CD3 binding proteins and methods of use thereof

Abstract

The present disclosure includes target binding proteins having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain and the light chain variable domain having specific sequences that bind to CD3 [epsilon] and wherein the heavy chain variable domain and the light chain variable domain are located within one or more polypeptides. The disclosure also includes compositions, nucleic acids, vectors, cells, and methods of making and using the proteins, compositions, and nucleic acids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. US 63 / 326,668, filed on April 1, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The sequence listing submitted by EFS together with this application is named "4862-124PCT.xml", created on March 31, 2023, with a size of 643,441 bytes, and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of biotechnology, and more particularly, to target binding molecules, including molecules that bind CD3. Background Art

[0006] Anti-CD3ε (CD3ε) antibodies have been used for several years and are intended to play a variety of roles and fight a variety of diseases. Many of these anti-CD3ε antibodies are derived from only a few variants, including SP-34. In order to further improve the efficacy, safety and manufacturability of anti-CD3ε molecules, additional anti-CD3ε binding proteins are needed. Anti-CD3ε binding proteins with different binding affinities from SP-34 antibodies, improved manufacturability, improved stability, and related therapeutic agents with customized CD3ε binding affinity to treat specific diseases are needed. Anti-CD3ε binding proteins are also needed, which show improved binding affinity, stability and manufacturability in the context of molecular structures other than full-length antibodies (including in the context of multivalent and monovalent binding proteins). Summary of the invention

[0007] The present disclosure provides target binding proteins having a CD3ε binding domain, as well as related compositions and methods.

[0008] In one aspect, the present disclosure provides a target binding protein, the target binding protein comprising: a heavy chain variable domain, the heavy chain variable domain comprising: a variable heavy chain complementary determining region 1 (VH CDR1) comprising a sequence of TYAMN (SEQ ID NO: 3); a variable heavy chain complementary determining region 2 (VH CDR2) comprising a sequence of RIRSKYNNYATYYADSV KD (SEQ ID NO: 5); and a variable heavy chain complementary determining region 3 (VH CDR 3) comprising a sequence of HGNFGNSYVSWX1AY (SEQ ID NO: 6), wherein X1 is W or F; and a light chain variable domain, the light chain variable domain comprising: a variable light chain complementary determining region 1 (VL CDR1) comprising a sequence of X2SSTGAVTTSNYX3N (SEQ ID NO: 10), wherein X2 is R or G, and X3 is P or V; a variable light chain complementary determining region 2 (VL CDR2) comprising GTNCRAP (SEQ ID NO: 11). NO:14); with a variable light chain complementary 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, wherein the heavy chain variable domain and the light chain variable domain are located in one or more polypeptides, wherein the target binding protein specifically binds to CD3ε.

[0009] 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 GTNCRAP (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).

[0010] In some embodiments, VH CDR1 comprises TYAMN (SEQ ID NO:3), VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), VLCDR1 comprises RSSTGAVTTSNYPN (SEQ ID NO:11), VL CDR2 comprises GTNKR AP (SEQ ID NO:14), and VLCDR3 comprises VLWYSNRWV (SEQ ID NO:16); VH CDR1 comprises TYAMN (SEQ ID NO:3), VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), VLCDR1 comprises GSSTG AVTTSNYVN (SEQ ID NO:13), VL CDR2 comprises GTNCRAP (SEQ ID NO:14), and VLCDR3 comprises VLWYSNRWV (SEQ ID NO:16). NO: 14), and VLCDR3 comprises VLWYSNRWV (SEQ ID NO: 16); VH CDR1 comprises TYAMN (SEQ ID NO: 3), VH CDR2 comprises RIRSK YNNYATYYADSVKD (SEQ ID NO: 5), VH CDR3 comprises HGNFGNSYVSWFAY (SEQ ID NO: 8), VLCDR1 comprises GSSTGAVTTSNYV N (SEQ ID NO: 13), VL CDR2 comprises GTNCRAP (SEQ ID NO: 14), and VLCDR3 comprises VLWYSNRWV (SEQ ID NO: 16); VH CDR1 comprises TYAMN (SEQ ID NO: 3), VH CDR2 comprises RIRSKYNNYATYY ADSVKD (SEQ ID NO: 5), VH CDR3 comprises HGNFGNSYVSWFAY (SEQ ID NO: 8), VLCDR1 comprises GSSTGAVTTSNYV N (SEQ ID NO: 13), VL CDR2 comprises GTNCRAP (SEQ ID NO: 14), and VLCDR3 comprises VLWYSNRWV (SEQ ID NO: 16); VH CDR1 comprises TYAMN (SEQ ID NO: 3), VH CDR2 comprises RIRSKYNNYATYY ADSVKD (SEQ ID NO: 5), VH CDR3 comprises HGNFGNSYVSWAY (SEQ ID NO: 7), VLCDR1 comprises RSSTGAVTTSNYVN (SEQ ID NO:12), VL CDR2 comprises GTNCRAP, and VL CDR3 comprises ILWYSNRWV (SEQ ID NO:18);VH CDR1 comprises TYAMN (SEQ ID NO:3), VH CDR2 comprises RIRSKYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 comprises HGNFGNSYVSWWAY (SEQ ID NO:7), VLCDR1 comprises GSSTGAVTTSNYVN (SEQ ID NO:13), VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and VLCDR3 comprises VLWYSNLWV (SEQ ID NO:17); 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), VLCDR1 comprises RSSTGAVTTSNYVN (SEQ ID NO:12), VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and VLCDR3 comprises VLWYSNLWV (SEQ ID NO:17). NO:14), and VLCDR3 comprises VLWYSNRWV (SEQ ID NO:16). ;

[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 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 GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the aforementioned VH CDRs 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 aforementioned VL CDRs 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.

[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 GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the aforementioned VH CDRs 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 aforementioned VL CDRs 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.

[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), and 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 GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the aforementioned VH CDRs 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 aforementioned VL CDRs 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.

[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 GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18). In some embodiments, the heavy chain variable domain comprises the aforementioned VH CDRs 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 aforementioned VL CDRs 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.

[0015] 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 GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO:17). In some embodiments, the heavy chain variable domain comprises the aforementioned VH CDRs 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 aforementioned VL CDRs 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.

[0016] 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 GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16). In some embodiments, the heavy chain variable domain comprises the aforementioned VH CDRs 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 aforementioned VL CDRs 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.

[0017] In another aspect, the present disclosure provides a target binding protein comprising: a heavy chain variable domain comprising the sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKG LEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLK TEDTAVYYCVRHGNFGNSYVSWX1AYWGX6GTLVTVSS (SEQ ID NO: 20); and a light chain variable domain comprising the sequence QTVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWVQQKPGX7APRGLIGGTNKRAPGTPARFSGSLJGGKAALTLSGX9QPEDEAEYY CX4LWYSNX5WVFGGGTKLTVL (SEQ ID NO: 21). NO:21), wherein: X1 is a) W or F; X2 is R or G; X3 is P or V; X4 is V or I; X5 is R or L, b) X6 is Q and X7 is Q; or X6 is C and X7 is C, c) when X9 is V, J is L; or when X9 is A, J is I, and d) the target binding protein specifically binds to CD3ε.

[0018] In some embodiments, X6 is Q and X7 is Q. In some embodiments, X6 is C and X7 is C.

[0019] 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; 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; 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; 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; 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.

[0020] In some embodiments, the heavy chain variable domain and the light chain variable domain are located in the same polypeptide. In some embodiments, the heavy chain variable domain and the light chain variable domain are coupled via a joint. In some embodiments, the length of the joint is 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.

[0021] In some embodiments, the target binding protein comprises a 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 comprises a BiTE, (scFv)2, Nanobodies - HSA VHH-scAb, VHH-Fab, double scFab, F(ab')2, double-chain antibody, DAF (two-in-one), DAE (four-in-one), DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, 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 TM , DVI-IgG, diabody-CH3, tribody, minibody, minibody, TriBi minibody, scFv-CH3KIH, Fab-scFv, F(ab')2-scFv2, scFv-KI'H, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, L'HH-Fc KiH, Fab-VHH-Fc, intracellular antibody, docking and locking, (monoclonal TCR (T cell receptor) for immune mobilization against cancer), IgG-IgG conjugate, Cov-X-Body, scFvl-PEG-scFv2, Adnectin, DARPin or fibronectin, IgG, IgM, IgA, IgE, IgD or DEP conjugate, TMEAbody TM , Or SHIELD antibodies.

[0022] 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.

[0023] In some embodiments, the target binding protein further comprises a masking portion that inhibits the binding of the target binding protein to CD3 in an inactive state. In some embodiments, the masking portion is coupled to the target binding protein via a cleavable portion (directly or indirectly, for example, via one or more joints), and the cleavable portion 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, apoptotic protease, apoptotic protease 1, apoptotic protease 2, apoptotic protease 3, apoptotic protease 4, apoptotic protease 5, apoptotic protease 6, apoptotic protease 7, apoptotic protease 8, apoptotic protease apoptosis proteinase 9, apoptosis proteinase 10, apoptosis proteinase 14, cysteine ​​cathepsins, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteinases, Cruzipain, Legumain, Otubain-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, metalloproteinases, transmembrane peptidases (Mepr in), Neprilysin, PSMA, BMP-1, MMPs, 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, chymotrypsin, coagulation factor protease, FVIIa, FIXa, FXa, F XIa, FXIIa, Elastase, Granzyme B, Guanidinobenzoic Acidase, HtrA1, Human Neutrophil Elastase, Lactoferrin, Marapsin, NS3 / 4A, PACE4, Plasmin, PSA, tPA, Thrombin, Tryptase, uPA, Type II Transmembrane, Serine Protease, TTSP, DESC1, DPP-4, FAP, Hepsin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3 or TMPRSS4.

