nM to about 20 nM, from about 2 nM to about 15 nM, from about 2 nM to about 10 nM, from about 2 nM to about 8 nM, from about 2 nM to about 6 nM, from about 2 nM to about 5 nM, from about 2 nM to about 4 nM, from about 2 nM to about 3 nM, from about 5 nM to about 500 nM, from about 5 nM to about 450 nM, from about 5 nM to about 400 nM, from about 5 nM to about 350 nM, from about 5 nM to about 300, from about 5 nM to about 250 nM, from about 5 nM to about 200 nM, from about 5 nM to about 150 nM, from about 5 nM to about 100 nM, from about 5 nM to about 80 nM, from about 5 nM to about 60 nM, from about 5 nM to about 50 nM, from about 5 nM to about 40 nM, from about 5 nM to about 25 nM, from about 5 nM to about 20 nM, from about 5 nM to about 15 nM, from about 5 nM to about 10 nM, from about 5 nM to about 8 nM, from about 5 nM to about 6 nM, from about 10 nM to about 500 nM, from about 10 nM to about 450 nM, from about 10 nM to about 400 nM, from about 10 nM to about 350 nM, from about 10 nM to about 300, from about 10 nM to about 250 nM, from about 10 nM to about 200 nM, from about 10 nM to about 150 nM, from about 10 nM to about 100 nM, from about 10 nM to about 80 nM, from about 10 nM to about 60 nM, from about 10 nM to about 50 nM, from about 10 nM to about 40 nM, from about 10 nM to about 25 nM, from about 10 nM to about 20 nM, from about 10 nM to about 15 nM, from about 20 nM to about 500 nM, from about 20 nM to about 450 nM, from about 20 nM to about 400 nM, from about 20 nM to about 350 nM, from about 20 nM to about 300, from about 20 nM to about 250 nM, from about 20 nM to about 200 nM, from about 20 nM to about 150 nM, from about 20 nM to about 100 nM, from about 20 nM to about 80 nM, from about 20 nM to about 60 nM, from about 20 nM to about 50 nM, from about 20 nM to about 40 nM, from about 50 nM to about 500 nM, from about 50 nM to about 450 nM, from about 50 nM to about 400 nM, from about 50 nM to about 350 nM, from about 50 nM to about 300, from about 50 nM to about 250 nM, from about 50 nM to about 200 nM, from about 50 nM to about 150 nM, from about 50 nM to about 100 nM, from about 50 nM to about 80 nM, from about 100 nM to about 500 nM, from about 100 nM to about 450 nM, from about 100 nM to about 400 nM, from about 100 nM to about 350 nM, from about 100 nM to about 300, from about 100 nM to about 250 nM, from about 100 nM to about 200 nMIt can specifically bind to CD3 with a Kd of about 100 nM to about 150 nM, about 250 nM to about 500 nM, about 250 nM to about 450 nM, about 250 nM to about 400 nM, about 250 nM to about 350 nM, about 250 nM to about 300, about 400 nM to about 500 nM, or about 400 nM to about 450 nM.

[0080] Linker The target binding protein may comprise one or more linkers. A linker may comprise a stretch of amino acid sequence that connects two components (e.g., between a heavy chain variable domain and a light chain variable domain, between a target binding domain and a cleavable moiety, between a target binding domain and a masking moiety, between a masking moiety and a cleavable moiety, or between a half-life extending moiety and another component in the target binding protein. The linker may not be cleavable by any protease or may not be cleavable by any protease naturally occurring in humans. In some embodiments, the linker(s) may be flexible linkers that may be introduced into the target binding protein to provide flexibility at one or more junctions between domains, between moieties, between moieties and domains, or any other junction where a linker is beneficial. In some embodiments, when the target binding protein is provided as a conformationally constrained construct, a linker may be inserted to facilitate formation and maintenance of the structure.

[0081] Any of the linkers described herein may provide the desired flexibility to facilitate inhibition of target binding or facilitate cleavage of the cleavable moiety by a protease. In some embodiments, the linker may be fully or partially flexible, so that it may include a flexible linker and one or more moieties that provide a less flexible structure to provide the desired target binding protein. Some linkers may contain cysteine ​​residues, which may form disulfide bonds and reduce the flexibility of the construct.

[0082] In some embodiments, when the target binding protein includes a masking moiety, the linker attached to the masking moiety may have a length that allows the masking moiety to be located in a tertiary or quaternary structure to effectively mask the target binding domain in the protein, allowing the masking moiety to mask the target binding domain in the target binding protein. For example, in the tertiary or quaternary structure, the masking moiety may be adjacent to the target binding domain to be masked.

[0083] In most cases, the length of a linker can be determined by counting the number of amino acids in the N to C direction from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous component to the C-terminus of the linker adjacent to the N-terminal amino acid of the next component (i.e., the length of the linker does not include either the C-terminal amino acid of the previous component or the N-terminal amino acid of the next component).

[0084] In some embodiments, the linkers comprise a total of 1 to 50, 1 to 40, 1 to 30, 1 to 25 (e.g., 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 15, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 25, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 15, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 8 ... 5, 2-4, 2-3, 4-25, 4-24, 4-22, 4-20, 4-18, 4-16, 4-15, 4-14, 4-12, 4-10, 4-8, 4-6, 4-5, 5-25, 5-24, 5-22, 5-20, 5-18, 5-16, 5-15, 5-14, 5-12, 5-10, 5-8, 5-6, 6-25, 6-24, 6-22, 6-20, 6-18, 6-16, 6-15, 6-14, 6-1 2, 6-10, 6-8, 8-25, 8-24, 8-22, 8-20, 8-18, 8-16, 8-15, 8-14, 8-12, 8-10, 10-25, 10-24, 10-22, 10-20, 10-18, 10-16, 10-15, 10-14, 10-12, 12-25, 12-24, 12-22, 12-20, 12-18, 12-16, 12-15, 12-14, 14-25, 14-2 In some embodiments, the linker may comprise a total 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 amino acids.

[0085] In some embodiments, the linker can be rich in glycine (Gly or G) residues. In some embodiments, the linker can be rich in serine (Ser or S) residues. In some embodiments, the linker can be rich in glycine and serine residues. In some embodiments, the linker can have one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs).

[0086] In some embodiments, a linker may have one or more Gly-Gly-Gly-Ser (GGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences). In some embodiments, a linker may have one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some embodiments, a linker may have one or more Gly-Gly-Ser-Gly (GGSG) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences). Exemplary linkers may include glycine polymers (G), glycine-serine polymers (e.g., (GS), (GGS), (GSGGS), and (GGGS), where n is at least an integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be relatively unstructured and thus may function as neutral linkages between components. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include GGSG, GGSGG (SEQ ID NO:667), GSGSG (SEQ ID NO:668), GSGGG (SEQ ID NO:669), GGGSG (SEQ ID NO:670), GSSSG (SEQ ID NO:671), GSSGGSGGSGG (SEQ ID NO:672), GGGS (SEQ ID NO:673), GGGSGGGS (SEQ ID NO:674), GGGSGGGSGGGS (SEQ ID NO:675), GGGGSGGGGSGGGGGS (SEQ ID NO:676), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:677), GGGGSGGGGS (SEQ ID NO:678), GGGGS (SEQ ID NO:679), GS, GGGGSGS (SEQ ID NO:680), GGG The sequences include one or a combination of one or more of GSGGGGSGGGGSGS (SEQ ID NO: 681), GGSLDPKGGGGS (SEQ ID NO: 682), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 683), SKYGPPCPPCPAPEFLG (SEQ ID NO: 684), GKSSGSGSESKS (SEQ ID NO: 685), GSTGSSGKSSEGKG (SEQ ID NO: 686), GSTGSSGKSSEGSGSTKG (SEQ ID NO: 687), GSTGSSGKPGSGEGSTKG (SEQ ID NO: 688), and GSTGSSGKPGSSEGST (SEQ ID NO: 689), or GSTGSSGKPGSSEGST (SEQ ID NO: 690).

[0087] Exemplary linkers may further include sequences that are at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the exemplary linkers described herein. One of skill in the art will recognize that a design may include a fully or partially flexible linker, such that the linker may include not only a flexible linker, but one or more moieties that confer a less flexible structure to provide a desired target binding protein structure.

[0088] In some embodiments, the target binding protein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker(s) (e.g., linker sequences the same or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linker may include non-amino acid based linkers, including but not limited to sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl) aminobenzoate), SMPB (succinimidyl 4-(N-maleimidophenyl) butyrate), and sulfo-SMPB (sulfosuccinimidyl 4-(N-maleimidophenyl) butyrate), which react with sulfhydryls of primary amines.

[0089] Half-life extender (EM) The target binding protein may further comprise a half-life extending moiety (EM). In some examples, the half-life extending moiety may be a serum half-life extending moiety, i.e., it can extend the serum half-life of a molecule bound to the EM.

[0090] In some examples, the EM may comprise a fragment crystallizable region (Fc domain) of an antibody. For example, the EM may be an Fc domain of an IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some examples, the EM may comprise a dimer formed by two Fc domains. The Fc domain may be a wild type peptide or a mutant. For example, the EM may comprise a dimer formed by two Fc domain mutants. In such a case, the two Fc domain mutants may be an Fc domain hole mutant and an Fc domain knob mutant. The knob and hole mutants may interact with each other to promote dimerization of the two Fc domains. In some embodiments, the knob and hole mutants may comprise one or more amino acid modifications within the interface between the two Fc domains (e.g., in the CH3 domain). In one example, the modifications include the amino acid substitutions T366W and, optionally, S354C in one IgG Fc domain, and the amino acid substitutions T366S, L368A, Y407V and, optionally, Y349C (numbered according to the EU numbering system) in the other IgG Fc domain. Examples of Fc variants also include SEQ ID NOs: 26-27.

[0091] Examples of Fc domain variants also include those described in U.S. Patent No. 7,695,936, which is incorporated herein by reference in its entirety. In one example, the modification comprises the amino acid substitution T366Y in one IgG Fc domain and the amino acid substitution Y407T in the other IgG Fc domain. In one example, the modification comprises the amino acid substitution T366W in one IgG Fc domain and the amino acid substitution Y407A in the other IgG Fc domain. In one example, the modification comprises the amino acid substitution F405A in one IgG Fc domain and the amino acid substitution T394W in the other IgG Fc domain. In one example, the modification comprises the amino acid substitutions T366Y and F405A in one IgG Fc domain and the amino acid substitutions T394W and Y407T in the other IgG Fc domain. In one example, the modification comprises the amino acid substitutions T366W and F405W in one IgG Fc domain and the amino acid substitutions T394S and Y407A in the other IgG Fc domain. In one example, the modification comprises amino acid substitutions F405W and Y407A in one IgG Fc domain and amino acid substitutions T366W and T394S in the other IgG Fc domain. In one example, the modification comprises amino acid substitution F405W in one IgG Fc domain and amino acid substitution T394S in the other IgG Fc domain. The mutation positions in the Fc domains are numbered according to the EU numbering system. The IgG Fc domain may comprise the sequence of SEQ ID NO: 34-37 (IgG1, IgG2, IgG3, or IgG4). In these sequences, amino acids 1-107 correspond to EU numbers 341-447.

[0092] In some examples, the Fc domain variants may have reduced effector function. Examples of such Fc domains include those disclosed in US20190135943, which is incorporated herein by reference in its entirety. Further examples of Fc domains include SEQ ID NOs: 38-42.

[0093] Further examples of EMs include immunoglobulins (e.g., IgG), serum albumins (e.g., human serum albumin (HSA), hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin-binding peptide (CBP), Strep-tag, cellulose binding domain, maltose binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitopes, epitope tags, E2 tags, HA epitope tags, Myc epitopes, FLAG epitopes, AU1 and AU5 epitopes, Glu-Glu epitopes, KT3 epitopes, IRS epitopes, B tag epitopes, protein kinase-C epitopes, and VSV epitopes.

[0094] In some embodiments, the serum half-life of the target binding protein may be longer than the serum half-life of a reference protein (e.g., a substantially identical target binding protein without the half-life extending moiety), e.g., the pK of the target binding protein is longer than the pK of the reference protein. In some examples, a target binding protein with an EM may have a serum half-life that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2x, 4x, 6x, 8x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x longer than the serum half-life of the identical reference target binding protein but without the EM. In some embodiments, the serum half-life of the target binding protein when administered to an organism can be at least 15 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour.

[0095] Additional target binding domains The target binding protein may comprise one or more target binding domains in addition to the CD3 binding domain described herein. In some embodiments, the target binding protein may comprise an additional light chain variable domain and an additional heavy chain variable domain. The additional light chain variable domain and the additional heavy chain variable domain may form an additional target binding domain. In some examples, the CD3 binding domain and the additional target binding domain may be identical. In some examples, the CD3 binding domain and the additional target binding domain may be different from each other (e.g., may specifically bind the same or different antigens or epitopes).

[0096] In some embodiments, the CD3 binding domain and the additional target binding domain may both be Fv fragments or at least one may be an Fv fragment. In some embodiments, the CD3 binding domain and the additional target binding domain may both be Fab fragments or at least one may be a Fab fragment. In some embodiments, the CD3 binding domain and the additional target binding domain may be Fab' fragments or at least one may be a Fab' fragment.