[0024] In some embodiments, the target binding protein further comprises a second target binding domain that specifically binds to a second target.

[0025] In some embodiments, the heavy chain variable domain and / or the light chain variable domain are conjugated to a toxin, a radioisotope, a small molecule, a diagnostic agent, a therapeutic macromolecule, a targeting moiety, or a detectable moiety via a conjugated moiety. In some embodiments, the conjugated moiety is cleavable by a protease. In some embodiments, the conjugated moiety is not cleavable by a protease.

[0026] In another aspect, the present disclosure provides a composition comprising a target binding protein herein and a carrier. In some embodiments, the composition is a pharmaceutical composition, wherein the carrier is a pharmaceutically acceptable carrier.

[0027] In another aspect, the present disclosure provides a container, a vial, a syringe, an injection pen or a kit comprising at least one dose of the composition herein.

[0028] In another aspect, the disclosure provides a nucleic acid comprising a sequence encoding the target binding protein herein.

[0029] In another aspect, the present disclosure provides a vector comprising the nucleic acid described herein.

[0030] In another aspect, the present disclosure provides a cell comprising the nucleic acid described herein or the vector described herein.

[0031] In another aspect, the disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a target binding protein herein or a composition herein. In some embodiments, the subject has been identified or diagnosed as having cancer.

[0032] In another aspect, the present disclosure provides a method of producing a target binding protein, the method comprising: culturing the cells herein in a culture medium under conditions sufficient to produce the target binding protein; and recovering the target binding protein from the cells or the culture medium. In some embodiments, the method further comprises isolating the target binding protein recovered from the cells or the liquid culture medium. In some embodiments, the method further comprises formulating the target binding protein into a pharmaceutical composition.

[0033] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments utilizing the principles of the invention. DETAILED DESCRIPTION

[0034] Provided herein are target binding proteins that specifically bind to cluster of differentiation 3 (CD3) (e.g., CD3ε). In one aspect, the target binding protein may 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., CD3ε). In some embodiments, the CD3 binding molecule herein comprises one or more mutations in its amino acid sequence compared to known anti-CD3 proteins produced from non-human species (e.g., sp34 mouse anti-human CD3 antibodies), which mutations make it more humanized, e.g., increase its similarity to naturally occurring CD3 binding molecules in humans.

[0035] In some embodiments, the target binding protein can be a single-chain protein, such as a single-chain antibody. For example, a 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.

[0036] In some embodiments, the target binding protein can be an activatable molecule, such as an activatable CD3 binding molecule comprising a masking portion coupled to a CD3 binding domain via a cleavable portion (directly or indirectly, such as via one or more linkers). The cleavable portion can be cleaved under certain conditions (e.g., when exposed to a protease in the tumor microenvironment), thereby releasing the masking portion from the CD3 binding domain.

[0037] In some embodiments, the target binding protein can be a multispecific (e.g., bispecific) binding protein that binds to one or more additional targets in addition to CD3. For example, a multispecific protein can specifically bind to CD3 and a tumor-associated antigen, such as HER2, Jagged, EGFR, etc.

[0038] Also provided herein are related compositions, kits, nucleic acids, vectors, and recombinant cells, as well as related methods, including methods of using any of the targetable binding proteins described herein and methods of producing any of the target binding proteins described herein.

[0039] definition

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present disclosure are described herein; 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 herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail.

[0041] The terms "a" or "an" refer to one or more (ie, at least one) grammatical object of the article. By way of example, "a cell" encompasses one or more cells.

[0042] As used herein, the terms "about" and "approximately" when used to modify a numerical value or an amount specified in a range, represent the numerical value and reasonable deviations from the value known to those skilled in the art. For example, ±20%, ±10% or ±5% are within the intended meaning of the stated value, where appropriate.

[0043] Concentration, amount and other numerical data can be expressed or presented in range format herein.It should be understood that this type of range format is used only for convenience and simplicity, and should therefore be flexibly interpreted as not only including the numerical value of the limit value of the range explicitly stated, but also including all individual numerical values ​​or sub-ranges contained within the range, as if each numerical value and sub-range were explicitly stated.As an illustration, the numerical range of "about 0.01 to 2.0" should be interpreted as not only including the value of about 0.01 to about 2.0 explicitly stated, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values, such as 0.5, 0.7 and 1.5, and sub-ranges, such as 0.5 to 1.7, 0.7 to 1.5 and 1.0 to 1.5, etc., are included in this numerical range.In addition, no matter how the width of the range or the features described, this explanation should apply.In addition, it should be noted that, unless otherwise specified, all percentages are by weight.

[0044] In understanding the scope of the present disclosure, the term "comprising" or "including" and their derivatives as used herein are intended to specify the existence of stated features, elements, components, groups, integers and / or steps, but do not exclude the open-ended terms of the existence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words with similar meanings, such as the terms "comprising", "having" and their derivatives. The term "consisting of" and its derivatives as used herein are intended to specify the existence of stated features, elements, components, groups, integers and / or steps, but exclude the closed terms of the existence of other unstated features, elements, components, groups, integers and / or steps. The term "consisting essentially of" as used herein is intended to specify the existence of stated features, elements, components, groups, integers and / or steps and those features, elements, components, groups, integers and / or steps that do not substantially affect one or more basic and novel features of features, elements, components, groups, integers and / or steps. It should be understood that reference to any of these transitional terms (i.e., "comprising," "consisting of," or "consisting essentially of") provides direct support for substitution with any other transitional term not specifically used. For example, since this definition is directed to any element disclosed throughout this disclosure, modifying the term from "comprising" to "consisting essentially of" or "consisting of" will be directly supported. Based on this definition, any element disclosed or incorporated by reference herein may be included in or excluded from the claimed invention.

[0045] As used herein, for convenience, multiple compounds, elements or steps may be presented in a common list. However, these lists should be interpreted as if each member of the list is individually identified as a separate and unique member. Therefore, individual members of such a list should not be interpreted as de facto equivalents of any other members of the same list based solely on their presentation in a common group without contrary indication.

[0046] The term "exemplary" as used herein means serving as an example, instance or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as superior or preferred to other aspects or designs. On the contrary, the word "exemplary" is used to present concepts in a more specific way.

[0047] In addition, certain molecules, constructs, compositions, elements, parts, excipients, diseases, illnesses, properties, steps, etc. may be discussed in the context of a specific embodiment or aspect or in a separate paragraph or chapter of the disclosure. It should be understood that this is only for convenience and brevity, and any such disclosure is equally applicable to any other embodiment or aspect found anywhere in the disclosure and claims, and is intended to be combined with any other embodiment or aspect described, all of which form the present application and the invention required on the filing date. For example, the list of constructs, molecules, method steps, kits, or compositions described with respect to constructs, compositions, or methods is intended to and does find direct support for the embodiments related to the constructs, compositions, preparations, and methods described in any other part of the disclosure, even if those method steps, activating agents, kits, or compositions are not re-listed in the context or chapter of the embodiment or aspect.

[0048] Target binding protein

[0049] The target binding protein herein may comprise a heavy chain variable domain and a light chain variable domain, which form a target binding domain that specifically binds to CD3 (e.g., CD3ε). An exemplary CD3ε molecule that can be bound by the protein herein is wild-type human CD3ε (SEQ ID NO: 1).

[0050] 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 coupled together by one or more joints. The joint can be a peptide joint described in the joint section below. In some instances, the target binding domain in the single-chain polypeptide can be a scFv. Alternatively, the heavy chain variable domain and the light chain variable domain are located in two different polypeptides.

[0051] In some embodiments, the target binding protein can be a protein complex comprising a plurality of polypeptides (e.g., two, three, four, five, six, seven, eight, nine, ten or more polypeptides). In some instances, some or all of the plurality of polypeptides (e.g., two, three, four, five, six, seven, eight, nine, ten or more polypeptides) can be identical. In some instances, each of the plurality of polypeptides in the target binding complex is different from one another.

[0052] The target binding domain may be present in any of a variety of different constructs, including antibodies or fragments thereof, VH domains, VHH domains, VNAR domains, and single chain variable fragments (scFv), BiTEs or components thereof, (scFv)2, Nanobodies-HSA, VHH-scAb, VHH-Fab, double scFab, F(ab')2, double-chain antibodies, DAF (two-in-one), DAE (four-in-one), DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, 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 TM , DVI-IgG, diabody-CH3, tribody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KI'H, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, L'HH-Fc KiH, Fab-VHH-Fc, intracellular antibody, docking and locking, (monoclonal TCR (T cell receptor) for immune mobilization against cancer), IgG-IgG conjugate, Cov-X-Body, scFvl-PEG-scFv2, Adnectin, Fibronectin, IgG, IgM, IgA, IgE, IgD, DEP conjugate, TMEAbodyTM, or SHIELD antibodies. VHH domains are single monomeric variable antibody domains found in camelids. VNAR domains are single monomeric variable antibody domains found in cartilaginous fish. Non-limiting aspects of VHH domains and VNAR domains are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Biotechnol 33:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biocheni. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al. Methods Mol, Biol, 911: 15-26, 2012; and Wesolowski et al. Med. Microbiol. Immunol. 198: 157-174, 2009, each of which is herein incorporated by reference in its entirety.