[0097] In some embodiments, the target binding protein may be multispecific (e.g., bispecific, trispecific, tetraspecific, and other multispecific target binding proteins), e.g., may bind to CD3 and one or more additional targets. In some embodiments, the multispecific target binding protein may be multivalent, e.g., includes multiple target binding sites, whether the binding sites recognize the same target or different targets. In some embodiments, the target binding protein may be bispecific. The term "bispecific" means that the target binding protein can specifically bind to two different targets. Typically, a bispecific target binding protein includes two target binding domains, each of which can specifically bind to a different target. In some embodiments, the bispecific target binding protein can simultaneously bind to two targets, e.g., two target proteins expressed on two different cells.

[0098] In some embodiments, the target binding protein may comprise a CD3 binding domain and an additional target binding domain capable of binding to a molecule on the surface of a cell associated with a disease (e.g., a tumor cell). Such a target binding protein may simultaneously bind to an immune cell (e.g., a T cell) and a cell associated with a disease (e.g., a tumor cell), thus activating the immune cell and crosslinking the activated immune cell to the cell associated with the disease. In some embodiments, the target binding protein may be formulated as part of a chimeric antigen receptor (CAR), a T cell-engaging antibody (e.g., a T cell-engaging bispecific antibody), a pro-bispecific T cell-engaging (pro-BITE) molecule, a pro-chimeric antigen receptor (pro-CAR) modified T cell, or other engineered receptor or other immune effector cell, e.g., a CAR-modified NK cell.

[0099] In some examples, the target binding protein may be a monovalent bispecific antibody comprising a CD3 binding domain and an additional target binding domain as described herein. As used herein, the term "monovalent bispecific antibody" refers to a bispecific antibody in which only one antigen binding domain is directed to a specific target. The inventors have surprisingly discovered that certain CD3 binding domains as described herein exhibit improved stability, manufacturability, and / or CD3 binding affinity in the context of monovalent CD3 binding proteins, including monovalent bispecific antibodies that specifically bind to CD3 (e.g., anti-CD3 scFv).

[0100] The targets of the additional target binding domains can be proteins or other types of molecules, such as cell surface receptors, secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), and the like.

[0101] In some examples, the additional target binding domain may bind to a target that is a molecule on or within a cell associated with a disease. For example, the additional target binding domain may bind to a tumor cell. In such a case, the additional target binding domain may bind to a tumor-associated antigen. As used herein, the term "tumor-associated antigen" refers to any antigen, including proteins, glycoproteins, gangliosides, carbohydrates, lipids, associated with cancer. Such antigens may be expressed on tumor cells (e.g., malignant cells) or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. In some embodiments, the tumor-associated antigen may be human epidermal growth factor receptor 2 (HER2). For example, the additional target binding domain may be trastuzumab or a fragment thereof. In another example, the additional target binding domain may be pertuzumab or a fragment thereof.

[0102] Activatable target-binding proteins In one aspect, the target binding protein herein includes an activatable target binding protein. In general, an activatable target binding protein may include a prodomain, which refers to a polypeptide that includes an amino acid sequence that, when bound to a target binding protein, functions to inhibit target binding by the target binding protein and forms a protease-cleavable substrate. The portion of the prodomain that inhibits target binding is referred to as the masking portion (MM), and the amino acid sequence that is a protease-cleavable substrate is referred to as the cleavable portion (CM). The prodomain may include a linker (L) between the MM and CM and / or at the terminus of the prodomain (e.g., at the carboxyl and / or amino terminus to facilitate binding of the prodomain to the target binding protein). In certain embodiments, the prodomain may comprise one of the following formulas (representing the amino acid sequence from N-terminus to C-terminus): MM-CM, MM-L-CM, MM-CM-L, MM-L-CM-L, CM-MM, CM-L-MM, L-CM-MM, or L-CM-L-MM (where each "-" represents a direct or indirect bond (e.g., via a linker)).

[0103] As used herein, the term "activatable target binding protein" refers to a target binding protein in its inactive (uncleaved or native) state. It will be apparent to one of skill in the art that in some embodiments, a cleaved activatable target binding protein may be associated with a MM that does not reduce, inhibit, or interfere with binding between the target binding domain and its target. In some embodiments, a cleaved activatable target binding protein may lack a MM due to cleavage of the CM (e.g., by a protease), resulting in the release of the MM. As used herein, the term "cleaved state" or "active state" refers to the state of an activatable target binding protein after cleavage of the CM by at least one protease. The term "uncleaved state" or "inactive state" refers to the state of an activatable target binding protein in the absence of cleavage of the CM by a protease.

[0104] By activatable, it is meant that when the activatable target binding protein is in an inhibited, masked, or uncleaved state (i.e., a first conformation), it exhibits a first level of binding to the target, and when in an uninhibited, unmasked, and / or cleaved state (i.e., a second conformation), it exhibits a second level of binding to the target, the second level of target binding being greater than the first level of binding. In general, the access of the target to the target binding domain of the activatable target binding protein is greater in the presence of a cleavage agent, i.e., a protease, that can cleave the CM than in the absence of such a cleavage agent. Thus, when the activatable target binding protein is in an uncleaved state, the target binding domain can be inhibited from target binding or masked from target binding (i.e., the first conformation is such that the target binding domain cannot bind to the target or is inhibited from binding to the target), and in the cleaved state, the target binding domain is not inhibited or is not masked from target binding.

[0105] In some embodiments, an activatable target binding protein can be designed by selecting a target binding domain of interest and constructing the remainder of the activatable target binding protein such that, when conformationally constrained, the MM results in masking of the target binding domain or reduced binding of the target binding domain to its target. To provide this functional feature, structural design criteria can be taken into account.

[0106] The activatable target binding protein herein can exhibit a desired dynamic range of activatable phenotypes with respect to target binding in inhibited and uninhibited conformations. Dynamic range generally refers to the ratio of (a) the maximum detectable level of a parameter under a first set of conditions to (b) the minimum detectable value of that parameter under a second set of conditions. For example, in the context of an activatable target binding protein, dynamic range refers to the ratio of (a) the maximum detectable level of target protein binding to the activatable target binding protein in the presence of a protease capable of cleaving CM in the activatable target binding protein to (b) the minimum detectable level of target protein binding to the activatable target binding protein in the absence of the protease. The dynamic range of an activatable target binding protein can be calculated as the ratio of the dissociation constant of the activatable target binding protein cleavage agent (e.g., enzyme) treatment to the dissociation constant of the activatable target binding protein cleavage agent treatment. The greater the dynamic range of an activatable target binding protein, the better the activatable phenotype of the activatable target binding protein. An activatable target binding protein having a relatively high dynamic range value (e.g., greater than 1) exhibits a more desirable activatable phenotype, such that binding of a target protein by the activatable target binding protein occurs to a greater extent (e.g., occurs predominantly) in the presence of a cleaving agent (e.g., an enzyme) that can cleave the CM of the activatable target binding protein than in the absence of the cleaving agent.

[0107] An activatable target binding protein herein may comprise a target binding domain (TB), one or more masking moieties (MM) that reduce, inhibit or interfere with binding of the target binding domain to its target(s), one or more cleavable moieties (CM) that bind one or more MMs to the TB, and optionally one or more half-life extending moieties (EM). In some embodiments, an activatable target binding protein may comprise a TB that specifically binds CD3, and a masking moiety (MM) that inhibits binding of the TB and CD3, where the MM is bound to the TB via the cleavable moiety (CM) (directly or indirectly, e.g., via one or more linkers). As used herein, unless otherwise stated, the components of an activatable target binding protein that are "linked" may be linked either via a direct covalent bond or an indirect covalent bond, e.g., via one or more binding peptides (also referred to as "linkers"), cleavable moieties, or other components of the activatable protein.

[0108] In some embodiments, the activatable target binding protein may comprise more than one target binding domain (TB). For example, the activatable target binding protein may comprise a first TB that specifically binds to CD3, a first MM (MM1) that inhibits the binding of TB1 and CD3, and that binds to TB1 (directly or indirectly, e.g., via one or more linkers) via a first cleavable moiety (CM1), a second target binding domain (TB2) that specifically binds to a second target, TB2, and a second masking moiety (MM2) that inhibits the binding of the second target, and that binds to TB2 (directly or indirectly, e.g., via one or more linkers) via a second cleavable moiety (CM2). The activatable target binding protein may further comprise a half-life extending moiety (EM). In one example, the activatable target binding protein comprises an scFv that comprises TB1.

[0109] Masking part (MM) The activatable target binding proteins herein may contain one or more masking moieties (MM) that can interfere with the binding of TB to its target. The masking moieties (not yet activated) in the activatable molecule "mask" or reduce or otherwise inhibit the binding of the target binding domain to its target. In some embodiments, the binding or modification of the target binding protein with MM may inhibit the ability of the protein to specifically bind to its target by inhibition means known in the art (e.g., conformational changes and competition for the antigen binding domain). In some embodiments, the binding or modification of the target binding protein with MM may result in a conformational change that reduces or inhibits the ability of the protein to specifically bind to its target. In some embodiments, the binding or modification of the target binding protein with MM sterically blocks, reduces or inhibits the ability of the antigen binding domain to specifically bind to its target.

[0110] The MM may be linked to the TB by a CM and, optionally, one or more linkers described herein. In some embodiments, when the activatable target binding protein is not activated, the MM prevents TB from binding to the target, but when the activatable target binding protein is activated (when the CM is cleaved by a protease), the MM does not substantially or significantly interfere with TB binding to the target.

[0111] In the activatable target binding protein, the MM that interferes with the target binding of the TB can be bound to the TB (directly or indirectly, for example, via one or more linkers). Alternatively, the MM that interferes with the target binding of the TB can be bound directly or indirectly to a component of the activatable target binding protein that is not a TB. For example, the MM can be bound directly or indirectly to a different TB. In another example, the MM can be bound directly or indirectly to an EM. In either case, in the tertiary or quaternary structure of the activatable structure, the MM can be in a position that allows the MM to mask the TB (for example, proximal to the TB to be masked).

[0112] In some embodiments, the MM may interact with TB and thus reduce or inhibit the interaction between TB and its binding partner. In some embodiments, the MM may comprise at least a partial or complete amino acid sequence of a naturally occurring binding partner of TB. For example, the MM may be a fragment of a naturally occurring binding partner. The fragment may retain 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25% or 20% or less of nucleic acid or amino acid sequence homology to the naturally occurring binding partner. In some embodiments, the MM may be a cognate peptide of TB. For example, the MM may comprise a sequence of an epitope of TB or a fragment thereof. The term "naturally occurring" as applied to an object herein refers to the fact that the subject may occur in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in a laboratory is naturally occurring.

[0113] In some embodiments, the MM may comprise an amino acid sequence that is not naturally occurring or does not include the amino acid sequence of a naturally occurring binding partner or target protein. In certain embodiments, the MM is not a natural binding partner of TB. The MM may be a modified binding partner of TB that contains amino acid changes that reduce the binding affinity and / or avidity to TB. In some embodiments, the MM may contain no or substantially no nucleic acid or amino acid homology to a natural binding partner of TB. In other embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to a natural binding partner of TB.

[0114] In some embodiments, the MM does not specifically bind to the TB, but may interfere with the binding of the TB to its binding partner through non-specific interactions such as steric hindrance. For example, the MM may be positioned within an activatable target binding protein such that the tertiary or quaternary structure of the activatable target binding protein allows the MM to mask the TB through charge-based interactions, thereby holding the MM in place and interfering with the access of binding partners to the TB.

[0115] In some embodiments, the MM may have a dissociation constant for binding to TB that is equal to or less than the dissociation constant of TB for its target, hi some embodiments, the MM may not interfere with or compete with TB for binding to its target in the cleaved state.

[0116] The structural features of the MM can be selected according to factors such as the minimum amino acid sequence required to interfere with target protein binding, the target protein-protein binding pair of interest, the size of the TB, the presence or absence of a linker, and the like.

[0117] In some embodiments, the MM may be unique to the TB to which it is bound. Examples of MM include MMs (e.g., affinity masked) that have been specifically screened for binding to the binding domain of TB or a fragment thereof. Methods for screening MMs to obtain MMs that are unique to TB and that specifically and / or selectively bind to the binding domain of a binding partner / target are provided herein and can include protein display methods.

[0118] As used herein, the term "masking efficiency" refers to the activity of an activatable target-binding protein in an inactivated state (e.g., EC 50) divided by the activity of a control antibody, which can be either a cleavage product of the activatable target binding protein, or an antibody or fragment thereof used as a TB for the activatable target binding protein. Activatable target binding proteins with reduced levels of TB activity may have a masking efficiency of greater than 10. In some embodiments, the activatable target binding proteins described herein may have a masking efficiency of greater than 10, 100, 1000, or 5000.

[0119] In some embodiments, the MM can be a polypeptide between about 2 and 50 amino acids in length, for example, the MM can be a polypeptide between 2 and 40, 2 and 30, 2 and 20, 2 and 10, 5 and 15, 10 and 20, 15 and 25, 20 and 30, 25 and 35, 30 and 40, 35 and 45, 40 and 50 amino acids in length. For example, the MM can be a polypeptide having a length 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 amino acids. In some examples, the MM can be a polypeptide of more than 50 amino acids in length, e.g., 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0120] In some embodiments, in the inactive state of the activatable target binding protein having TB and interfering MM, in the presence of a TB target, e.g., as measured by an in vitro immunosorbent assay as described in US20200308243A1, the activatable target binding protein exhibits a TB binding activity of at least 0.1, 0.5, 1, 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 140, 160, 180, 200, 220, 240, 260, 280, 290, 300, 360, 480, 500, 600, 720, 840, 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 160, 200, 220, 240, 260, 280, 290, 300, 360, 480, 500, 600, 720, 840, 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 140, 200, 220, 240, 260, 28 ... There is no or substantially no binding of TB to its target for 50, 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or there is no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of TB to its target compared to binding of the corresponding antibody without the interfering MM.

[0121] The binding affinity of TB with an interfering MM to a target or binding partner is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times lower than the binding affinity of TB without the interfering MM to its binding partner, or is 5-10, 10-100, 10-1,000, 10-10,000, 10~100,000, 10~1,000,000, 10~10,000,000, 100~1,000, 100~10,000, 100~1,000,000, 100~10,000,000, 1,000~10,000, 1,000~100,000, 1, 000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower.

[0122] The dissociation constant of the MM to mask TB may be greater than the dissociation constant of the TB to the target. The dissociation constant of the MM to masked TB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the dissociation constant of the TB to the target. Conversely, the binding affinity of the MM to masked TB may be lower than the binding affinity of the TB to the target. The binding affinity of the MM to TB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times lower than the binding affinity of TB to the target.

[0123] In some embodiments, the MM may comprise genetically encoded amino acids or non-genetically encoded amino acids. Examples of non-genetically encoded amino acids include, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In certain embodiments, the MM comprises no more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% non-genetically encoded amino acids.

[0124] In some embodiments, when released from activatable target binding protein and in a free state, MM can have biological activity or therapeutic effect, for example, binding ability. For example, free peptide can bind to the same or different binding partner. In certain embodiments, free MM can exert therapeutic effect and provide secondary function to the composition disclosed herein. In some embodiments, when separated from activatable target binding protein and in a free state, MM can have the advantage of not showing biological activity. For example, in some embodiments, free MM does not induce immune response in a subject.

[0125] A suitable MM may be identified and / or further optimized through a screening procedure from a library of candidate activatable target binding proteins with variable MM. For example, the TB and CM may be selected to provide a desired enzyme / target combination, and the amino acid sequence of the MM may be identified by a screening procedure described below to identify a MM that provides a switchable phenotype. For example, a random peptide library (e.g., of peptides containing 2-40 or more amino acids) may be used in the screening methods disclosed herein to identify a suitable MM.

[0126] In some embodiments, MMs with specific binding affinity to TB can be identified by a screening procedure that includes providing a library of peptide scaffolds that contain candidate MMs, each scaffold being composed of a transmembrane protein and a candidate MM. The library can then be contacted with a whole or part of a protein, such as a full-length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment that contains the protein (which can also bind to a binding partner of interest), to identify one or more candidate MMs with detectable binding proteins. Screening can be performed, for example, by one or more rounds of magnetic activated sorting (MACS) or fluorescence activated sorting (FACS), as described in WO2009025846 and US20200308243A1, which are incorporated herein by reference in their entirety, and determining the binding affinity of the MMs to TB, and then determining the masking efficiency.

[0127] In some embodiments, a MM may be selected for use with a particular antibody or antibody fragment. For example, a suitable MM for use with TB that binds an epitope may include the sequence of the epitope. In some examples, suitable MMs for masking the anti-CD3 binding proteins disclosed herein include MMs that include the sequence GYLWGCEWNCGGITT (SEQ ID NO: 691), NAFRCWWDPPCQPMT (SEQ ID NO: 692), ARGLCWWDPPCTHDL (SEQ ID NO: 693), or NHSLCYWDPPCEPST (SEQ ID NO: 694). Additional masking moieties for anti-CD3 binding proteins include the sequences MMYCGGNEVLCGPRV (SEQ ID NO: 695), GYRWGCEWNCGGITT (SEQ ID NO: 696), MMYCGGNEIFCEPRG (SEQ ID NO: 697), GYGWGCEWNCGGSSP (SEQ ID NO: 698), and MMYCGGNEIFCGPRG (SEQ ID NO: 699).

[0128] Examples of suitable MMs are WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO20190 No. 75405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated by reference in their entireties.

[0129] Cuttable Part (CM) An activatable target binding protein may contain one or more cleavable moieties (CMs). "Cleavable moiety" and "CM" are used interchangeably herein to refer to a peptide whose amino acid sequence comprises a substrate for a sequence-specific protease. In some embodiments, the CM may be located between the TB and the MM.

[0130] The CM and TB of an activatable target binding protein can be selected such that the TB represents a binding portion of a particular target and the CM represents a substrate for one or more proteases, where the protease is co-localized with the target in a tissue (e.g., at a therapeutic or diagnostic site of a subject). The protease can cleave the CM in the activatable target binding protein when the activatable target binding protein is exposed to the protease. In some embodiments, an activatable target binding protein may find particular use when, for example, one or more proteases capable of cleaving a site in the CM are present at relatively higher levels in target-containing tissue at a therapeutic or diagnostic site than in tissue at a non-therapeutic site (e.g., in healthy tissue).

[0131] In some embodiments, the CM herein may include substrates for proteases that have known substrates reported in multiple cancers. See, e.g., La Roca et al., British J. Cancer 90(7):1414-1421, 2004. Substrates suitable for use in the CM components utilized herein include substrates that are more prevalent in cancer cells and tissues. Thus, in certain embodiments, the CM may include substrates for proteases that are more prevalent in diseased tissues associated with cancer. Examples of cancer include gastric cancer, breast cancer, osteosarcoma, esophageal cancer, breast cancer, HER2-positive cancer, Kaposi's sarcoma, hairy cell leukemia, chronic myelogenous leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, ovarian cancer, bladder cancer, BCG-resistant non-muscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colon cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer, etc. In some embodiments, the CM component comprises a substrate for a protease(s) that is more abundant in tumor tissue. For example, the protease(s) may be produced by the tumor of the subject.

[0132] In some embodiments, the activatable target binding protein may comprise two CMs (e.g., for binding a MM to multiple TBs). In some examples, the first CM and the second CM may comprise a substrate for the same protease. In some examples, the first and second CM may comprise substrates for different proteases. In some examples, the first CM and the second CM may comprise or consist of the same sequence. In some examples, the first and second CM may comprise or consist of different sequences.

[0133] Suitable CMs for use with the activatable target binding proteins herein include any of the protease substrates known in the art. In some examples, the CM may include a substrate for a serine protease (e.g., u-type plasminogen activator (uPA, also referred to as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1). In some examples, the CM may include a substrate for a matrix metalloprotease (MMP). In some examples, the CM may include a substrate for a cysteine ​​protease (CP) (e.g., legumain).

[0134] In some embodiments, the CM is a disintegrin and metalloproteinase (ADAM) or a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5), an aspartic protease (e.g., BACE, renin), an aspartic cathepsin (e.g., cathepsin D, cathepsin E), caspases (e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14), cysteine ​​cathepsins (e.g., cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P), cysteine ​​proteinases (e.g., cruzipain, legumain, oocyte stimulatory system, etc.), tubein-2), chymase, DESC1, DPP-4, FAP, elastase, FVIIa, FIXA, FXa, FXIa, FXIIa, granzyme B, guanidinobenzoatase, hepsin, HtrA1, human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), metalloproteinases (e.g., meprin, neprilysin, PSMA, BMP-1), lactoferrin, marapsin, matripsin In some embodiments, the substrates may include substrates for tryptase-2, MT-SP1 / matriptase, NS3 / 4A, PACE4, plasmin, PSA, MMPs (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27), TMPRSS2, TMPRSS3, TMPRSS4, tPA, thrombin, tryptase, and uPA.

[0135] In some embodiments, a protease substrate in a CM may comprise a peptide sequence that is not substantially identical (e.g., less than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence that is naturally cleaved by the same protease.

[0136] In some embodiments, the CM may be or may include a sequence encompassed by the consensus of any one of the sequences in Table 6 below. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

[0137] Examples of CM include WO2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO2020176672 ...76672, WO2020176672, WO2020176672, WO2020176672, WO2020176672, WO2020176672, WO2020176672, WO2020176672, WO2020176672, WO2020176672, WO2020176672, WO202017667 019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974, which are incorporated by reference in their entireties for all purposes.

[0138] In some embodiments, the CM may be or include combinations, C-terminal truncation variants, or N-terminal truncation variants of the above exemplary sequences. Truncation variants of the aforementioned amino acid sequences suitable for use in the CM may be any that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncation variants that include at least three consecutive amino acids of the aforementioned amino acid sequences, or at least 4, 5, 6, 7, 8, 9, or 10 amino acids of the aforementioned amino acid sequences that retain the recognition site of the protease. In certain embodiments, the truncation variants of the aforementioned amino acid sequences may be amino acid sequences corresponding to any of the above, but truncated at the C-terminus and / or N-terminus by 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, or 1-3 amino acids, (1) having at least three amino acid residues, and (2) retaining the recognition site of the protease. In some of the aforementioned embodiments, the truncated CM is an N-terminally truncated CM. In some embodiments, the truncated CM is a CM truncated at the C-terminus. In some embodiments, the truncated CM is a CM truncated at the C-terminus and the N-terminus.

[0139] In some embodiments, the CM may comprise a total of 3 amino acids to 25 amino acids, in some embodiments, the CM may comprise a total of 3-25, 3-20, 3-15, 3-10, 3-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, 10-15, 15-25, 15-20, or 20-25 amino acids.

[0140] In some embodiments, the C is about 0.001 to 1500x10 4 M -1 S -1 , or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500x104 M -1 S -1 The rate can be determined by the substrate cleavage kinetics (k cat / K m )

[0141] Conjugation Agents In some embodiments, the target binding protein (including the activatable target binding protein) may further comprise one or more additional agents, such as a targeting moiety that facilitates delivery to a cell or tissue of a subject, a therapeutic agent (e.g., a chemotherapeutic agent or an anti-tumor agent, such as an anti-tumor agent), a toxin, a radioisotope, a small molecule, a diagnostic agent, a targeting moiety, or a detectable moiety, or a fragment thereof. The additional agent may be conjugated to the target binding protein. "Agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0142] In some embodiments, the target binding protein may be conjugated to a cytotoxic agent, e.g., a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope. In some embodiments, the target binding protein may be conjugated to a T cell activator, such as, for example, a small molecule agonist of a Toll-like receptor (TLR), including, for example, TLR3, TLR7, TLR8, and TLR9. Non-limiting examples of such activators include TLR3 agonists, such as (Riboxxol), RGC100, ARNAX, and Poly-IC; TLR7 / 8 agonists, such as resiquimod (R848), and motolimod (VTX-2337) (a second generation experimental derivative of imiquimod, an imidazoquinoline), PF-4878691, BDC-1001, LHC165, NKTR-262, TQ-A3334, RO7119929, DSP-0509, BNT411, and NJH395; TLR9 agonists, such as Bacillus Calmette-Guerin (BCG), cavrotolimod / AST-008 (Exicure), CMP-001 (Checkmate), CpG-28 (University of Paris), EnanDIM (Mologen AG), IMO-2055 (Idera), IMO-2125 / tilsotolimod (Idera), MGN1703 / lefitolimod (Mologen AG), NZ-TLR9 (LIDDS), PF-3512676 (Pfizer), SD-101 (Dynavax), and S-540956 (Shionogi).

[0143] Examples of cytotoxic agents that can be conjugated to the target binding protein include dolastatins and derivatives thereof (e.g., auristatin E, AFP, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), desmethylauristatin E (DMAE), auristatin F, desmethylauristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolones), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycins, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, s These include ponzistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted campothecins, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaplysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0144] Examples of enzymatically active toxins that can be conjugated to the target-binding proteins include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii protein, dianfhin protein, Phytoiaca Americana protein (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geionin, mitogeliin, restrictocin, phenomycin, neomycin, and trichothecin.

[0145] Examples of anti-neoplastic agents that can be conjugated to the target binding proteins include adriamycin, cervidin, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabidine, and cytarabine.

[0146] Examples of antiviral agents that can be conjugated to the target binding protein include acyclovir, Virra A, and Symmetrel. Examples of antifungal agents that can be conjugated to the target binding protein include nystatin. Examples of detection reagents that can be conjugated to the target binding protein include fluorescein and its derivatives, fluorescein isothiocyanate (FITC). Examples of antibacterial agents that can be conjugated to the target binding protein include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. Examples of 3beta,16beta,17alpha-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-alpha-L-arabinopyranoside) (OSW-1) that can be conjugated to target binding proteins include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterin, cephalotaxine, homoharringionine, pyrrol obenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepenes, calcicheamicin, podophyiitoxin, taxanes, and vinca alkoids. Examples of radiopharmaceuticals that can be conjugated to target binding proteins include: 123 I, 89 Zr, 125 I, 131 I, 99 mTc, 201 T1, 62 Cu, 18 F, 68 Ga, 13 N, 15 O. 38 K, 82 Rb, 111 In, 133 Xe, 11 C, and 99Examples of heavy metals that can be conjugated to the target binding protein include barium, gold, and platinum. Examples of anti-mycoplasma agents that can be conjugated to the target binding protein include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0147] In some embodiments, the target binding protein may include a signal peptide. When including multiple peptides, the target binding protein may include multiple signal peptides, e.g., one signal peptide for each of the multiple peptides. The signal peptide may be a peptide (e.g., 10-30 amino acids in length) present at the end (e.g., N-terminus or C-terminus) of a newly synthesized protein destined for the secretory pathway. In some embodiments, the signal peptide may be conjugated to the target binding protein via a spacer. In some embodiments, the spacer may be conjugated to the target binding protein in the absence of a signal peptide.