[0053] The term "antibody" is used herein in its broadest sense to include certain types of immunoglobulin molecules that include 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 antibodies, and multispecific antibodies. An example of a target binding domain is represented by V H -V LDimer formation. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0054] A "light chain" consists of a variable domain (VL) and a constant domain (CL). There are two different light chains, called kappa or lambda. A "heavy chain" consists of a 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 several subtypes, and these 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).

[0055] "Fragment antigen binding" (Fab) includes a complete light chain paired with the VH domain and CH1 domain of the heavy chain. When an antibody is cleaved by pepsin below the hinge region (or truncated in other ways), an F(ab')2 fragment is formed, in which case the two fragment target binding domains (Fab) of the antibody molecule remain connected. The F(ab')2 fragment contains two complete light chains paired with the two VH and CH1 domains of the heavy chain joined together by the hinge region. The "crystallizable fragment" (Fc) fragment (also referred to herein as F C The "single-chain variable" (scFv) fragment contains only the light chain variable domain (VL) connected to the heavy chain variable domain (VH) by a peptide. The name single-chain Fv comes from the variable fragment. The "hinge region" or "interdomain" is a flexible amino acid segment that joins or connects the Fab fragment to the Fc domain. The "synthetic hinge region" is an amino acid sequence that joins or connects the Fab fragment to the Fc domain.

[0056] "Fv" fragments include a non-covalently linked dimer of a heavy chain variable domain and a light chain variable domain. "Dual variable domain immunoglobulin G" or "DVD-IgG" refers to multivalent and multispecific target binding proteins, such as those described in: DiGiammarino et al., Methods Mol.Biol.899:145-156, 2012; Jakob et al., MABs5:358-363, 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, all of which are incorporated herein by reference in their entirety. Examples of DARTs are described in, for example, Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0057] In some embodiments, the target binding protein can be a mouse, rat, rabbit, goat, camel, donkey, primate, chimeric, human or humanized protein. In one example, the target binding protein can be a human protein. In one example, the target binding protein can be a humanized (e.g., fully humanized) protein.

[0058] The term "humanized" refers to a target binding protein having an amino acid sequence that includes the VH and VL region sequences of a reference protein produced in a non-human species (e.g., a mouse), but also includes modifications to these sequences relative to the reference protein, intended to make them more "human-like," i.e., more similar to human germline variable sequences. In some embodiments, a "humanized" target binding protein is a protein that immunospecifically binds to an antigen of interest and has a framework (FR) region having substantially the same amino acid sequence as a human protein, and a complementarity determining region (CDR) having substantially the same amino acid sequence as a non-human protein contains humanized VH and VL regions.

[0059] 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 "human" even if its amino acid sequence, for example in one or more CDRs, includes residues or elements that are not encoded by human germline immunoglobulin sequences (e.g., including sequence variations, such as may be (initially) introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo).

[0060] 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, VLCDR2, and VL CDR3). VH CDR1 may comprise the sequence SEQ ID NO:3. VH CDR2 may comprise the sequence SEQ ID NO:5. VHCDR3 may comprise the sequence SEQ ID NO:6. For example, VH CDR3 may comprise the sequence SEQ ID NO:7 or 8. VL CDR1 may comprise the sequence SEQ ID NO:10. For example, VL CDR1 may comprise the sequence SEQ ID NO:11, 12, or 13. VL CDR2 may comprise the sequence SEQ ID NO:14. VL CDR3 may comprise the sequence SEQ ID NO:15. For example, VL CDR3 may comprise the sequence SEQ ID NO:16, 17, or 18.

[0061] The target binding protein may comprise: a heavy chain variable domain comprising VH CDR1, VH CDR2 and VH CDR3; and a light chain variable domain comprising VL CDR1, VL CDR2 and VL CDR3. In some examples, in the target binding protein, VH CDR1 may comprise the sequence of TYAMN (SEQ ID NO:3); VH CDR2 may comprise the sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:5); VH CDR3 may comprise the sequence of HGNFGNSYVSWWAY (SEQ ID NO:7) or HGNFGNSYVSWFAY (SEQ ID NO:8); VL CDR1 may comprise the sequence of RSSTGAVTTSNYPN (SEQ ID NO:11), RSSTGAVTTSNYVN (SEQ ID NO:12), or GSSTGAVTTSNYVN (SEQ ID NO:13); VLCDR2 may comprise the sequence of GTNCRAP (SEQ ID NO:14); and VL CDR3 may comprise the sequence of VLWYSNRWV (SEQ ID NO:16), VLWYSNLWV (SEQ ID NO:17), or ILWYSNRWV (SEQ ID NO:18).

[0062] In one example, in the 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 GTNCRAP (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 RIRSKYNNYA TYYADSVKD (SEQ ID NO:5), VH CDR3 may comprise HGNFGNSYVS WWAY (SEQ ID NO:7), VL CDR1 may comprise GSSTGAVTTSNYVN (SEQ ID NO:13), VLCDR2 may comprise GTNCRAP (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 GTNCRAP (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 RSSTGAVTTSNYVN (SEQ ID NO:12), VL CDR2 may comprise GTNCRAP (SEQ ID NO:14), and VL CDR3 may comprise ILWY SNRWV (SEQ ID NO:18).In another example, VH CDR1 may comprise TYAMN (SEQ ID NO:3), VH CDR2 may comprise RIRSKYNNYATYYADSV KD (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 GTNCRAP (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 RIRS KYNNYATYYADSVKD (SEQ ID NO:5), VH CDR3 may comprise HGNF GNSYVSWWAY (SEQ ID NO:7), VL CDR1 may comprise RSSTGAVTTS NYVN (SEQ ID NO:12), VL CDR2 may comprise GTNCRAP (SEQ ID NO:14), and VL CDR3 may comprise VLWYSNRWV (SEQ ID NO:16).

[0063] Other examples of VH CDR1 include the amino acid sequences at positions 31-35 of the heavy chain variable domain in Table 9B. Other examples of VH CDR2 include the amino acid sequences at positions 50-68 of the heavy chain variable domain in Table 9B. Other examples of VHCDR3 include the amino acid sequences at positions 101-114 of the heavy chain variable domain in Table 9B. Other examples of VL CDR1 include the amino acid sequences at positions 23-36 of the light chain variable domain in Table 9B. Other examples of VH CDR2 include the amino acid sequences at positions 52-58 of the light chain variable domain in Table 9B. Other examples of VH CDR3 include the amino acid sequences at positions 91-99 of the light chain variable domain in Table 9B.

[0064] 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.

[0065] In some examples, the heavy chain variable domain and the light chain variable domain may comprise one or more cysteines that form disulfide bonds. In some examples, disulfide bonds can improve stability while not interfering with target binding of the molecule. For example, the heavy chain domain may comprise the sequence SEQ ID NO:24 and the light chain domain may comprise the sequence SEQ ID NO:25. In some examples, the heavy chain and light chain variable domains do not have such cysteine ​​mutations. For example, the heavy chain domain may comprise the sequence SEQ ID NO:22 and the light chain domain may comprise the sequence SEQ ID NO:23.

[0066] For example, the heavy chain variable domain may comprise the sequence of SEQ ID NO: 46 or 128, and the light chain variable domain in 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 includes 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, and 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 NO: 46 or 128; and the light chain variable domain includes 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 includes an LH CDR1 comprising the sequence of SEQ ID NO: 16, 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 of NO: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, and the heavy chain variable domain comprises a sequence at least 95% identical to any one of SEQ ID NO: 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, 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 includes 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 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 includes a VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO:11), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light chain variable domain includes 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. NO:49 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 a sequence.

[0067] In one example, the heavy chain variable domain in the heavy chain variable domain includes 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 heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46; and the light chain variable domain includes a VL CDR1 comprising RSSTGAVTTSNYPN (SEQ ID NO:11), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:49.

[0068] 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 includes 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 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 includes a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light chain variable domain includes 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. NO:64 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 a sequence. In one example, the heavy chain variable domain includes 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 heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46; and the light chain variable domain includes a VLCDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), a VLCDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:64.

[0069] 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 includes 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 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 includes a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18), and the light chain variable domain includes 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. NO:113 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 a sequence. In one example, the heavy chain variable domain includes 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 heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46; and the light chain variable domain includes a VLCDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18), and the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:113.

[0070] 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 includes 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), and 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 includes a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VLCDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO:17), and the light chain variable domain includes 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. NO:98 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). In one example, the heavy chain variable domain includes 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), and the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46; and the light chain variable domain includes a VLCDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNLWV (SEQ ID NO:17), and the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:98.

[0071] 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 includes 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 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 includes a VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light chain variable domain includes 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. NO:107 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 a sequence. In one example, the heavy chain variable domain includes 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 heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:46; and the light chain variable domain includes a VLCDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:107.

[0072] 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 includes 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 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:128; and the light chain variable domain includes a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light 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:128. 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 ID NO: 122. In one example, the heavy chain variable domain includes 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 heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:128; and the light chain variable domain includes a VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the light chain variable domain comprises a sequence at least 95% identical to SEQ ID NO:122.

[0073] Additional examples of heavy chain variable domains and light chain variable domains include those in Table 9B.

[0074] In the case 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 includes 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, 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%, at least 99% or 100% identical) to any one of SEQ ID NO: 50, 28, 114, 99, 188 or 32. For example, the single-chain target binding protein includes 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, and the single-chain target binding protein comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 50, 28, 114, 99, 188 or 32.

[0075] 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 GTNCRAP (SEQ ID NO: 14), and 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: 50. For example, the single-chain target binding protein includes 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 GTNCRAP (SEQ ID NO:14), and a VLCDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the single-chain target binding protein comprises a sequence at least 95% identical to SEQ ID NO:50.