[0148] Those skilled in the art will recognize that a wide variety of possible agents can be conjugated to any of the target binding proteins described herein. The agent can be conjugated to another component of the target binding protein by a conjugation moiety that can be a linker, CM, or other molecule or fragment thereof that can connect the two molecules. In some examples, the conjugation moiety can be cleaved by an enzyme (e.g., a protease). In some examples, the conjugation moiety may not be cleavable by an enzyme* (e.g., a protease).

[0149] Conjugation can include any chemical reaction that links two molecules together, so long as the target binding protein and the other moiety retain their respective activities. Conjugation can include many chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In some embodiments, the binding can be a covalent bond. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of an external cross-linking molecule. Many bivalent or multivalent binding agents can be useful for conjugating any of the target binding proteins described herein. For example, conjugation can include organic compounds such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene, hexamethylenediamine, and the like. In some embodiments, the target binding protein can include or be otherwise introduced with one or more non-natural amino acid residues to provide suitable sites for conjugation.

[0150] In some embodiments, the drug and / or conjugate may be attached to the antigen-binding domain by a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Since many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancer tissue microenvironment can reduce the disulfide bond, followed by release of the drug and / or conjugate at the delivery site.

[0151] In some embodiments, when the conjugate binds to its target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancer tissue)), the amide or ester bond linking the conjugate and / or agent to the linker is cleaved, resulting in release of the conjugate and / or agent in its active form. These conjugates and / or agents, when administered to a subject, can achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancer tissue)). These conjugates and / or agents can be effective for in vivo delivery of any of the conjugates and / or agents described herein.

[0152] In some embodiments, the conjugate moiety may not be cleavable by the enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, which ultimately lyses the target cell. In such embodiments, the conjugate and / or agent is to be delivered to the target cell (e.g., a hormone, enzyme, corticosteroid, neurotransmitter, or gene). Additionally, the conjugate moiety may be mildly susceptible to cleavage by serum proteases, and the conjugate and / or agent is released slowly at the target site.

[0153] In some embodiments, the conjugate and / or agent may be designed such that the conjugate and / or agent is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or agent is not released.

[0154] In some embodiments, the conjugate and / or agent may be attached directly to the antigen-binding domain or via an amino acid (e.g., D-amino acid), peptide, thiol-containing moiety, or other organic compound that can be modified by the methods described herein to contain a functional group that can then be utilized for attachment to the antigen-binding domain.

[0155] In some embodiments, the target binding protein may include at least one conjugation point of an agent. In some embodiments, all possible conjugation points are available for conjugation with an agent. In some embodiments, one or more conjugation points may include sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues that contain sulfur atoms. In such cases, the residues may be naturally occurring in the protein construct structure or may be incorporated into the protein construct using methods including site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0156] The present disclosure also provides methods and materials for preparing target binding proteins with one or more conjugation agents. In some embodiments, target binding proteins can be modified to include one or more interchain disulfide bonds. For example, disulfide bonds can be reduced after exposure to a reducing agent, such as, but not limited to, TCEP, DTT, or β-mercaptoethanol. In some cases, reduction of disulfide bonds can only be partial. As used herein, the term partial reduction refers to a situation in which a target binding protein is contacted with a reducing agent and a portion of all possible conjugation sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, a target binding protein may be partially reduced if, after contact with a reducing agent, less than 99% (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of all possible conjugation sites are reduced. In some embodiments, target binding proteins having one or more interchain disulfide bonds reduced may be conjugated with a drug that reacts with free thiols.

[0157] The present disclosure also provides methods and materials for conjugating a therapeutic agent to a specific location on a target binding protein. In some embodiments, the target binding protein may be modified such that a therapeutic agent may be conjugated to the target binding protein at a specific location on the target binding protein. For example, the target binding protein may be partially reduced in a manner that facilitates conjugation to the target binding protein. In such cases, partial reduction of the target binding protein may occur such that the conjugation site within the target binding protein is not reduced. In some embodiments, the conjugation site(s) on the target binding protein may be selected to facilitate conjugation of an agent at a specific location on the protein construct. Various factors may affect the "reduction level" of the target binding protein upon treatment with a reducing agent. For example, but not limited to, the ratio of reducing agent to target binding protein, the length of incubation, the incubation temperature, and / or the pH of the reduction reaction solution may require optimization to achieve partial reduction of the target binding protein using the methods and materials described herein. Any suitable combination of factors (e.g., the ratio of reducing agent to target binding protein, the length and temperature of incubation with the reducing agent, and / or the pH of the reducing agent) can be used to achieve partial reduction of the target binding protein (e.g., general reduction of potential conjugation sites or reduction at specific conjugation sites).

[0158] An effective ratio of reducing agent to target binding protein can be any ratio that at least partially reduces the target binding protein in a manner that allows for conjugation with an agent (e.g., general reduction of potential conjugation sites or reduction at specific conjugation sites). In some embodiments, the ratio of reducing agent to target binding protein can be in the range of about 20:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 20:1 to 1:1.5, 10:1 to 1:1.5, 9:1 to 1:1.5, 8:1 to 1:1.5, 7:1 to 1:1.5, 6:1 to 1:1.5, 5:1 to 1:1.5, 4:1 to 1:1.5, 3:1 to 1:1.5, 2:1 to 1:1.5, 1.5:1 to 1:1.5, or 1:1 to 1:1.5.

[0159] Effective incubation times and temperatures for treating a target binding protein with a reducing agent can be any times and temperatures that at least partially reduce the target binding protein in a manner that allows for conjugation of an agent to the target binding protein (e.g., general reduction of potential conjugation sites or reduction at specific conjugation sites). In some embodiments, incubation times and temperatures for treating the target binding protein can range from about 1 hour at 37° C. to about 12 hours at 37° C. (or any subrange therein).

[0160] An effective pH for a reduction reaction for treating a target binding protein with a reducing agent can be any pH that at least partially reduces the target binding protein in a manner that allows for conjugation of the target binding protein with an agent (e.g., general reduction of potential conjugation sites or reduction at specific conjugation sites).

[0161] When the partially reduced target binding protein is contacted with a thiol-containing agent, the agent can be conjugated with the interchain thiol in the target binding protein. The agent can be modified to contain a thiol using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, the target binding protein can be partially reduced after incubation with a reducing agent (e.g., TEPC) at about 37°C for about 1 hour at a desired ratio of reducing agent to target binding protein. The effective ratio of reducing agent to target binding protein can be any ratio that partially reduces at least two interchain disulfide bonds located in the target binding protein in a manner that allows conjugation of the thiol-containing agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0162] In some embodiments, the target binding protein may be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds. In some embodiments, the target binding protein may be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0163] In some embodiments, the agent (e.g., an agent conjugated to a target binding protein) may be a detectable moiety, such as, for example, a label or other marker. For example, the agent may be or may include a radiolabeled amino acid, one or more biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety may be attached by a spacer molecule. In some embodiments, the detectable label may include an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent may include a radioisotope. In some embodiments, the radioisotope may be indium or technetium. In some embodiments, the contrast agent may include iodine, gadolinium, or iron oxide. In some embodiments, the enzyme may include horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label may include yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein t dimer 2 (RFP t dimer 2), HCRED, or europium derivatives. In some embodiments, the luminescent label may include N-methylacridium derivatives. In some embodiments, the label may include an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label may include biotin, avidin, streptavidin, or one or more haptens.

[0164] Further examples of detectable labels also include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Contains H.

[0165] In some embodiments, the agent may be conjugated to the target binding protein using a carbohydrate moiety, a sulfhydryl group, an amino group, or a carboxylate group. In some embodiments, the agent may be conjugated to the target binding protein via a linker and / or CM described herein. In some embodiments, the agent may be conjugated to a cysteine ​​or lysine in the target binding protein. In some embodiments, the agent may be conjugated to another residue of the target binding protein, such as a residue disclosed herein.

[0166] In some embodiments, various bifunctional protein binding agents can be used to conjugate the agent to the target binding protein, including N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, radionucleotides can be conjugated to target binding proteins using carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agents (see, e.g., WO94 / 11026).

[0167] Suitable conjugation moieties include those described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to target binding proteins via oligopeptides. In some embodiments, suitable conjugation moieties include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co. catalog number 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co. catalog number 24510) conjugated with EDC. Additional exemplary conjugation moieties include SMCC, sulfo-SMCC, SPDB, and sulfo-SPDB.

[0168] The conjugation moieties may contain components with different properties, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. In addition, SMPT can form conjugates with increased stability that contain sterically hindered disulfide bonds. Disulfide bonds are generally less stable than other bonds because disulfide bonds are cleaved in vitro, resulting in less available conjugates. Sulfo-NHS can particularly increase the stability of carbodiimide bonds. When carbodiimide bonds (e.g., EDC) are used in conjugation with sulfo-NHS, they form esters that are more resistant to hydrolysis than the carbodiimide conjugation reaction alone.

[0169] Those of skill in the art will recognize that a wide variety of possible moieties can be conjugated to the target-binding proteins of the present disclosure. (See, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.) In general, effective conjugation of an agent (e.g., a cytotoxic agent) to a target-binding protein can be achieved by any chemical reaction that bonds the agent to the target-binding protein while allowing the agent and target-binding protein to retain functionality.

[0170] Nucleic Acids and Vectors In some aspects, the disclosure further provides a nucleic acid comprising a sequence encoding a target binding protein, or a component or fragment thereof. The nucleic acid may comprise coding sequences for the heavy chain variable domain, the light chain variable domain, the TB, the CM, the MM, the EM, and the linker(s) in the target binding protein. If the target binding protein comprises multiple peptides (e.g., multiple TBs on different peptides, or one TB comprises multiple peptides), the nucleic acid may comprise coding sequences for multiple peptides. In some examples, the coding sequence for one of the peptides is located in one nucleic acid and the coding sequence for another of the peptides is located in another nucleic acid. In some examples, the coding sequences for two or more of the multiple peptides are located in the same nucleic acid. The disclosure includes polynucleotides encoding the proteins or portions thereof described herein, and the use of such polynucleotides to produce proteins and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.) encoding the proteins defined herein. The disclosure includes compositions comprising such polynucleotides. In some aspects, such compositions may be used therapeutically or prophylactically.

[0171] Unless otherwise specified, a "nucleic acid sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and therefore code for the same amino acid sequence. The term "nucleic acid" refers to deoxyribonucleic acid (DNA), or ribonucleic acid (RNA), or combinations thereof, in single- or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses complementary sequences in addition to the sequence explicitly indicated. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid includes mixtures or hybrids of DNA and RNA.

[0172] The term "N-terminally positioned", when referring to the position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, means that the first domain is positioned closer to the N-terminus of the polypeptide primary amino acid sequence. In some embodiments, there may be additional sequences and / or domains between the first domain or sequence and the second domain or sequence. The term "C-terminally positioned", when referring to the position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, means that the first domain is positioned closer to the C-terminus of the polypeptide primary amino acid sequence. In some embodiments, there may be additional sequences and / or domains between the first domain or sequence and the second domain or sequence.

[0173] Modifications can be introduced into nucleotide sequences by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include the aliphatic hydroxyamino acids (e.g., serine and threonine), the amide family (e.g., asparagine and glutamine), the aliphatic family (e.g., alanine, valine, leucine and isoleucine), and the aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0174] The present disclosure further provides vectors and sets of vectors comprising any of the nucleic acids described herein. One skilled in the art would be able to select a suitable vector or set of vectors (e.g., expression vectors) for making any of the target binding proteins described herein, and use the vector or set of vectors to express any of the target binding proteins described herein. For example, in selecting a vector or set of vectors, a cell type can be selected such that the vector(s) can be integrated into the chromosome of the cell and / or replicate therein. Exemplary vectors that can be used to produce target binding proteins are also described herein. As used herein, the term "vector" refers to a polynucleotide that can induce expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any of the cells described herein). A "vector" can deliver nucleic acids and fragments thereof into a host cell, and includes control sequences (e.g., promoters, enhancers, poly(A) signals). An exogenous polynucleotide can be inserted into an expression vector for expression. The term "vector" also encompasses artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0175] Methods for constructing suitable vectors containing any of the nucleic acids described herein and suitable for transforming cells (e.g., mammalian cells) are well known in the art. See, for example, Sambrook et al., Eds. "Molecular Cloning: A Laboratory Manual," 2 nd Ed., Cold Spring Harbor Press, 1989, and Ausubel et al., Eds. "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0176] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vectors), adeno-associated viral (AAV) vectors, lentiviral vectors, and retroviral vectors), and any Gateway® vectors. A vector can, for example, contain sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. One of skill in the art will be able to select suitable vectors and mammalian cells for making any of the target binding proteins described herein.

[0177] In some embodiments, target binding proteins can be made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0178] cell In some aspects, the disclosure provides a host cell comprising any of the vectors or nucleic acids described herein. The cell can be used to produce the target binding protein described herein. In some embodiments, the cell can be an animal cell, a mammalian cell (e.g., a human cell), a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, or a guinea pig cell), a non-human primate cell, an insect cell, a bacterial cell, a fungal cell, or a plant cell. In some embodiments, the cell can be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell that has a distinct, membrane-bound nucleus. Such cells can include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, avian, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some embodiments, the cell may be a prokaryotic cell.

[0179] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or sets of vectors described herein) into cells are known in the art. Examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0180] In some embodiments, the introducing step comprises introducing into the cell a vector (e.g., any vector or set of vectors described herein) that contains nucleic acids encoding monomers that make up any of the target binding proteins described herein.

[0181] Compositions and kits The present disclosure also provides compositions and kits that include the target binding proteins described herein. The compositions and kits may further include one or more excipients, carriers, reagents, and instructions required for use of the target binding proteins.

[0182] In some embodiments, the composition may be a pharmaceutical composition comprising the target binding protein, its derivatives, fragments, analogs, and homologs. The pharmaceutical composition may comprise the target binding protein and a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils may also be used. The use of such media and agents for pharma- ceutical active substances is well known in the art. Insofar as any conventional media or agent is incompatible with the active compound, their use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition.

[0183] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain one or more of the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetic acid, citric acid, or phosphate, and agents for adjusting osmotic pressure such as sodium chloride or dextrose. PH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials. In some cases, any of the target binding proteins described herein are prepared with carriers that protect against rapid elimination from the body, such as sustained release and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such pharmaceutical compositions and formulations are clear to those skilled in the art. For example, the target binding protein can be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, colloid drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, prepared, for example, by coacervation techniques or by interfacial polymerization.

[0184] A sustained release preparation can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and y-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time.

[0185] In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL □(BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The composition may be sterile and should be fluid to the extent that easy syringability exists. It is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, can be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0186] In some embodiments, pharmaceutical compositions may include sterile injectable solutions.Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersions can be prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and other required components from the above-listed components.In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which allows the powder of active ingredient plus any additional desired ingredients to be obtained from the solution that has been previously sterilized and filtered.

[0187] In some embodiments, pharmaceutical compositions may include oral compositions. Oral compositions may include inert diluents or edible carriers. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, active compounds may be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, where the fluid carrier containing the compound is poured into the mouth, swished, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor, or compounds of a similar nature.

[0188] In some embodiments, the pharmaceutical composition can be formulated for administration by inhalation. For example, the compound can be delivered in the form of an aerosol spray from a pressured container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0189] In some embodiments, the pharmaceutical composition can be formulated for systemic administration. For example, systemic administration can be intravenous administration, or transmucosal or transdermal administration. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound can be generally formulated into ointments, salves, gels, or creams, as is known in the art.

[0190] In some embodiments, pharmaceutical compositions can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0191] In one embodiment, pharmaceutical compositions can be prepared with carriers that protect the compound from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparing such formulations will be clear to those skilled in the art.

[0192] For ease of administration and uniformity of dosage, oral or parenteral compositions can be advantageously formulated in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined and can directly depend on the unique characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the art of compounding such active compound for the treatment of individuals.

[0193] In some embodiments, the compositions (eg, pharmaceutical compositions) may be included in a container, vial, syringe, injector pen, pack, or dispenser, optionally together with instructions for administration.

[0194] Also provided herein is a kit that includes any of the target binding proteins described herein, any of the compositions that include any of the target binding proteins described herein, or any of the pharmaceutical compositions that include any of the target binding proteins described herein.Also provided is a kit that includes one or more second therapeutic agent(s) in addition to the target binding proteins described herein.The second therapeutic agent(s) can be provided in a dosage form that is separate from the target binding protein.Alternatively, the second therapeutic agent(s) can be formulated together with the target binding protein.

[0195] Any of the kits described herein may include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the target binding proteins described herein. In some embodiments, the kits may include instructions for performing any of the methods described herein. In some embodiments, the kits may include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits may provide a syringe for administering any of the pharmaceutical compositions described herein.

[0196] Also provided herein are target binding proteins produced by any of the methods described herein. Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the target binding proteins produced by any of the methods described herein. Also provided herein are kits comprising at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0197] Methods for Producing Target-Binding Proteins Provided herein is a method of producing any of the target binding proteins described herein, comprising: (a) culturing any of the recombinant host cells described herein in a liquid culture medium under conditions sufficient to produce the target binding protein; and (b) recovering the target binding protein from the host cells and / or the liquid culture medium.

[0198] Methods for culturing cells are well known in the art. In some embodiments, cells can be maintained in vitro under conditions that favor cell proliferation, cell differentiation, and cell growth. For example, cells can be cultured by contacting cells (e.g., any of the cells described herein) with a cell culture medium that contains the necessary growth factors and nutritional supplements sufficient to support cell viability and growth.

[0199] In some embodiments, the method may further include isolating the recovered target binding protein. Isolation of the target binding protein can be performed using any protein separation or purification technique, for example, exemplary isolation methods include isolation using a protein purification tag (e.g., His tag), ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand-affinity chromatography, ion exchange chromatography (e.g., anion or cation), and hydrophobic interaction chromatography.

[0200] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow for the formation of a disulfide bond between the MM and TB of the target binding protein.

[0201] In some embodiments, the method further comprises formulating the isolated target binding protein into a pharmaceutical composition.Various formulations are known in the art and described herein.Any isolated target binding protein described herein can be formulated for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0202] Methods of Using Target Binding Proteins In some aspects, the disclosure further provides methods of using the target binding proteins herein. In some embodiments, the disclosure provides a method of treating a disease in a subject (e.g., cancer (e.g., any of the cancers described herein)) comprising administering to the subject a therapeutically effective amount of any of the target binding proteins described herein. In some embodiments, the disclosure provides a method of preventing, delaying the progression of, treating, alleviating symptoms, or ameliorating a disease in a subject by administering to a subject in need thereof a therapeutically effective amount of a target binding protein described herein. The term "treatment" means improving at least one symptom of a disorder. The disorder can be cancer, an autoimmune disease, an infectious disease, chronic inflammation, or transplant rejection (e.g., in kidney, liver, or heart transplants). In some embodiments, the disorder being treated is cancer, an autoimmune disease (e.g., type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease), or an infectious disease (e.g., chickenpox, common cold, diphtheria, E. coli, giardiasis, rheumatoid arthritis ... The following diseases are considered to be causing the disease: A, HIV / AIDS, Infectious Mononucleosis, Influenza (Influenza (flu)), Lyme Disease, Malaria, Measles, Meningitis, Mumps, Polio, Pneumonia, Rocky Mountain Spotted Fever, 3-day Rubella (German Rubella), Salmonella Infection, Severe Acute Respiratory Syndrome (SARS), Sexually Transmitted Diseases, Shingles (Shigs), Tetanus, Toxic Shock Syndrome, Tuberculosis, Viral Hepatitis, West Nile Virus, Whooping cough (Pertussis), and the treatment is to improve at least one symptom of the cancer, autoimmune disease, or infectious disease.

[0203] As used herein, the term "subject" refers to any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster, or a guinea pig), a non-human primate (e.g., an ape (e.g., a monkey (e.g., a baboon, a marmoset), or a tailless ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some embodiments, the subject is a human. The terms subject and patient are used interchangeably herein. In some embodiments, the subject has been previously identified or diagnosed as having a disease (e.g., a cancer (e.g., any of the cancers described herein)).

[0204] In some embodiments, a subject may be identified as having a mutation in the HER2 gene that increases the expression and / or activity of HER2 in a mammalian cell (e.g., any of the mammalian cells described herein). For example, a mutation in the HER2 gene that increases the expression and / or activity of HER2 in a mammalian cell may be a mutation that results in expression of HER2 with gene duplication, one or more amino acid substitutions (e.g., one or more amino acid substitutions selected from the group consisting of G309A, G309E, S310F, R678Q, L755S, L755W, I767M, D769H, D769Y, V777L, Y835F, V842I, R896C, and G1201V) (compared to the wild-type protein). See, e.g., Weigelt and Reis-Filho, Cancer Discov. 2013, 3(2):145-147.

[0205] Non-limiting examples of the method for detecting HER2-related disease in a subject include immunohistochemistry, fluorescent in situ hybridization (FISH), and chromogenic in situ hybridization (CISH).See, for example, Yan et al., Cancer Metastasis Rev. 2015, 34:157-164.

[0206] The therapeutically effective amount of the target binding protein of the present disclosure generally relates to the amount required to achieve a therapeutic purpose. As mentioned above, this may be a binding interaction between an antibody and its target antigen, which in certain cases interferes with the function of the target. The amount required for administration will further depend on the binding affinity of the target binding protein to its specific target, and also on the rate at which the administered target binding protein is depleted from the free volume of the other subject to which it is administered. The general range of therapeutically effective dosage of the target binding protein of the present disclosure may be, as a non-limiting example, about 0.001, 0.01, 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / kg body weight or more. The structure of the target binding protein of the present disclosure allows the dosage of the target binding protein to be reduced to be administered to a subject, compared to conventional target binding proteins and compared to conventional antibodies. For example, the dose administered on a unit dosage basis or the total dose over a dosing regimen can be reduced by 10, 20, 30, 40, or 50% compared to the corresponding dose of a corresponding conventional target binding protein or corresponding conventional antibody.

[0207] Typical dosing frequencies can range, for example, once or twice daily, weekly, biweekly, or monthly.

[0208] The efficacy of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods for screening target-binding proteins with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immunologically mediated techniques known in the art.

[0209] In another embodiment, target binding proteins directed to two or more targets are used in methods known in the art related to target localization and / or quantification (e.g., for use in measuring the levels of one or more targets in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc.) In a given embodiment, target binding proteins directed to two or more targets that contain an antigen-binding domain derived from an antibody, or derivatives, fragments, analogs, or homologs thereof, are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0210] The target binding proteins used in any of these method and use embodiments may be administered at any stage of disease. For example, such target binding proteins may be administered to patients suffering from any stage of cancer, from early stages to metastatic stages. In some embodiments, the target binding proteins and formulations thereof may be administered to subjects suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity.

[0211] Subjects suffering from or susceptible to diseases or disorders associated with abnormal target expression and / or activity can be identified using any of a variety of methods known in the art.For example, subjects suffering from cancer or other neoplastic conditions can be identified using any of a variety of clinical and / or laboratory tests, such as physical examination and blood, urine, and / or stool analysis, to assess health status.For example, subjects suffering from inflammation and / or inflammatory disorders can be identified using any of a variety of clinical and / or laboratory tests, such as physical examination and / or body fluid analysis, such as blood, urine, and / or stool analysis, to assess health status.

[0212] In some embodiments, administration of a target binding protein to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if any of a variety of laboratory or clinical objectives are achieved. For example, administration of a target binding protein to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if one or more of the symptoms associated with the disease or disorder are alleviated, relieved, inhibited, or do not progress to a further, i.e., worse state. Administration of a target binding protein to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if the disease or disorder goes into remission or does not progress to a further, i.e., worse state.