[0076] 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 GTNCRAP (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 includes 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 GTNCRAP (SEQ ID NO:14), a VLCDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the single-chain target binding protein comprises a sequence at least 95% identical to SEQ ID NO:28.

[0077] In some examples, the single-chain target binding protein can comprise the sequence of SEQ ID NO:114. In some examples, the single-chain target binding protein includes 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 CDR 1 comprising RSSTGAVTTSNYVN (SEQ ID NO:12), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSN RWV (SEQ ID NO:18), 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:114. For example, the single-chain target binding protein includes a VH CDR1 comprising TYAMN (SEQ ID NO:3), a VH CDR2 comprising RIRSKYNNYAT YYADSVKD (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 GTNCRAP (SEQ ID NO:14), and a VL CDR3 comprising ILWYSNRWV (SEQ ID NO:18), and the single-chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:114.

[0078] 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 GTNCRAP (SEQ ID NO: 14), and a VLCDR3 comprising VLWYSNLWV (SEQ ID NO: 17), 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: 99. For example, the single-chain target binding protein includes 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 VLCDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO:13), a VL CDR2 comprising GTNCRAP (SEQ ID NO:14), and a VLCDR3 comprising VLWYSNLWV (SEQ ID NO:17), and the single-chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:99.

[0079] In some examples, the single-chain target binding sequence is SEQ ID NO: 108. In some examples, the single-chain target binding protein includes 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 GTNCRAP (SEQ ID NO: 14), and 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: 108. For example, the single-chain target binding protein includes a VHCDR1 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 GTNCRAP (SEQ ID NO:14), and a VLCDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the single-chain target binding protein comprises a sequence at least 95% identical to SEQ ID NO:108.

[0080] 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 GTNCRAP (SEQ ID NO: 14), and 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: 32. For example, the single-chain target binding protein includes 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 GTNCRAP (SEQ ID NO:14), and a VLCDR3 comprising VLWYSNRWV (SEQ ID NO:16), and the single-chain target binding protein comprises a sequence that is at least 95% identical to SEQ ID NO:32.

[0081] Additional examples of single-chain target binding proteins include those disclosed in Table 9B.

[0082] The target binding protein can specifically bind to CD3 (e.g., CD3ε). As used herein, the terms "specific binding" and "immunologically bind" refer to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin has specificity. The strength or affinity of the immunological binding interaction can be measured based on the dissociation constant (K) of the interaction. d or K D ) is used to represent the dThe smaller the value, the greater the affinity. The strength or affinity of a binding interaction can be expressed in terms of an association rate (Kon) or a 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, "binding affinity" as used herein 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 conventional methods known in the art, including those described herein. For example, surface plasmon resonance (SPR) technology (e.g., ) or biolayer interferometry (e.g. ) to determine affinity. Additional methods for determining the affinity of a target binding domain to its corresponding antigen or epitope are known in the art.

[0083] As used herein, the statement that a CD3 binding protein "specifically binds" to CD3 means that the dissociation constant (Kd) of the protein binding to CD3 is less than 100 μM (eg, less than 5 μM or 10 μM). The target binding protein can be about 0.01 nM to about 500 nM (e.g., about 0.01 nM to about 450 nM, about 0.01 nM to about 400 nM, about 0.01 nM to about 350 nM, about 0.01 nM to about 300, about 0.01 nM to about 250 nM, about 0.01 nM to about 200 nM, about 0.01 nM to about 150 nM, about 0.01 nM to about 100 nM, about 0.01 nM to about 80 nM, about 0.01 nM to about 60 nM, about 0.01 nM to about 50 nM, about 0.01 nM to about 40 nM, about 0.01 nM to about 250 nM, about 0.01 nM to about 300 nM, about 0.01 nM to about 250 nM, about 0.01 nM to about 200 nM, about 0.01 nM to about 150 nM, about 0.01 nM to about 100 nM, about 0.01 nM to about 80 nM, about 0.01 nM to about 60 nM, about 0.01 nM to about 50 nM, about 0.01 nM to about nM 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 2nM, 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 0.05nM to about 500nM, about 0.05nM to about 450nM, about 0.05nM to about 50 ... nM, about 0.05 nM to about 400 nM, about 0.05 nM to about 350 nM, about 0.05 nM to about 300, about 0.05 nM to about 250 nM, about 0.05 nM to about 200 nM, about 0.05 nM to about 150 nM, about 0.05 nM to about 100 nM, about 0.05 nM to about 80 nM, about 0.05 nM to about 60 nM, about 0.05 nM to about 50 nM, about 0.05 nM to about 40 nM, about 0.05 nM to about 25 nM, about 0.05 nM to about 20 nM, about 0.05 nM to about 15 nM, about 0.05 nM to about 10 nM, about 0.05 nM to about 80 nM, about 0.05 nM to about 60 nM, about 0.05 nM to about 50 nM, about 0.05 nM to about 40 nM, about 0.05 nM to about 25 nM, about 0.05 nM to about 20 nM, about 0.05 nM to about 15 nM, about 0.05 nM to about 10 nM, about 0.05 nM to 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 0.05nM 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, about 0.1nM to about 350nM, about 0.1nM to about 300, about 0.1nM to about 250nM,about 0.1 nM to about 200 nM, about 0.1 nM to about 150 nM, about 0.1 nM to about 100 nM, about 0.1 nM to about 80 nM, about 0.1 nM to about 60 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 40 nM, about 0.1 nM to about 25 nM, about 0.1 nM to about 20 nM, about 0.1 nM to about 15 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.8 nM, about 0.1 nM to about 0.6nM, about 0.1nM to about 0.4nM, about 0.1nM to about 0.2nM, about 0.2nM to about 500nM, about 0.2nM to about 450nM, about 0.2nM to about 400nM, about 0.2nM to about 350nM, about 0.2nM to about 300, about 0.2nM to about 250nM, about 0.2nM to about 200nM, about 0.2nM to about 150nM, about 0.2nM to about 100nM, about 0.2nM to about 80nM, about 0.2nM to about 60nM, about 0.2nM to about 50nM, about 0.2nM to about 40nM, about 0.2nM to about 25nM, about 0.2nM to about 20nM, about 0.2nM to about 150nM, about 0.2nM to about 100nM, about 0.2nM to about 80nM, about 0.2nM to about 60nM, about 0.2nM to about 50nM, about 0.2nM to about 40nM, about 0.2nM to about 25nM, about 0.2nM to about 20nM, about 0.2nM to about 1 5nM, about 0.2nM to about 10nM, about 0.2nM to about 8nM, about 0.2nM to about 6nM, about 0.2nM to about 5nM, about 0.2nM to about 4nM, about 0.2nM to about 3nM, about 0.2nM to about 2nM, about 0.2nM to about 1nM, about 0.2nM to about 0.8nM, about 0.2nM to about 0.6nM, about 0.2nM to about 0.4nM, about 0.4nM to about 500nM, about 0.4nM to about 450nM, about 0.4nM to about 400nM, about 0.4nM to about 350nM, about 0.4nM to about 300, about 0.4nM to about 250nM, about 0.4nM to about 200nM, about 0.4nM to about 1 about 0.4 nM to about 5 nM, about 0.4 nM to about 4 nM, about 0.4 nM to about 50 nM, about 0.4 nM to about 100 nM, about 0.4 nM to about 80 nM, about 0.4 nM to about 60 nM, about 0.4 nM to about 50 nM, about 0.4 nM to about 40 nM, about 0.4 nM to about 25 nM, about 0.4 nM to about 20 nM, about 0.4 nM to about 15 nM, about 0.4 nM to about 10 nM, about 0.4 nM to about 8 nM, about 0.4 nM to about 6 nM, about 0.4 nM to about 5 nM, about 0.4 nM to about 4 nM, about 0.4 nM to about 3 nM, about 0.4 nM to about 2 nM, about 0.4 nM to about 1 nM, about 0.4 nM to about 0.8 nM, about 0.4 nM to about 0.6 nM, about 0.5 nM to about 500 nM,about 0.5 nM to about 450 nM, about 0.5 nM to about 400 nM, about 0.5 nM to about 350 nM, about 0.5 nM to about 300, about 0.5 nM to about 250 nM, about 0.5 nM to about 200 nM, about 0.5 nM to about 150 nM, about 0.5 nM to about 100 nM, about 0.5 nM to about 80 nM, about 0.5 nM to about 60 nM, about 0.5 nM to about 50 nM, about 0.5 nM to about 40 nM, about 0.5 nM to about 250 nM, about 0.5 nM to about 20 nM, about 0.5 nM to about 150 nM, about 0.5 nM to about 100 nM, about 0.5 nM to about 80 nM, about 0.5 nM to about 60 nM, about 0.5 nM to about 50 nM, about 0.5 nM to about 40 nM, about 0.5 nM to about 250 nM, about 0.5 nM to about 10 nM, about 0.5 nM to about 8 nM, about 0.5 nM to about 6 nM, about 0.5 nM to about 5 nM, about 0.5 nM to about 4 nM, about 0.5 nM to about 3 nM, about 0.5 nM to about 2 nM, about 0.5 nM to about 1 nM, about 0.5 nM to about 0.8 nM, about 0.5 nM to about 0.6 nM, about 0.6 nM to about 500 nM, about 0.6 nM to about 450 nM, about 0.6 nM to about 400 nM, about 0.6 nM to about 350 nM, about 0.6 nM to about 300, about 0.6 nM to about 250 nM, about 0.6 nM to about 200 0 nM, about 0.6 nM to about 150 nM, about 0.6 nM to about 100 nM, about 0.6 nM to about 80 nM, about 0.6 nM to about 60 nM, about 0.6 nM to about 50 nM, about 0.6 nM to about 40 nM, about 0.6 nM to about 25 nM, about 0.6 nM to about 20 nM, about 0.6 nM to about 15 nM, about 0.6 nM to about 10 nM, about 0.6 nM to about 8 nM, about 0.6 nM to about 6 nM, about 0.6 nM to about 5 nM, about 0.6 nM to about 4 nM, about 0.6 nM to about 3 nM, about 0.6 nM to about 2 nM, about 0.6nM to about 1nM, about 0.6nM to about 0.8nM, about 1nM to about 500nM, about 1nM to about 450nM, about 1nM to about 400nM, about 1nM to about 350nM, about 1nM to about 300, about 1nM to about 250nM, about 1nM to about 200nM, about 1nM to about 150nM, about 1nM to about 100nM, about 1nM to about 80nM, about 1nM to about 60nM, about 1nM to about 50nM, about 1nM to about 40nM, about 1nM to about 25nM, about 1nM to about 20nM, about 1nM to about 15nM, about 1nM to about 100nM, about 1nM to about 80nM, about 1nM to about 60nM, about 1nM to about 50nM, about 1nM to about 40nM, about 1nM to about 25nM, about 1nM to about 20nM, about 1nM to about 15nM, about 1 nM to about 10 nM, about 1 nM to about 8 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 500 nM, about 2 nM to about 450 nM, about 2 nM to about 400 nM, about 2 nM to about 350 nM, about 2 nM to about 300, about 2 nM to about 250 nM, about 2 nM to about 200 nM,about 2nM to about 150nM, about 2nM to about 100nM, about 2nM to about 80nM, about 2nM to about 60nM, about 2nM to about 50nM, about 2nM to about 40nM, about 2nM to about 25nM, about 2nM to about 20nM, about 2nM to about 15nM, about 2nM to about 10nM, about 2nM to about 8nM, about 2nM to about 6nM, about 2nM to about 5nM, about 2nM to about 4nM, about 2nM to about 3nM, about 5nM to about 500nM, about 5nM to about 450nM, about 5nM to about 400nM, about 5nM to about 350nM, about 5nM to about 300nM, about 5nM to about 250nM, about 5nM to about 200nM, about 5nM to about nM to about 150nM, about 5nM to about 100nM, about 5nM to about 80nM, about 5nM to about 60nM, about 5nM to about 50nM, about 5nM to about 40nM, about 5nM to about 25nM, about 5nM to about 20nM, about 5nM to about 15nM, about 5nM to about 10nM, about 5nM to about 8nM, about 5nM to about 6nM, about 10nM to about 500nM, about 10nM to about 450nM, about 10nM to about 400nM, about 10nM to about 350nM, about 10nM to about 300, about 10nM to about 250nM, about 10nM to about 200nM, about 10nM to about 150nM, about 10nM to about 100nM, about 10 nM to about 80nM, about 10nM to about 60nM, about 10nM to about 50nM, about 10nM to about 40nM, about 10nM to about 25nM, about 10nM to about 20nM, about 10nM to about 15nM, about 20nM to about 500nM, about 20nM to about 450nM, about 20nM to about 400nM, about 20nM to about 350nM, about 20nM to about 300, about 20nM to about 250nM, about 20nM to about 200nM, about 20nM to about 150nM, about 20nM to about 100nM, about 20nM to about 80nM, about 20nM to about 60nM, about 20nM to about 50nM, about 20nM to about 40nM, about 50 nM to about 500nM, about 50nM to about 450nM, about 50nM to about 400nM, about 50nM to about 350nM, about 50nM to about 300, about 50nM to about 250nM, about 50nM to about 200nM, about 50nM to about 150nM, about 50nM to about 100nM, about 50nM to about 80nM, about 100nM to about 500nM, about 100nM to about 450nM, about 100nM to about 400nM, about 100nM to about 350nM, about 100nM to about 300, about 100nM to about 250nM, about 100nM to about 200nM, about 100nM to about 150nM, about 250nM to about 500nM,The invention specifically binds to CD3 with a Kd of 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).

[0084] Connectors

[0085] The target binding protein may include one or more joints. The joint may include an amino acid sequence connecting two components (e.g., between a heavy chain variable domain and a light chain variable domain, between a target binding domain and a cleavable portion, between a target binding domain and a shielding portion, between a shielding portion and a cleavable portion, or between a half-life extension portion and another component in the target binding protein). The joint may be one that cannot be cleaved by any protease, or one that cannot be cleaved by any human naturally occurring protease. In some embodiments, one or more joints may be flexible joints that may be introduced into the target binding protein to provide flexibility at one or more junctions between domains, at junctions between parts, at junctions between parts and domains, or at any other junctions where the joint will be beneficial. In some embodiments, when the target binding protein is provided in the form of a conformationally constrained construct, a joint may be inserted to facilitate the formation and maintenance of the structure.

[0086] Any linker described herein can provide the desired flexibility to help inhibit the binding of the target, or to facilitate the cleavage of the cleavable portion by a protease. In some embodiments, the linker can be fully or partially flexible, such that the linker can include a flexible linker and one or more portions that impart less flexible structure to provide the desired target binding protein. Some linkers can include cysteine ​​residues, which can form disulfide bonds and reduce the flexibility of the construct.

[0087] In some embodiments, when the target binding protein comprises a masking moiety, the linker coupled to the masking moiety can have a certain length so that the masking moiety is in a position in the tertiary or quaternary structure that effectively masks the target binding domain in the protein, thereby 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 located proximal to the target binding domain to be masked.

[0088] In most cases, the linker length is determined by counting the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the preceding component to the C-terminus of the linker adjacent to the N-terminal amino acid of the following component in the N to C direction (i.e., wherein the linker length does not include the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component).

[0089] In some embodiments, the linkers may comprise 1 to 50, 1 to 40, 1 to 30, 1 to 25 total (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 5, 2 to 4, 2 to 3, 4 to 25, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 15, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 5 to 25, 5 to 24, 5 to 22, 5 to 20, 5 to 18, 5 to 16, 5 to 15, 5 to 14, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 25, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 15, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 25, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 15, 8 to 14, 8 to 12, 8 to 10, 10 to 25, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 15, 10 to 14, 10 to 12, 12 to 25, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 15, 12 to 14, 14 to 25, 20 to 24, 20 to 22, 22 to 25, 22 to 24, or 24 to 25 amino acids). In some embodiments, the linker may comprise 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 total.

[0090] In some embodiments, the linker may be rich in glycine (Gly or G) residues. In some embodiments, the linker may be rich in serine (Ser or S) residues. In some embodiments, the linker may be rich in glycine and serine residues. In some embodiments, the linker may have one or more pairs of glycine-serine residues (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more pairs of GS).

[0091] In some embodiments, the 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, the 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, the 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). Examples of linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GGS)n, (GSGGS)n, and (GGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to act as neutral links between components. Glycine even approaches significantly greater affinity than alanine. sterically, and are much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include one or a combination of one or more of the following: 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), GGGGSGGGGSGGGGS (SEQ ID NO: 676), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 677), GGGGSGGGGS (SEQ ID NO: 678), GGGGS (SEQ ID NO: 679), GS, GGGGSGS (SEQ ID NO: 680), NO: 680), GGGGSGGGGSGGGGSGS (SEQ ID NO: 681), GGSLDPKGGGGS (SEQ ID NO: 682), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 683), SKYGPPCPPCPAPEFLG (SEQ ID NO: 684), GKSGSGSESKS (SEQ ID NO: 685), GSTSGSGK SSEGKG (SEQ ID NO:686), GSTSGSGKSSEGSGSTKG (SEQ ID NO:687), GSTGSSGKPGSGEGSTKG (SEQ ID NO:688), and GSTGSGSKPGSSEGST (SEQ ID NO:689) or GSTSGSGKPGSSEGST (SEQ ID NO:690).

[0092] Examples of linkers can also 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 ordinary skill will recognize that designs can include fully or partially flexible linkers such that a linker can include a flexible linker as well as one or more portions that impart a less flexible structure to provide a desired target binding protein structure.

[0093] In some embodiments, the target binding protein may include one, two, three, four, five, six, seven, eight, nine, or ten linkers (e.g., the same or different linker sequences of any exemplary linker sequences described herein or known in the art). In some embodiments, the linker may include a linker that is not based on amino acids, 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), wherein the linker reacts with a primary amine thiol.

[0094] Half-life extension component (EM)

[0095] The target binding protein may also comprise a half-life extending moiety (EM). In some examples, the half-life extending moiety may be a serum half-life extending moiety, ie, capable of extending the serum half-life of a molecule to which the EM is attached.

[0096] In some instances, 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 instances, 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 this case, the two Fc domain mutants may be an Fc domain hole mutant and an Fc domain knob mutant. The knob mutant and the hole mutant may interact with each other to promote the dimerization of the two Fc domains. In some embodiments, the knob mutant and the hole mutant 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 modification comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one IgG Fc domain, and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in another IgG Fc domain (numbered according to the EU numbering system). Examples of Fc mutants also include SEQ ID NOs: 26-27.