[0213] As used herein, the term "treatment" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer (e.g., any of the cancers described herein)) in a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treatment results in a reduction in cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0214] In some embodiments, the disease may be cancer. In some embodiments, the subject may have been identified or diagnosed with cancer. Examples of cancer include solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B cell neoplasms, multiple myeloma, lymphomas (e.g., B cell lymphoma, B cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T cell lymphoma), leukemias (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloma, myeloma, leukemia ... The cancers include myeloma, myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is Burkitt's lymphoma. In some aspects, the subject has been identified or diagnosed as having a familial cancer syndrome, such as Li-Fraumeni syndrome, familial breast-ovarian cancer (BRCA1 or BRCA2 mutation) syndrome, and the like. The disclosed methods are also useful for treating non-solid cancers. Exemplary solid tumors include malignant tumors (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as the lung, breast, lymphatic system, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., renal, urothelial, or testicular tumors), pharynx, prostate, and ovary. Exemplary adenocarcinomas include colorectal carcinoma, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine. Further examples of cancers that may be treated by the compositions and methods herein include acute lymphocytic leukemia, adult; acute lymphocytic leukemia, pediatric; acute myeloid leukemia, adult; adrenocortical carcinoma; adrenocortical carcinoma, pediatric; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma, pediatric cerebellum; astrocytoma, pediatric cerebral; cholangiocarcinoma, extrahepatic; bladder cancer; bladder cancer, pediatric; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma, pediatric; brain tumor, adult; brain tumor, brain stem glioma, pediatric; brain tumor, cerebellar astrocytoma, pediatric; brain tumor, cerebral astrocytoma / malignant glioma, pediatric; brain tumor, ependymoma, pediatric;Brain tumors, medulloblastoma, children;Brain tumors, supratentorial primitive neuroectodermal tumor, children;Brain tumors, visual pathway and hypothalamic glioma, children;Brain tumors, children (other);Breast cancer;Breast cancer and pregnancy;Breast cancer, children;Breast cancer, male;Bronchial adenoma / carcinoid, children;Carcinoid tumors, children;Carcinoid tumors, gastrointestinal;Cytoma, adrenal cortical;Cytoma, islet cell;Carcinoma of unknown primary;Central nervous system lymphoma, primary;Cerebellar astrocytoma, children;Cerebral astrocytoma / malignant glioma, children;Cervical cancer;Childhood cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Clear cell sarcoma of tendon sheath;Colon cancer;Colorectal cancer, small pediatric;cutaneous T-cell lymphoma;endometrial cancer;ependymoma, pediatric;epithelial carcinoma, ovarian;esophageal cancer;esophageal cancer, pediatric;Ewing's family of tumors;extracranial germ cell tumors, pediatric;extragonadal germ cell tumors;extrahepatic bile duct cancer;eye cancer, intraocular melanoma;eye cancer, retinoblastoma;gallbladder cancer;gastric (stomach) cancer;gastric (stomach) cancer, pediatric;gastrointestinal carcinoid tumors;germ cell tumors, extracranial, pediatric;germ cell tumors, extragonadal;germ cell tumors, ovarian;gestational trophoblastic tumors;glioma, pediatric brainstem;glioma, pediatric visual pathway and hypothalamus;hairy cell leukemia;head and neck cancer;hepatocellular (liver) carcinoma, adult ( primary);hepatocellular (liver) cancer, children (primary);Hodgkin's lymphoma, adults;Hodgkin's lymphoma, children;Hodgkin's lymphoma in pregnancy;hypopharyngeal cancer;hypothalamic and visual pathway glioma, children;intraocular melanoma;islet cell carcinoma (pancreatic islet);Kaposi's sarcoma;renal cancer;laryngeal cancer;laryngeal cancer, children;leukemia, acute lymphocytic leukemia, adults;leukemia, acute lymphocytic leukemia, children;leukemia, acute myeloid leukemia, adults;leukemia, acute myeloid leukemia, children;leukemia, chronic lymphocytic;leukemia, chronic myeloid;leukemia, hairy cell;lip and oral cavity cancer;liver cancer, adults (primary);liver cancer, children (primary);lung Cancer, non-small cell;Lung cancer, small cell;Lymphocytic leukemia, adult acute;Lymphoblastic leukemia, childhood acute;Lymphocytic leukemia, chronic;Lymphoma, AIDS-related;Lymphoma, central nervous system (primary);Lymphoma, cutaneous T cell;Lymphoma, Hodgkin lymphoma, adult;Lymphoma, Hodgkin lymphoma, childhood;Lymphoma during pregnancy, Hodgkin lymphoma;Lymphoma, non-Hodgkin lymphoma, adult;Lymphoma, non-Hodgkin lymphoma, childhood;Lymphoma during pregnancy, non-Hodgkin lymphoma;Lymphoma, primary central nervous system;Macroglobulinemia, Waldenstrom's disease, male breast cancer;Malignant mesothelioma, adult;Malignant mesothelioma, childhood;Malignant thymoma;Medulloblastoma, childhood;Malignant melanoma;Malignant melanoma, intraocular;Merkel cell carcinoma;Mesothelioma, malignant;Metastatic squamous cell carcinoma of the neck of unknown primary;Multiple endocrine neoplasia syndrome, childhood;Multiple myeloma / plasma cell neoplasm;Mycosis fungoides;Myelodysplastic syndrome;Myeloid leukemia, chronic;Myeloid leukemia, childhood acute;Myeloma, multiple;Myeloproliferative disorders, chronic;Nasal and paranasal sinus cancer;Nasopharyngeal carcinoma;Nasopharyngeal carcinoma, childhood;Neuroblastoma;Non-Hodgkin's lymphoma, adult;Non-Hodgkin's lymphoma, childhood;Non-Hodgkin's lymphoma in pregnancy;Non-small cell lung cancer;Oral Cancer, pediatric;Oral cavity and lip cancer;Oropharyngeal cancer;Osteosarcoma / malignant fibrous histiocytoma of bone;Ovarian cancer, pediatric;Ovarian epithelial cancer;Ovarian germ cell tumor;Ovarian low malignant potential tumor;Pancreatic cancer;Pancreatic cancer, pediatric;Pancreatic cancer, islet cell;Sino-nasal and nasal cancer;Parathyroid cancer;Penile cancer;Pheochromocytoma;Pineal and supratentorial primitive neuroectodermal tumors, pediatric;Pituitary tumors;Plasma cell neoplasms / multiple myeloma;Pleuropulmonary blastoma;Pregnancy and breast cancer;Pregnancy and Hodgkin's lymphoma;Pregnancy and non-Hodgkin's lymphoma;Primary central nervous system lymphoma;Primary liver cancer, adult;Primary liver cancer, pediatric;Prostate cancer;Rectal cancer;Renal cell alveolar (kidney) cancer;renal cell carcinoma, children;renal pelvis and ureter, transitional cell carcinoma;retinoblastoma;rhabdomyosarcoma, children;salivary gland cancer;salivary gland cancer, children;sarcoma, Ewing's family of tumors;sarcoma, Kaposi's sarcoma;sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;sarcoma, rhabdomyosarcoma, children;sarcoma, soft tissue, adults;sarcoma, soft tissue, children;Sezary syndrome;skin cancer;skin cancer, children;skin cancer (melanoma);skin cancer, Merkel cell;small cell lung cancer;small intestine cancer;soft tissue sarcoma, adults;soft tissue sarcoma, children;squamous cell carcinoma of the neck of unknown primary, metastatic;stomach (gastric) cancer;stomach (gastric) cancer Cancer, pediatric; supratentorial primitive neuroectodermal tumor, pediatric; T-cell lymphoma, skin; testicular cancer; thymoma, pediatric; thymoma, malignant; thyroid cancer; thyroid cancer, pediatric; transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, gestational; primary site unknown, pediatric cancer; rare cancer of childhood; ureter and renal pelvis, transitional cell carcinoma; urethral cancer; uterine sarcoma; vaginal cancer; visual pathway and hypothalamic glioma, pediatric; vulvar cancer; Waldenstrom's macroglobulinemia; Wilms' tumor; diffuse large B-cell lymphoma (DLBCL); and mantle cell lymphoma (MCL). Metastasis of the aforementioned cancers can also be treated or prevented according to the methods described herein.;

[0215] In some embodiments, the methods herein may result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., compared to the number, severity, or frequency of one or more symptoms of cancer in a subject prior to treatment).

[0216] The method may further include administering one or more additional agents to the subject. In some embodiments, the additional agent(s) may be a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine. In some embodiments, the additional agent(s) may be a checkpoint inhibitor, a kinase inhibitor, an agent that targets inhibitors in the tumor microenvironment, and / or a T cell or NK agonist. In some embodiments, the additional agent(s) may be radiation therapy alone or in combination with another additional agent(s), such as a chemotherapeutic agent or an anti-tumor agent. In some embodiments, the additional agent(s) may be a vaccine, an oncovirus, and / or a DC activator, such as, by way of non-limiting example, a Toll-like receptor (TLR) agonist and / or α-CD40. In some embodiments, the additional agent(s) may be a tumor-targeting antibody (e.g., an antibody drug conjugate (ADC)) designed to kill tumors via ADCC or via direct conjugation with a toxin.

[0217] In some embodiments, the checkpoint inhibitor can be an inhibitor of a protein such as CTLA-4, LAG-3, PD-1, PD-1, TIGIT, TIM-3, B7H4, BTLA, or Vista. In some embodiments, the kinase inhibitor can be B-RAFi, MEKi, Btk inhibitor, ibrutinib, or crizotinib. In some embodiments, the tumor microenvironment inhibitor can be IDO inhibitor, alpha-CSF1R inhibitor, alpha-CCR4 inhibitor, TGF-beta, myeloid-derived suppressor cell, or regulatory T cell. In some embodiments, the agonist can be Ox40, GITR, CD137, ICOS, CD27, or HVEM.

[0218] In some embodiments, the target binding protein may be administered during and / or after treatment in combination with one or more additional agents. In some embodiments, the target binding protein may be formulated into a single therapeutic composition, and the target binding protein and the additional agent(s) may be administered simultaneously. Alternatively, the target binding protein and the additional agent(s) may be separated from each other, e.g., each formulated into a separate therapeutic composition, and the target binding protein and the additional agent(s) are administered simultaneously, or the target binding protein and the additional agent(s) are administered at different times during the treatment regimen. For example, the target binding protein may be administered before the administration of the additional agent, after the administration of the additional agent, or alternately. The target binding protein and the additional agent(s) may be administered in a single dose or multiple doses.

[0219] One or more of the target binding proteins herein may be co-formulated with and / or co-administered with one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzyme inhibitors. Examples of drugs or inhibitors that may be used include nonsteroidal anti-inflammatory drug(s) (NSAIDs), such as ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such as prednisolone; cytokine suppressive anti-inflammatory drug(s) (CSAIDs); inhibitors of nucleotide biosynthesis, such as inhibitors of purine biosynthesis, folate antagonists (e.g., methotrexate (N-[4-[[(2,4 -diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid); and inhibitors of pyrimidine biosynthesis, such as dihydroorotate dehydrogenase (DHODH) inhibitors. Suitable therapeutic agents for use in combination with the antibodies of the present disclosure include one or more of: NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g., methotrexate). Examples of additional inhibitors include corticosterone. and mTOR inhibitors, such as sirolimus; rapamycin or rapamycin derivatives, such as soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); agents that interfere with signaling by proinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors, such as celecoxib, rofecoxib, and variants thereof; phosphodiesterase inhibitors, such as R973401 (phosphodiesterase IV inhibitors); phospholipase inhibitors, such as inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone analogs); inhibitors of vascular endothelial growth factor or growth factor receptors, such as VEGF inhibitors and / or VEGF-R inhibitors; and inhibitors of angiogenesis.Suitable therapeutic agents for use in combination with the antibodies of the disclosure are immunosuppressants, e.g., cyclosporine, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus (rapamycin) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2 inhibitors, e.g., celecoxib and variants thereof, and phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g., trifluoromethyl ketone analogues. Additional examples of therapeutic agents that can be combined with the antibodies of the present disclosure include one or more of the following: 6-mercaptopurine (6-MP); azathioprine sulphasalazine; mesalazine; olsalazine; chloroquine / hydroxychloroquine; pencilamine; aurothiornalate (intramuscular and oral); azathioprine; colchicine; beta-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeteral); xanthines (theophylline, arninophilin); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

[0220] The disclosure also provides methods for detecting the presence or absence of a cleavage agent and / or a target in a subject or sample. Such methods may include: (i) contacting the subject or biological sample with a target binding protein, where the target binding protein includes a detectable label disposed on a portion of the target binding protein that is released after cleavage of the CM substrate, and (ii) measuring a level of activated target binding protein in the subject or biological sample, where a detectable level of activated target binding protein in the subject or biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and / or sufficiently absent in the subject or biological sample such that target binding and / or protease cleavage of the target binding protein cannot be detected in the subject or biological sample, and a reduced detectable level of activated target binding protein in the subject or biological sample indicates that the cleavage agent and the target are present in the subject or biological sample.

[0221] The reduced level of detectable label can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction, or substantially 100% reduction. In some embodiments, the detectable label can be conjugated to a component of the target binding protein, such as TB. In some embodiments, measuring the level of the target binding protein in a subject or sample can be achieved using a secondary reagent that specifically binds to the activating antibody, the reagent comprising a detectable label. The secondary reagent can be an antibody comprising a detectable label.

[0222] In some embodiments, the target binding protein is also useful for detecting targets in patient samples, and thus is useful as a diagnostic agent. For example, the target binding protein can be used in an in vitro assay, such as an ELISA, to detect target levels in patient samples. For example, the target binding protein can be immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized target binding protein can function as a capture antibody for any target that may be present in a test sample. Before contacting the immobilized antibody with a patient sample, the solid support can be rinsed and treated with a blocking agent, such as milk protein or albumin, to prevent non-specific adsorption of the analyte.

[0223] In some embodiments, based on the results obtained using the target binding protein in an in vitro diagnostic assay, the stage of disease in a subject can be determined based on the expression level of the target antigen. For a particular disease, blood samples can be taken from subjects diagnosed as being at different stages of disease progression and / or at different points in the therapeutic treatment of the disease. A population of samples that provides statistically significant results for each stage of progression or treatment is used to specify a range of concentrations of the antigen that can be considered characteristic of each stage.

[0224] The target binding proteins herein may also be used in diagnostic and / or imaging methods. In some embodiments, such methods may be in vitro methods. In some embodiments, such methods may be in vivo methods. In some embodiments, such methods may be in situ methods. In some embodiments, such methods may be ex vivo methods. For example, a target binding protein having a CM may be used to detect the presence or absence of an enzyme capable of cleaving the CM. Such target binding proteins may be used in diagnostics, which may include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as may provide for reduction of disulfide bonds) via the measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of the target binding protein) in specific cells or tissues of a particular host organism. Such accumulation of activated antibodies indicates not only that the tissue expresses enzyme activity (or increased reduction potential depending on the nature of the CM), but also that the tissue expresses the target to which the activated antibody binds.

[0225] For example, the CM can be selected to be a protease substrate for proteases found at the site of a tumor, at the site of a viral or bacterial infection in a biologically restricted site (e.g., in an abscess, in an organ, etc.), and the like. The TB can be one that binds to a target antigen. Using methods well known to those skilled in the art, a detectable label (e.g., a fluorescent label or a radioactive label or a radioactive tracer) can be conjugated to the TB or other region of the target binding protein. Suitable detectable labels can be considered in the context of the screening methods described above, with additional specific examples provided below. By using a TB specific for a disease state protein or peptide together with a protease with increased activity in the diseased tissue of interest, the target binding protein can show an increased rate of binding to the diseased tissue compared to tissue in which the CM-specific enzyme is not present at detectable levels, is present at a lower level than in the diseased tissue, or is inactive (e.g., in the form of a proenzyme or complexed with an inhibitor). Because small proteins and peptides are rapidly cleared from the blood by the renal filtration system and because enzymes specific for CM are not present at detectable levels (or are present at lower levels or in an inactive conformation in non-diseased tissue), accumulation of activating antibodies in diseased tissue may be enhanced compared to non-diseased tissue.