[0097] Examples of Fc domain mutants 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 is contained in an amino acid substitution T366Y in an IgG Fc domain, and an amino acid substitution Y407T in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution T366W in an IgG Fc domain, and an amino acid substitution Y407A in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution F405A in an IgG Fc domain, and an amino acid substitution T394W in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution T366Y and F405A in an IgG Fc domain, and an amino acid substitution T394W and Y407T in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution T366W and F405W in an IgG Fc domain, and an amino acid substitution T394S and Y407A in another IgG Fc domain. In one example, modification is included in the amino acid replacement F405W and Y407A in an IgG Fc domain, and the amino acid replacement T366W and T394S in another IgG Fc domain. In one example, modification is included in the amino acid replacement F405W in an IgG Fc domain, and the amino acid replacement T394S in another IgG Fc domain. The mutation position in the Fc domain is numbered according to the EU numbering system. The IgG Fc domain can include the sequence of SEQ ID NO:34-37 (IgG1, IgG2, IgG3 or IgG4). In these sequences, amino acid 1-107 corresponds to EU numbering 341-447.

[0098] In some instances, the Fc domain mutant may have reduced effector function. Examples of such Fc domains include those disclosed in US20190135943, which are incorporated herein by reference in their entirety. Other examples of Fc domains include SEQ ID NO: 38-42.

[0099] Other examples of EMs include immunoglobulins (e.g., IgG), serum albumins (e.g., human serum albumin (HSA)), six-capped GST (glutathione S-transferase) glutathione affinity, calmodulin (CBP) binding peptide (CBP), Strep-tag, cellulose binding domain, maltose binding protein, S peptide tag, chitin binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope and VSV epitope.

[0100] 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 a half-life extending moiety), e.g., the pK of the target binding protein is longer than the pK of the reference protein. In some instances, the serum half-life of a target binding protein with an EM may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold longer than the serum half-life of the same reference target binding protein without the EM. In some embodiments, when administered to an organism, the serum half-life of the target binding protein 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.

[0101] Additional target binding domains

[0102] In addition to the CD3 binding domains described herein, the target binding protein may also include one or more target binding domains. In some embodiments, the target binding protein may include additional light chain variable domains and additional heavy chain variable domains. Additional light chain variable domains and additional heavy chain variable domains can form additional target binding domains. In some instances, the CD3 binding domain and the additional target binding domain can be the same. In some instances, the CD3 binding domain and the additional target binding domain can be different from each other (e.g., can specifically bind to the same or different antigens or epitopes).

[0103] 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 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.

[0104] In some embodiments, the target binding protein can be multispecific (e.g., bispecific, trispecific, tetraspecific, and other multispecific target binding proteins), for example, binding CD3 and one or more additional targets. In some embodiments, the multispecific target binding protein can be multivalent, for example, comprising multiple target binding sites, regardless of whether the binding sites recognize the same or different targets. In some embodiments, the target binding protein can be bispecific. The term "bispecific" means that the target binding protein is able to specifically bind to two different targets. Typically, a bispecific target binding protein comprises two target binding domains, each of which is able to specifically bind to different targets. In some embodiments, a bispecific target binding protein is able to simultaneously bind to two targets, for example, two target proteins expressed on two different cells.

[0105] In some embodiments, the target binding protein may include a CD3 binding domain and an additional target binding domain capable of binding to a molecule on the surface of a cell (e.g., a tumor cell) associated with a disease. Such target binding proteins can be simultaneously combined with immune cells (e.g., T cells) and cells associated with a disease (e.g., tumor cells), thereby activating immune cells and cross-linking activated immune cells with cells associated with the disease. In some embodiments, the target binding protein can be formulated as a part of a chimeric antigen receptor (CAR), a T cell engagement antibody (e.g., a T cell engagement bispecific antibody), a former bispecific T cell engager (former BITE) molecule, a former chimeric antigen receptor (former CAR) modified T cell or other engineered receptors or other immune effector cells (e.g., CAR modified NK cells).

[0106] In some examples, the target binding protein can be a monovalent bispecific antibody comprising a CD3 binding domain and an additional target binding domain described herein. As used herein, the term "monovalent bispecific antibody" refers to a bispecific antibody having only one antigen binding domain directed against a given target. The inventors unexpectedly discovered that certain CD3 binding domains 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).

[0107] The targets of the additional target binding domains can be proteins or other types of molecules, such as cell surface receptors and secretory binding proteins (eg, growth factors), soluble enzymes, structural proteins (eg, collagen, fibronectin), and the like.

[0108] In some examples, the additional target binding domain can bind to a target of a molecule on or in a cell associated with the disease. For example, the additional target binding domain can bind to a tumor cell. In such cases, the additional target binding domain may bind to a tumor-associated antigen. As used herein, the term "tumor-associated antigen" refers to any antigen associated with cancer, including proteins, glycoproteins, gangliosides, carbohydrates, lipids. Such antigens may be expressed on tumor cells (e.g., malignant cells) or in a tumor microenvironment (e.g., tumor-associated blood vessels, extracellular matrix, mesenchymal matrix, 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.

[0109] Activates target binding protein

[0110] In one aspect, the target binding protein herein includes an activatable target binding protein. In general, the activatable target binding protein may include a prodomain, which refers to a polypeptide that inhibits the target binding of the target binding protein when connected to the target binding protein, and includes an amino acid sequence that forms a protease cleavable substrate. The prodomain portion that inhibits target binding is called a masking portion (MM), and the amino acid sequence that is a protease cleavable substrate is called a cleavable portion (CM). The prodomain may include a connector (L) located between MM and CM and / or at the end of the prodomain (e.g., carboxyl end and / or amino end, to promote the connection of the prodomain to the target binding protein). In certain embodiments, the prodomain may include one of the following formulas (representing an amino acid sequence from the N-terminal to the C-terminal direction): 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, wherein each "-" represents direct or indirect (e.g., via a connector) connection.

[0111] As used herein, the term "activatable target binding protein" refers to a target binding protein in an inactive (uncleaved or native) state. It will be apparent to one of ordinary skill that in some embodiments, a cleaved activatable target binding protein may be linked to a MM that does not reduce, inhibit, or interfere with the binding between the target binding domain and its target. In some embodiments, the cleaved activatable target binding protein may lack a MM due to the release of the MM caused by the cleavage of the CM (e.g., cleavage by a protease). As used herein, the term "cleaved state" or "active state" refers to the condition 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 condition of an activatable target binding protein in the absence of cleavage of the CM by a protease.

[0112] Activatable means that when the activatable target binding protein is in an inhibited, masked or uncleaved state (i.e., a first conformation), the activatable target binding protein exhibits a first binding level to the target, and when in an uninhibited, unmasked and / or cleaved state (i.e., a second conformation), the activatable target binding protein exhibits a second binding level to the target, wherein the second target binding level is greater than the first binding level. Generally speaking, in the presence of a cleaving agent (i.e., a protease) that is capable of cleaving the CM, the target is closer to the target binding domain of the activatable target binding protein than in the absence of such a cleaving agent. Thus, when the activatable target binding protein is in an uncleaved state, the binding of the target binding domain to the target is inhibited and its target binding may be masked (i.e., the first conformation renders the target binding domain unable to bind to the target or inhibited from binding to the target), and in the cleaved state, the target binding of the target binding domain is not inhibited or masked.

[0113] 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 so that when conformationally constrained, the MM provides masking of the target binding domain or reduced binding of the target binding domain to its target. Structural design criteria can be considered to provide this functional feature.

[0114] The activatable target binding protein herein exhibits an activatable phenotype of the desired dynamic range of target binding in a suppressed conformation versus an unsuppressed conformation. Dynamic range generally refers to the ratio of the maximum detection level of a parameter under (a) a first set of conditions to the minimum detection value of the parameter under (b) a second set of conditions. For example, in the context of an activatable target binding protein, dynamic range refers to the ratio of the maximum detection level of a target protein bound to an activatable target binding protein (a) in the presence of a protease capable of cleaving the CM in an activatable target binding protein to the minimum detection level of a target protein bound to an activatable target binding protein (b) 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 an activatable target binding protein cleaving agent (e.g., enzyme) to the dissociation constant of an activatable target binding protein cleaving agent. The larger the dynamic range of an activatable target binding protein, the better the activatable phenotype of an 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 the activatable target binding protein to the target protein occurs to a greater extent (e.g., predominantly) in the presence of a cleavage agent (e.g., an enzyme) capable of cleaving the CM of the activatable target binding protein than in the absence of the cleavage agent.

[0115] The activatable target binding protein herein may comprise a target binding domain (TB), one or more masking moieties (MM) that reduce, inhibit or interfere with the binding of the target binding domain to its target, one or more cleavable moieties (CM) that couple one or more MMs to TB, and optionally one or more half-life extension moieties (EM). In some embodiments, the activatable target binding protein may comprise a TB that specifically binds to CD3, and a masking moiety (MM) that inhibits the binding of TB to CD3, wherein the MM is coupled to TB via a cleavable moiety (CM) (directly or indirectly, for example, via one or more linkers). As used herein and unless otherwise indicated, the "coupled" component of the activatable target binding protein may be coupled via direct covalent bonds or indirect covalent bonds, for example, via one or more connecting peptides (also referred to as "linkers"), cleavable moieties, or other components of the activatable protein.

[0116] In some embodiments, the activatable target binding protein may include more than one target binding domain (TB). For example, the activatable target binding protein may include a first TB that specifically binds to CD3, a first MM (MM1) that inhibits the binding of TB1 to CD3, a second target binding domain (TB2) that specifically binds to a second target, and a second masking portion (MM2) that inhibits the binding of TB2 to the second target, wherein MM1 is coupled to TB1 via a first cleavable portion (CM1) (directly or indirectly, such as via one or more connectors), and wherein MM2 is coupled to TB2 via a second cleavable portion (CM2) (directly or indirectly, such as via one or more connectors). The activatable target binding protein may also include a half-life extension portion (EM). In one example, the activatable target binding protein includes a scFv containing TB1.