[0226] In some embodiments, the target binding protein may be useful for in vivo imaging, where detection of a fluorescent signal in a subject, e.g., a mammal, including a human, indicates that a disease site contains a target and a protease specific for the CM of the target binding protein. In vivo imaging may be used to identify or narrow down a patient population suitable for treatment with the target binding protein of the present disclosure. For example, a patient who tests positive for both the target and a protease that cleaves a substrate in the CM of the target binding protein being tested (e.g., accumulates activating antibodies at the disease site) may be identified as a suitable candidate for treatment with such target binding protein that contains such CM. Similarly, a patient who tests negative may be identified as a suitable candidate for another form of therapy (i.e., not suitable for treatment with the target binding protein being tested). In some embodiments, such patients who test negative for a first target binding protein may be tested with other target binding proteins that contain different CMs (e.g., a target binding protein that contains a CM that is cleaved by the patient at the site of disease) until a suitable target binding protein for treatment is identified.

[0227] In some embodiments, in situ imaging can be useful in methods to identify which patients to treat. For example, in situ imaging can use target binding proteins to screen patient samples to identify patients with the appropriate protease(s) and target(s) in the appropriate location, e.g., tumor site. In some embodiments, in situ imaging is used to identify or narrow a patient population suitable for treatment with the target binding proteins of the present disclosure. For example, patients who test positive for both the target and the protease that cleaves the substrate in the CM of the target binding protein being tested (e.g., accumulate activating antibodies at the disease site) are identified as suitable candidates for treatment with such target binding protein containing such CM. Similarly, patients who test negative for either or both the target and the protease that cleaves the substrate in the CM substrate of the target binding protein being tested using these methods are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the target binding protein being tested). In some embodiments, such patients who test negative for a first target binding protein may be tested with other target binding proteins that comprise different CMs (e.g., activatable target binding proteins that comprise a CM that is cleaved by the patient at the site of disease) until an appropriate target binding protein for treatment is identified.

[0228] The present disclosure includes any combination of the following numbered items: 1. A target binding protein comprising: Variable heavy chain complementarity determining region 1 (VH CDR1) comprising the sequence TYAMN (SEQ ID NO: 3), A variable heavy chain complementarity determining region 2 (VH CDR2) comprising the sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), and a heavy chain variable domain comprising a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the sequence HGNFGNSYVSWX1AY (SEQ ID NO: 6), wherein X1 is W or F; A variable light chain complementarity determining region 1 (VL CDR1) comprising the sequence of X2SSTGAVTTSNYX3N (SEQ ID NO: 10), wherein X2 is R or G and X3 is P or V; A variable light chain complementarity determining region 2 (VL CDR2) comprising the sequence GTNKRAP (SEQ ID NO: 14), and a light chain variable domain comprising a variable light chain complementarity determining region 3 (VL CDR3) comprising the sequence of X4LWYSNX5WV (SEQ ID NO: 15), wherein X4 is V or I and X5 is R or L; the heavy chain variable domain and the light chain variable domain are arranged in one or more polypeptides; The target binding protein, wherein said target binding protein specifically binds to CD3 epsilon.

[0229] 2.a. the VH CDR1 comprises the sequence of TYAMN (SEQ ID NO:3); b. the VH CDR2 comprises the sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:5); c. the VH CDR3 comprises the sequence HGNFGNSYVSWWAY (SEQ ID NO:7) or HGNFGNSYVSWFAY (SEQ ID NO:8); d. the VL CDR1 comprises the sequence RSSTGAVTTSNYPN (SEQ ID NO:11), RSSTGAVTTSNYVN (SEQ ID NO:12), or GSSTGAVTTSNYVN (SEQ ID NO:13); e. the VL CDR2 comprises the sequence of GTNKRAP (SEQ ID NO: 14); f. The target binding protein of claim 1, wherein the VL CDR3 comprises the sequence of VLWYSNRWV (SEQ ID NO: 16), VLWYSNLWV (SEQ ID NO: 17), or ILWYSNRWV (SEQ ID NO: 18).

[0230] 3. the VH CDR1 comprises TYAMN (SEQ ID NO:3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), the VL CDR1 comprises RSSTGAVTTSNYPN (SEQ ID NO:11), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16); or the VH CDR1 comprises TYAMN (SEQ ID NO:3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), the VL CDR1 comprises GSSTGAVTTSNYVN (SEQ ID NO:13), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16); or the VH CDR1 comprises TYAMN (SEQ ID NO:3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises HGNFGNSYVSWFAY (SEQ ID NO:8), the VL CDR1 comprises GSSTGAVTTSNYVN (SEQ ID NO:13), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises VLWYSNRWV (SEQ ID NO:16); or the VH CDR1 comprises TYAMN (SEQ ID NO:3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), the VL CDR1 comprises RSSTGAVTTSNYVN (SEQ ID NO:12), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises ILWYSNRWV (SEQ ID NO:18); or the VH CDR1 comprises TYAMN (SEQ ID NO:3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), the VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), the VL CDR1 comprises GSSTGAVTTSNYVN (SEQ ID NO:13), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VL CDR3 comprises VLWYSNLWV (SEQ ID NO:17); or 2. The target binding protein of claim 1, wherein the VH CDR1 comprises TYAMN (SEQ ID NO: 3), the VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO: 7), the VL CDR1 comprises RSSTGAVTTSNYVN (SEQ ID NO: 12), the VL CDR2 comprises GTNKRAP (SEQ ID NO: 14), and the VL CDR3 comprises VLWYSNRWV (SEQ ID NO: 16).

[0231] 4. The target binding protein of item 3, wherein the heavy chain variable domain comprises the VH CDR1 comprising TYAMN (SEQ ID NO: 3), the VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises the VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO: 11), the VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0232] 5. The target binding protein of item 4, wherein the heavy chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:46, and the light chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:49.

[0233] 6. The target binding protein according to item 4, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 46 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 49.

[0234] 7. The target binding protein of item 3, wherein the heavy chain variable domain comprises the VH CDR1 comprising TYAMN (SEQ ID NO: 3), the VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises the VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), the VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0235] 8. The target binding protein of item 7, wherein the heavy chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:46, and the light chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:64.

[0236] 9. The target binding protein according to item 7, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 64.

[0237] 10. The target binding protein of item 3, wherein the heavy chain variable domain comprises the VH CDR1 comprising TYAMN (SEQ ID NO: 3), the VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNSYVSWFAY (SEQ ID NO: 8), and the light chain variable domain comprises the VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), the VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0238] 11. The target binding protein of item 10, wherein the heavy chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 128, and the light chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 122.

[0239] 12. The target binding protein according to item 10, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 128 and the light chain variable domain comprises the sequence of SEQ ID NO: 122.

[0240] 13. The target binding protein of item 3, wherein the heavy chain variable domain comprises the VH CDR1 comprising TYAMN (SEQ ID NO: 3), the VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises the VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO: 12), the VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and the VL CDR3 comprising ILWYSNRWV (SEQ ID NO: 18).

[0241] 14. The target binding protein of item 13, wherein the heavy chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 113.

[0242] 15. The target binding protein according to item 13, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 113.

[0243] 16. The target binding protein of item 3, wherein the heavy chain variable domain comprises the VH CDR1 comprising TYAMN (SEQ ID NO: 3), the VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises the VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), the VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNLWV (SEQ ID NO: 17).

[0244] 17. The target binding protein of item 16, wherein the heavy chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 98.

[0245] 18. The target binding protein according to item 16, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 98.

[0246] 19. The target binding protein of item 3, wherein the heavy chain variable domain comprises the VH CDR1 comprising TYAMN (SEQ ID NO: 3), the VH CDR2 comprising RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNSYVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises the VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO: 12), the VL CDR2 comprising GTNKRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0247] 20. The target binding protein of item 19, wherein the heavy chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain comprises a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 107.

[0248] 21. The target binding protein according to item 19, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 107.

[0249] 22. A target binding protein comprising: A heavy chain variable domain comprising the sequence of EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWX1AYWGX6GTLVTVSS (SEQ ID NO: 20); and a light chain variable domain comprising the sequence of QTVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWVQQKPGX7APRGLIGGTNKRAPGTPARFSGSLJGGKAALTLSGX9QPEDEAEYYCX4LWYSNX5WVFGGGTKLTVL (SEQ ID NO: 21), X1 is W or F, X2 is R or G, X3 is P or V, X4 is V or I, and X5 is R or L; X6 is Q and X7 is Q, or X6 is C and X7 is C; J is L when X9 is V, or J is I when X9 is A; The target binding protein, wherein said target binding protein specifically binds to CD3 epsilon.

[0250] 23. The target binding protein according to item 22, wherein X6 is Q and X7 is Q.

[0251] 24. The target binding protein according to item 22, wherein X6 is C and X7 is C.

[0252] 25. The heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 49; or the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 64; or the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 113; or the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 98; or the heavy chain variable domain comprises the sequence of SEQ ID NO: 46 and the light chain variable domain comprises the sequence of SEQ ID NO: 107; or 23. The target binding protein of claim 22, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 128 and the light chain variable domain comprises the sequence of SEQ ID NO: 122.

[0253] 26. The target binding protein according to any one or combination of items 1 to 25, wherein the heavy chain variable domain and the light chain variable domain are located within the same polypeptide.

[0254] 27. The target binding protein according to item 26, wherein the heavy chain variable domain and the light chain variable domain are linked via a linker.

[0255] 28. The target binding protein according to item 27, wherein the linker has a length of 5 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids.

[0256] 29. The target binding protein according to item 26, comprising the sequence of SEQ ID NO: 50, 28, 114, 99, 108, or 32.

[0257] 30. The target binding protein according to any one or combination of items 26 to 29, wherein the target binding protein comprises a single chain variable fragment (scFv).

[0258] 31. The target binding protein is selected from the group consisting of BiTE, (scFv)2, NANOBODY®, nanobody-HSA VHH-scAb, VHH-Fab, dual scFab, F(ab')2, diabody, CROSSMAB®, DAF(2-in-1), DAE(4-in-1), DUTAMAB®, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab-arm exchange, SEED body, LUZ-Y, FcAb, kl-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, ZYBODY™, DVI-IgG, diabody-CH3, triple body, mini antibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2,

number

number

[0259] 32. The target binding protein according to any one or combination of items 1 to 30, wherein the target binding protein comprises an IgG, IgM, IgA, IgE, or IgD antibody, or a fragment thereof.

[0260] 33. The target binding protein of item 32, wherein the target binding protein comprises an IgG1, IgG2, IgG3, or IgG4 antibody.

[0261] 34. The target binding protein according to any one or combination of items 1 to 33, wherein said target binding protein is humanized.

[0262] 35. The target binding protein according to any one or combination of items 1 to 34, further comprising a masking moiety that inhibits binding of the target binding protein to CD3 in an inactive state.

[0263] 36. The target binding protein of item 35, wherein the masking moiety is attached to the target binding protein via a cleavable moiety, the cleavable moiety being a substrate for a protease.

[0264] 37. The protease is ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartic protease, BACE, renin, aspartic cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, or caspase 8. , caspase 9, caspase 10, caspase 14, cysteine ​​cathepsin, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteinase, cruzipain, legumain, otubain-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, metalloproteinase, meprin, neprilysin, PSMA, BMP-1, MMP , MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine protease, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, granzyme B, guanidinobenzoic acid 37. The target binding protein of any one or combination of items 36, which is tPA, thrombin, tryptase, uPA, type II transmembrane protein, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, or TMPRSS4.

[0265] 38. The target binding protein according to any one or combination of items 1 to 37, wherein the heavy chain variable domain and / or the light chain variable domain is conjugated to a toxin, a radioisotope, a small molecule, a diagnostic agent, a therapeutic macromolecule, a targeting moiety, or a detectable moiety via a conjugate moiety.

[0266] 39. The target binding protein according to item 38, wherein the conjugate moiety is cleavable by a protease.

[0267] 40. The target binding protein according to item 38, wherein the conjugate moiety cannot be cleaved by a protease.

[0268] 41. A composition comprising a target binding protein according to any one or combination of items 1 to 40 and a carrier.

[0269] 42. The composition according to item 41, wherein the composition is a pharmaceutical composition and the carrier is a pharma- ceutically acceptable carrier.

[0270] 43. A container, vial, syringe, injector pen, or kit comprising at least one dose of the composition according to item 41 or 42.

[0271] 44. A nucleic acid comprising a sequence encoding a target binding protein according to any one or combination of items 1 to 40.

[0272] 45. A vector comprising the nucleic acid according to item 44.

[0273] 46. ​​A cell comprising the nucleic acid according to item 44 or the vector according to item 45.

[0274] 47. A method for performing a treatment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a target binding protein according to any one or combination of items 1 to 40, or a composition according to item 41 or 42.

[0275] 48. The method of claim 47, wherein the subject has been identified or diagnosed as having cancer, an inflammatory condition, disorder, or disease, or an autoimmune condition, disorder, or disease.

[0276] 49. A method for producing a target binding protein, comprising: Cultivating the cell of item 46 in a culture medium under conditions sufficient to produce the target binding protein; and recovering said target binding protein from said cells or said culture medium.

[0277] 50. The method of claim 49, further comprising isolating the target binding protein recovered from the cell or the culture medium.

[0278] 51. The method of claim 50, further comprising formulating the target binding protein into a pharmaceutical composition. EXAMPLES

[0279] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0280] Example 1: Production of monovalent bispecific antibodies containing anti-CD3 scFv This example illustrates an exemplary method for producing a monovalent bispecific antibody comprising an anti-CD3 scFv and a Her2 binding domain. The monovalent bispecific antibody was prepared by recombinant methods. The protein was prepared by transforming a host cell with an expression vector of a polynucleotide comprising the coding sequence of the anti-CD3 scFv of SEQ ID NO: 50, 28, 114, 32, 99, 108, or 164, and other components of the monovalent bispecific antibody, followed by culturing the resulting recombinant host cell under conditions suitable for producing the monovalent bispecific antibody comprising the anti-CD3 scFv. The supernatant was harvested 5 days after transformation and the titer of Protein-A binders was measured using the Octet assay (Table 7), followed by purification of the monovalent bispecific antibody using affinity chromatography (Protein A and / or CH1) and size exclusion chromatography methods. Higher Protein A Octet titers generally correlated with the desired higher yields, and several of the CD3 variants exhibited this desired property (Table 7).