[0117] Masking part(MM)

[0118] The activatable target binding protein herein may comprise one or more masking moieties (MM) that are capable of interfering with the binding of TB to the target. The masking moiety in the activatable molecule (not yet activated) "masks" or reduces or otherwise inhibits the binding of the target binding domain to its target. In some embodiments, the coupling or modification of the target binding protein to the MM can inhibit the ability of the protein to specifically bind to its target by inhibition methods known in the art (e.g., structural changes and competing antigen binding domains). In some embodiments, the coupling or modification of the target binding protein to the MM may cause structural changes, thereby reducing or inhibiting the ability of the protein to specifically bind to its target. In some embodiments, the coupling or modification of the target binding protein to the MM sterically blocks, reduces or inhibits the ability of the antigen binding domain to specifically bind to its target.

[0119] MM is coupled to TB via CM and optionally one or more linkers described herein. In some embodiments, when the activatable target binding protein is not activated, MM prevents TB from binding to the target; however, when the activatable target binding protein is activated (when CM is cleaved by a protease), MM does not substantially or significantly interfere with the binding of TB to the target.

[0120] In the activatable target binding protein, the MM that interferes with TB target binding can be coupled to TB (directly or indirectly, for example, via one or more linkers). Alternatively, the MM that interferes with TB target binding can be directly or indirectly coupled to an activatable target binding protein component other than TB. For example, the MM can be directly or indirectly coupled to a different TB. In another example, the MM can be directly or indirectly coupled to the EM. In either case, in the tertiary or quaternary structure of the activatable structure, the MM may be in a position that allows the MM to mask the TB (for example, close to the TB to be masked).

[0121] In some embodiments, MM may interact with TB, thereby reducing or inhibiting the interaction between TB and its binding partner. In some embodiments, MM may comprise at least part or all of the amino acid sequence of a naturally occurring binding partner of TB. For example, MM may be a fragment of a naturally occurring binding partner. The fragment may retain up to 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25% or 20% nucleic acid or amino acid sequence homology with a naturally occurring binding partner. In some embodiments, MM may be a homologous peptide of TB. For example, MM may comprise a sequence of a TB epitope or a fragment thereof. As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and is not intentionally modified by a person in a laboratory or otherwise is naturally occurring.

[0122] In some embodiments, MM comprises a non-naturally occurring amino acid sequence, or an amino acid sequence that does not contain a naturally occurring binding partner or target protein. In certain embodiments, MM is not a natural binding partner of TB. MM may be a modified binding partner of TB that contains amino acid changes that reduce binding affinity and / or avidity to TB. In some embodiments, MM does not contain or substantially does not contain nucleic acid or amino acid homology to a natural binding partner of TB. In other embodiments, MM is at most 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.

[0123] In some embodiments, the MM may not bind specifically to the TB, but rather 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 located in the 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 TB in place to interfere with the binding partner's access to the TB.

[0124] In some embodiments, the dissociation constant of MM binding to TB does not exceed the dissociation constant of TB to the target. In some embodiments, MM does not interfere with or compete for the binding of TB to the target in the cleaved state.

[0125] The structural properties of the MM can be selected based on factors such as the minimal amino acid sequence required to interfere with protein binding to the target, the target protein-protein binding pair of interest, the size of the TB, the presence or absence of a linker, etc.

[0126] In some embodiments, the MM is unique to the coupled TB. Examples of MMs include MMs that are specifically screened to bind to a binding domain of TB or a fragment thereof (e.g., affinity masking). Methods for screening MMs to obtain MMs that are unique to TB and those that specifically and / or selectively bind to a binding domain of a binding partner / target are provided herein and may include protein display methods.

[0127] As used herein, the term "masking efficiency" refers to the ability of a target binding protein to activate activity in an inactive state (e.g., EC 50 ) divided by the activity of a control antibody, wherein the control antibody can be a cleavage product of the activatable target binding protein, or an antibody or fragment thereof of TB used as an activatable target binding protein. The activatable target binding protein with reduced levels of TB activity can have a masking efficiency greater than 10. In some embodiments, the activatable target binding protein described herein has a masking efficiency greater than 10, 100, 1000, or 5000.

[0128] In some embodiments, the MM can be a polypeptide of about 2 to 50 amino acids in length. For example, the MM can be a polypeptide of 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50 amino acids in length. For example, the MM can be a polypeptide 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 length. In some examples, the MM can be a polypeptide that is greater than 50 amino acids in length, such as 100, 200, 300, 400, 500, 600, 700, 800 or more amino acids.

[0129] In some embodiments, when measured in an in vitro immunosorbent assay, for example, as described in US20200308243A1, in an inactive state of an activatable target binding protein having TB and an interfering MM, in the presence of a TB target, TB has no binding or substantially no binding to the target, or TB has binding to the target of at most 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 101%, 10 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0130] The binding affinity of TB to the target or binding partner under the interfering MM may be 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 to its binding partner without the interfering MM, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 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, 1,000-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1000-10,000,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times.

[0131] The dissociation constant of MM for its masked TB may be greater than the dissociation constant of TB for the target. The dissociation constant of MM for 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 TB for the target. Conversely, the binding affinity of MM for masked TB may be lower than the binding affinity of TB for the target. The binding affinity of MM for 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 for the target.

[0132] In some embodiments, the MM may contain genetically encoded amino acids or genetically non-encoded amino acids. Examples of genetically non-encoded amino acids include, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In specific embodiments, the MM contains at most 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of genetically non-encoded amino acids.

[0133] In some embodiments, once released from the activatable target binding protein and in a free state, the MM may have a biological activity or therapeutic effect, such as binding ability. For example, the free peptide may bind to the same or different binding partners. In certain embodiments, the free MM may exert a therapeutic effect, thereby providing a secondary function for the compositions disclosed herein. In some embodiments, once uncoupled from the activatable target binding protein and in a free state, the MM may advantageously not exhibit biological activity. For example, in some embodiments, the MM in a free state does not induce an immune response in a subject.

[0134] Suitable MMs can be identified and / or further optimized from a library of candidate activatable target binding proteins with variable MMs by a screening procedure. For example, TB and CM can be selected to provide a desired enzyme / target combination, and the amino acid sequence of the MM can be identified by a screening procedure described below to identify MMs that provide a switchable phenotype. For example, a random peptide library (e.g., a random peptide library comprising peptides of 2 to 40 amino acids or more) can be used in the screening methods disclosed herein to identify suitable MMs.

[0135] In some embodiments, MMs with specific binding affinity for TB can be identified by a screening procedure comprising providing a library of peptide scaffolds comprising candidate MMs, wherein each scaffold consists of a transmembrane protein and a candidate MM. The library can then be contacted with the entire protein or a portion (e.g., a full-length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing a protein that is also capable of binding a binding partner of interest), and one or more candidate MMs with detectably bound proteins are identified. Screening can be performed by one or more rounds of magnetic activated sorting (MACS) or fluorescence activated sorting (FACS), and determining the binding affinity of the MMs for TB and subsequently determining the masking efficiency, such as described in WO2009025846 and US20200308243A1, which are incorporated herein by reference in their entirety.

[0136] In some embodiments, the MM can be selected for use with a specific antibody or antibody fragment. For example, a MM suitable for use with a TB that binds an epitope may comprise a sequence of the epitope. In some examples, MMs suitable for masking the anti-CD3 binding proteins disclosed herein include MMs comprising the sequence of GYLWGCEWNCGGITT (SEQ ID NO: 691), NAFRCW WDPPCQPMT (SEQ ID NO: 692), ARGLCWWDPPCTHDL (SEQ ID NO: 693), or NHSLCYWDPPCEPST (SEQ ID NO: 694). Additional masking portions of anti-CD3 binding proteins include sequences of MMYCGGNEVLCGP RV (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).

[0137] Examples of suitable MMs are disclosed in WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO201916 5143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201 and WO2016014974, which are incorporated herein by reference in their entirety.

[0138] Cleavable fraction (CM)

[0139] The activatable target binding protein may comprise one or more cleavable moieties (CM). The terms "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.

[0140] The CM and TB of the activatable target binding protein can be selected such that the TB represents a conjugated moiety for a given target, and the CM represents a substrate for one or more proteases, wherein the proteases are co-localized with the target in a tissue (e.g., at a treatment site or a diagnostic site in a subject). When the activatable target binding protein is exposed to the protease, the protease may cleave the CM in the activatable target binding protein. In some embodiments, the activatable target binding protein may have a special use, where, for example, one or more proteases capable of cleaving a site in the CM are present at relatively higher levels in target-containing tissues at a treatment site or a diagnostic site than in tissues at non-treatment sites (e.g., in healthy tissue).

[0141] In some embodiments, the CM herein may include substrates for proteases whose known substrates have been reported in various cancers. See, for example, La Roca et al., British J. Cancer 90(7):1414-1421, 2004. Substrates suitable for use in the CM components employed herein include those more commonly found in cancerous cells and tissues. Thus, in certain embodiments, the CM may include substrates for proteases more commonly found in diseased tissues associated with cancer. Examples of cancer include gastric cancer, breast cancer, osteosarcoma, esophageal cancer, breast cancer, HER2-positive cancer, Kaposi sarcoma, hairy cell leukemia, chronic myeloid 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), colorectal 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 that is more prevalent in tumor tissue. For example, the protease can be produced by a tumor of a subject.