[0281] Example 2: Characterization of monovalent bispecific antibodies containing anti-CD3 scFv CD3 binding of the monovalent bispecific antibody produced in Example 1 was determined using an Octet assay. The binding kinetics of anti-CD3 scFv with human CD3ε was analyzed using Biolayer Interferometry on an Octet system (Sartorius, Octet RED96). All proteins were diluted in kinetics buffer (PBS, 2% BSA, 0.1% Tween®-20). Superstreptavidin (SSA)-coated biosensors (Sartorius, 18-5057) were equilibrated with kinetics buffer for 10 min at room temperature before data acquisition, and the experiments were performed at 30° C. with stirring at 1000 rpm. An initial baseline level was performed for 60 s ("sec"). 10 nM of recombinant His-tagged and biotinylated CD3ε protein (AcroBiosystems, CDE-H8223) was loaded onto the SSA biosensor for 120 s. Prior to analyte association, another baseline level was established for 60 seconds. The loaded sensors were associated with 200 nM monovalent CD3 module in the off-rate screening, resulting in concentrations ranging from 200 nM to 3.125 nM in a full kinetic assay for 150 seconds, followed by a dissociation step in kinetic buffer for 300 seconds. A reference molecule containing V12_LH (SEQ ID NO: 164) was included in each off-rate screening assay. A reference sensor loaded with ligand but no analyte was subtracted from the data before fitting. The data were fitted using a mass transport model. Analysis was performed using ForteBio Data Analysis 10.0 software. The CD3 monovalent bispecific antibody was characterized by the K in Table 7 below. d and K off As shown by the data, specific binding to CD3 was demonstrated.

[0282] Monomer content of monovalent bispecific antibodies was analyzed using analytical size exclusion chromatography (SEC). Analysis was performed using an Agilent 1260 Infinity HPLC system with UV detection at 280 nm absorbance. Protein aliquots (approximately 25 μg) of affinity chromatography (Protein A or CH1) purified material were injected onto a 7.8 mm×15 cm TSKgel QC-PAK GFC300 column (TOSOH Biosciences, King of Prussia, PA) equilibrated with 0.1 M sodium phosphate, 0.1 M sodium sulfate pH 6.8, and a flow rate of 1.0 mL / min. A low monomer content % of the affinity purified material (prior to the size exclusion polishing step) is generally undesirable as it can negatively impact the overall yield in the polishing step. All CD3 variants listed in Table 7 had a monomer content of >=50%, which is acceptable for the affinity purification step.

[0283] Thermal denaturation was tested using a Nanotemper Prometheus NT.48 (NanoTemper Technologies, Munich). The concentrations of the samples were between 0.8 mg / ml and 1 mg / ml, and the heating rate was 1 °C / min. Stability data were recorded using the temperature-dependent change in tryptophan fluorescence at emission wavelengths of 330 nm and 350 nm. The intrinsic fluorescence of tryptophan and tyrosine (Trp / Try) was measured at both 330 nm and 350 nm wavelengths and plotted against temperature from 15 to 95 °C during unfolding at a heating rate of 1 °C / min. Unfolding / denaturation led to a change in the microenvironment polarity around the tryptophan residues, resulting in a change in fluorescence that is reflected in the melting curves. A plot of the fluorescence ratio F350 / F330 against temperature led to a clear melting transition that was used to analyze the melting temperature. The melting temperature was determined by detecting the maximum of the first derivative of the fluorescence ratio (F350 / F330). Melting temperatures (Tm) were calculated by the first derivative of the F350 / 330 plot. A higher Tm indicates a more stable protein. All CD3 variants listed in Table 7 had the desired Tm above 55°C, except for the reference v12_LH, which was significantly higher than the reference v12_LH. The monovalent bispecific antibodies are identified by their CD3 binding domain in Table 7 below. Koff data are reported as relative measurements, with "+++" indicating that the Koff rate of CD3 binding is similar to or better than the reference monovalent bispecific antibody comprising v12_LH (SEQ ID NO: 164) as the anti-CD3 scFv. [Table 7]

[0284] Example 3 Cytotoxicity of a Monovalent Bispecific Antibody Comprising an Anti-CD3 scFv and a Her2 Binding Domain The in vitro potency of the monovalent bispecific antibodies was determined by cytotoxicity assay. Briefly, SKOV3-luc2 target cells and human PBMC effector cells (Stemcell technologies) were seeded together in RPMI medium (Gibco catalog number 22400071) supplemented with 5% human serum (MP Bio catalog number 2930949) at a target to effector cell ratio of 1:10 and co-cultured. A titration of the monovalent bispecific antibody produced in Example 1 was added to the co-culture, and the plate was incubated at 37° C., 5% CO2 for approximately 48 hours. After incubation, cytotoxicity was assessed using the ONE-Glo™ Luciferase Assay System (Promega catalog number E6130) and luminescence was measured with a plate reader (Tecan). Percent cytotoxicity was calculated as follows: (1-(RLU experimental / mean RLU untreated))*100. Use GraphPad PRISM to plot percent cytotoxicity data and calculate EC 50 Values ​​were calculated. Results are shown below in Table 8 as relative EC50 values ​​compared to the EC50 of an internal control monovalent bispecific antibody comprising v12-LH as the anti-CD3 scFv (SEQ ID NO: 164). Each molecule was tested at least twice and the range of results obtained is reported in Table 8. Typical assay error is approximately 2-3 fold, so a ratio of about 0.5 to about 2 indicates that the tested monovalent bispecific antibody has similar potency as the reference monovalent bispecific antibody comprising v12-LH as the anti-CD3 binding protein. A ratio of less than about 0.5 indicates that the tested monovalent bispecific antibody is more potent than the reference monovalent bispecific antibody comprising v12-LH anti-CD3 scFv. Monovalent bispecific antibodies are identified by their CD3 binding domain. Most of the CD3 variants in Table 8 (all except v619_HLp2) had EC50 ratios suggesting superior potency to v12-LH, within the limits of assay variation. [Table 8]

[0285] Exemplary sequences are listed below in Tables 9A and 9B. [Table 9A-1] [Table 9A-2] [Table 9A-3] [Table 9A-4] [Table 9A-5] [Table 9A-6]

[0286] Table 9B. Exemplary sequences of heavy chain variable domains, light chain variable domains, and related molecules. The CDRs in the heavy chain variable domain respectively comprise the sequences of amino acids 31 to 35, 50 to 68, and 101 to 114 of the heavy chain variable domain. The CDRs in the light chain variable domain respectively comprise the sequences of amino acids 23 to 36, 52 to 58, and 91 to 99 of the light chain variable domain. [Table 9B-1] [Table 9B-2] [Table 9B-3] [Table 9B-4] [Table 9B-5] [Table 9B-6] [Table 9B-7]

Table 9B-8

Table 9B-9

Table 9B-10

Table 9B-11

Table 9B-12

Table 9B-13

Table 9B-14

Table 9B-15

Table 9B-16

Table 9B-17

Table 9B-18

Table 9B-19

Table 9B-20

Table 9B-21

Table 9B-22

Table 9B-23

Table 9B-24

[0287] Other embodiments While the invention has been described in connection with the above detailed description, it is to be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0288] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A target-binding protein, Variable heavy chain complementarity determination region 1 (VH CDR1), Variable heavy chain complementarity determination region 2 (VH CDR2), and A heavy chain variable domain including variable heavy chain complementarity determination region 3 (VH CDR3), Variable light chain complementarity determination region 1 (VL CDR1), Variable light chain complementarity determination region 2 (VL CDR2), and A light chain variable domain including a variable light chain complementarity determination region 3 (VL CDR3), The heavy chain variable domain and the light chain variable domain are arranged within one or more polypeptides. The aforementioned target-binding protein specifically binds to CD3 epsilon, (a) The heavy chain variable domain includes VH CDR1 containing TYAMN (SEQ ID NO: 3), VH CDR2 containing RIRSKYNNYATYYAADSVKD (SEQ ID NO: 5), VH CDR3 containing HGNFGNSYVSWWAY (SEQ ID NO: 7), and the light chain variable domain includes VL CDR1 containing GSSTGAVTTTSNYVN (SEQ ID NO: 13), VL CDR2 containing GTNKRAP (SEQ ID NO: 14), and VLWYSNRWV (SEQ ID NO: 16), The heavy chain variable domain contains a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain contains a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 64, or (b) The heavy chain variable domain includes VH CDR1 containing TYAMN (SEQ ID NO: 3), VH CDR2 containing RIRSKYNNYATYYADSVKD (SEQ ID NO: 5), VH CDR3 containing HGNFGNSYVSWWAY (SEQ ID NO: 7), and the light chain variable domain includes VL CDR1 containing RSSTGAVTTTSNYPN (SEQ ID NO: 11), VL CDR2 containing GTNKRAP (SEQ ID NO: 14), and VLWYSNRWV (SEQ ID NO: 16), The heavy chain variable domain contains a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46, and the light chain variable domain contains a sequence that is at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

49. The aforementioned target-binding protein.

2. (a) The heavy chain variable domain includes the sequence of SEQ ID NO: 46, and the light chain variable domain includes the sequence of SEQ ID NO: 64, or (b) The heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 46, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

49. The target-binding protein according to claim 1.

3. The target-binding protein according to claim 1, wherein the heavy chain variable domain and the light chain variable domain are located within the same polypeptide.

4. (a) The heavy chain variable domain and the light chain variable domain are linked via a linker, for example, the linker having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids, or (b) The target-binding protein comprises the sequence of SEQ ID NO: 28 or 50, The target-binding protein according to claim 3.

5. The target-binding protein according to claim 4, wherein the target-binding protein comprises a single-stranded variable fragment (scFv).

6. (a) The target binding protein is BiTE, (scFv) ２ NANOBODY, Nanobody-HSA VHH-scAb, VHH-Fab, Double scFab, F(ab')2, Diabody, CROSSMAB, DAF 2-in-1, DAE 4-in-1, DUTAMAB, DT-IgG, Knob-in-hole common light chain, Knob-in-hole assembly, Charge pair, Fab-arm exchange, SEED body, LUZ-Y, FcAb, kl-body, Orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, ZYBODY, DVI-IgG, Diabody-CH3, Triplebody, Miniantibody, Minibody, TriBiMinibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, [Number 7] Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, [Number 8] This may include Fab-VHH-Fc, intrabody, dock and lock, immune mobilization monoclonal T cell receptor against cancer, IgG-IgG conjugate, Cov-X-body, scFvl-PEG-scFv2, adonectin, DARPin, fibronectin, IgG, IgM, IgA, IgE, IgD, or DEP conjugate, TMEA body, SAFE body, TRITAC, or SHIELD antibody, or (b) The target-binding protein contains an IgG, IgM, IgA, IgE, or IgD antibody, or a fragment thereof, or the target-binding protein contains an IgG1, IgG2, IgG3, or IgG4 antibody, The target-binding protein according to claim 1.

7. (a) The target binding protein is humanized or (b) The target binding protein further comprises a masking portion that inhibits the binding of the target binding protein to CD3 in an inactive state. The target-binding protein according to claim 1.

8. The masking portion binds to the target-binding protein via a cleavable portion, and the cleavable portion binds to the substrate of the protease, for example, ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartate protease, BACE, renin, aspartate cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, cysteine ​​cathepsin, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteinase, cruzipain, regmain, otsubine-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Me Taloproteinase, Meprin, Neprilysin, PSMA, BMP-1, MMP, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, Serine protease, Activated protein C, Cathepsin A, Cathepsin G, Chymase, Coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane protein, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, or TMPRSS4, and / or The heavy chain variable domain and / or the light chain variable domain are conjugated via a conjugate moiety with a toxin, radioisotope, small molecule, diagnostic agent, therapeutic macromolecule, targeted moiety, or detectable moiety, or (i) the conjugate moiety is cleavable by a protease, or (ii) the conjugate moiety is not cleavable by a protease. The target-binding protein according to claim 7.

9. A composition comprising a target-binding protein according to any one of claims 1 to 8 and a carrier, wherein the composition is optionally a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

10. A container, vial, syringe, injector pen, or kit comprising at least one dose of the composition according to claim 9.

11. A nucleic acid comprising a sequence encoding a target-binding protein according to any one of claims 1 to 8.

12. A vector comprising the nucleic acid described in claim 11.

13. A cell comprising the nucleic acid described in claim 11.

14. A composition for use in a method of treating a disease, comprising a target-binding protein according to any one of claims 1 to 8, wherein the composition is administered to a subject in need of treatment for a disease.

15. The composition according to claim 14, characterized in that the subject is identified or diagnosed as having cancer, an inflammatory condition, disorder, or disease, or an autoimmune condition, disorder, or disease.

16. A method for producing a target-binding protein, The cells according to claim 13 are cultured in a culture medium under conditions sufficient to produce the target-binding protein, The method comprising recovering the target-binding protein from the cells or the culture medium.

17. (i) further comprising isolating the target-binding protein recovered from the cells or the culture medium, and / or (ii) Further comprising formulating the target-binding protein into a pharmaceutical composition, The method according to claim 16.

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