[0142] In some embodiments, the activatable target binding protein may comprise two CMs (e.g., for coupling a MM to multiple TBs). In some instances, the first and second CMs may comprise substrates for the same protease. In some instances, the first and second CMs may comprise substrates for different proteases. In some instances, the first and second CMs may comprise the same sequence or consist of the same sequence. In some instances, the first and second CMs may comprise different sequences or consist of different sequences.

[0143] CMs suitable for use herein that can activate target binding proteins include any protease substrates known in the art. In some examples, the CM may include substrates for serine proteases (e.g., u-type plasminogen activator (uPA, also known as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1). In some examples, the CM may include substrates for matrix metalloproteinases (MMPs). In some examples, the CM may include substrates for cysteine ​​proteases (CPs) (e.g., legumin).

[0144] In some embodiments, the CM may comprise substrates for a disintegrin and metalloproteinase (ADAM) or a disintegrin and metalloproteinase with a thrombospondin motif (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), an apoptotic protease (e.g., apoptotic cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P), cysteine ​​proteases (e.g., Cruzipain, legumin, Otubain-2), Protease, DESC1, DPP-4, FAP, Elastase, FVIIa, FIXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoic acid enzyme, Hepsin, HtrA1, Human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), Metalloproteinase (e.g., Meprin, Enkephalinase, PSMA, BMP-1), Lactoferrin, Marapsin , interstitial proteinase-2, MT-SP1 / interstitial proteinase, 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.

[0145] In some embodiments, the protease substrate in the CM may comprise a peptide sequence that is substantially non-identical (eg, no more than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence naturally cleaved by the same protease.

[0146] In some embodiments, the CM may be or include a sequence encompassed by the consensus sequence of any one of the sequences in Table 6 below.

[0147] Table 6. Exemplary CM sequences

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] Examples of CM also include CMs described in WO 2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO201 9165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974, which are incorporated herein by reference in their entirety for all purposes.

[0157] In some embodiments, the CM may be or include a combination of the above-mentioned exemplary sequences, a C-terminal truncated variant, or an N-terminal truncated variant. The truncated variants of the above-mentioned amino acid sequences suitable for use in the CM may be any truncated variants that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncated variants that retain the recognition site of the protease and contain at least 3 consecutive amino acids of the above-mentioned amino acid sequence or at least 4, 5, 6, 7, 8, 9 or 10 amino acids of the aforementioned amino acid sequence. In certain embodiments, the truncated variants of the above-mentioned amino acid sequence may correspond to any of the above-mentioned amino acid sequences, but the C-terminus and / or N-terminus are truncated by 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids or 1 to 3 amino acids, and: (1) have at least three amino acid residues; and (2) retain the recognition site of the protease. In some of the aforementioned embodiments, the truncated CM is an N-terminal truncated CM. In some embodiments, the truncated CM is a C-terminal truncated CM. In some embodiments, the truncated C is a C-terminally and N-terminally truncated CM.

[0158] 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 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids.

[0159] In some embodiments, CM is treated with at least one protease at a concentration of about 0.001-1500×10 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 1500×10 4 M -1 S -1 The rate of specific cleavage is determined by the substrate cleavage kinetics (k cat / K m ) is measured as disclosed in WO2016118629.

[0160] Conjugating agent

[0161] In some aspects, the target binding protein (including activatable target binding protein) can also include one or more additional agents, for example, a targeting moiety that facilitates delivery to a cell or tissue of interest, a therapeutic agent (e.g., an anti-tumor agent such as a chemotherapeutic agent or 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. Additional agents can be combined with the target binding protein. The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biomacromolecule, or an extract made from a biological material.

[0162] In some embodiments, the target binding protein can be conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof) or a radioactive isotope. In some embodiments, the target binding protein can be conjugated to a T cell activator, such as a small molecule agonist of a toll-like receptor (TLR), including 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-Guérin (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-TLR 9 (LIDDS), PF-3512676 (Pfizer), SD-101 (Dynavax) and S-540956 (Shionogi).

[0163] Examples of cytotoxic agents that can be conjugated to the target binding protein include: dolastatin and its derivatives (e.g., auristatin E, AFP, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), demethyl auristatin E (DMAE), auristatin F, demethyl auristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycin, alpha-amanitin, n), turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PE S8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracycline (anthr acycline, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecins, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoa plysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0164] Examples of enzymatically active toxins that can be conjugated to 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 proteins, dianthin proteins, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotins, sapaonaria officinalis inhibitors, gelonin, mitogeliin, restrictocin, phenomycin, neomycin, and tricothecene.

[0165] Examples of anti-tumor agents that can be conjugated to the target binding protein include: adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabizine, and cytarabine.

[0166] Examples of antiviral agents that can be conjugated to the target binding protein include: acyclovir, vira A, and symmetrel. Examples of antifungal agents that can be conjugated to the target binding protein include: nystatin. Examples of detectable agents 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. 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to target binding proteins include: s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepines Dimer (PBD), functionalized pyrrolobenzodiazepine , calcicheamicin, podophyllotoxin, taxanes, and vinca alkaloids. 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 99mTc (Technetium). Examples 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: tylosine, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0167] In some embodiments, the target binding protein may include a signal peptide. If multiple peptides are included, the target binding protein may include multiple signal peptides, for example, each of the multiple peptides has a signal peptide. The signal peptide can be a peptide (e.g., 10-30 amino acids long) located at the end (e.g., N-terminus or C-terminus) of a newly synthesized protein, the destination of which is the secretory pathway. In some embodiments, the signal peptide can be conjugated to the target binding protein via a spacer. In some embodiments, the spacer is conjugated to the target binding protein in the absence of a signal peptide.

[0168] Those of ordinary skill in the art will recognize that a large number of possible agents can be conjugated to any target binding protein described herein. The agent can be conjugated to another component of the target binding protein through a conjugated portion, which can be a linker, CM, or other molecules or fragments thereof that can connect two molecules. In some instances, the conjugated portion can be cleaved by an enzyme (e.g., a protease). In some instances, the conjugated portion cannot be cleaved by an enzyme (e.g., a protease).

[0169] Conjugation can include any chemical reaction that will bind two molecules, as long as the target binding protein and other parts retain their corresponding activities. Conjugation can include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. In some embodiments, the binding can be covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linkers can be used to conjugate any target binding protein described herein. For example, conjugation can include organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazonium and hexamethylenediamine. In some embodiments, the target binding protein may include or otherwise introduce one or more non-natural amino acid residues to provide a suitable conjugation site.

[0170] In some embodiments, the agent and / or conjugate can be attached to the antigen binding domain via a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Because many cancers naturally release high levels of the reducing agent glutathione, the glutathione present in the cancerous tissue microenvironment can reduce the disulfide bonds and subsequently release the agent and / or conjugate at the delivery site.

[0171] In some embodiments, when the conjugate binds to its target in the presence of complement at the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide bond or ester bond attaching the conjugate and / or agent to the linker is cleaved, thereby releasing the conjugate and / or agent in its active form. When administered to a subject, these conjugates and / or agents can achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These conjugates and / or agents can effectively deliver any conjugate and / or agent described herein in vivo.

[0172] In some embodiments, the conjugated moiety cannot be cleaved by enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, because complement activation will eventually dissolve the target cell. In such embodiments, the conjugate and / or agent will be delivered to the target cell (e.g., hormone, enzyme, corticosteroid, neurotransmitter or gene). In addition, the conjugated moiety is slightly susceptible to cleavage by serum proteases, and the conjugate and / or agent is slowly released at the target site.

[0173] In some embodiments, the conjugate and / or agent can be designed such that the conjugate and / or agent is delivered to a target site (eg, diseased tissue (eg, cancerous tissue)), but the conjugate and / or agent is not released.

[0174] In some embodiments, the conjugate and / or agent can be attached directly to the antigen binding domain, or via amino acids (e.g., D-amino acids), peptides, thiol-containing moieties, or other organic compounds that can be modified to include functional groups that can then be used to attach to the antigen binding domain by the methods described herein.

[0175] In some embodiments, the target binding protein may include at least one conjugation point of the agent. In some embodiments, all possible conjugation points can be used for conjugation with the 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 sulfur atoms involved in intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. In such cases, the residue may be naturally present 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.

[0176] The present disclosure also provides methods and materials for preparing target binding proteins with one or more conjugating agents. In some embodiments, the target binding protein can be modified to include one or more interchain disulfide bonds. For example, the 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, the reduction of the disulfide bonds is only partial. As used herein, the term partial reduction refers to a situation in which the 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 is partially reduced if 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 after contact with a reducing agent. In some embodiments, a target binding protein with one or more interchain disulfide bonds reduced can be conjugated to a drug reactive with free thiols.

[0177] The present disclosure also provides methods and materials for conjugating therapeutic agents to specific positions on target binding proteins. In some embodiments, the target binding protein can be modified so that the therapeutic agent can be conjugated to the target binding protein at a specific position of the target binding protein. For example, the target binding protein can be partially reduced in a manner that is conducive to binding to the target binding protein. In such cases, the partial reduction of the target binding protein occurs in a manner in which the conjugation site in the target binding protein is not reduced. In some embodiments, one or more conjugation sites on the target binding protein are selected to facilitate the conjugation of the agent at a specific position on the protein construct. Various factors can affect the "reduction level" of the target binding protein after treatment with a reducing agent. For example, without limitation, the ratio of the reducing agent to the target binding protein, the duration of incubation, the incubation temperature, and / or the pH value of the reduction reaction solution may need to be optimized to achieve partial reduction of the target binding protein with 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 possible conjugation sites or reduction at a specific conjugation site).

[0178] The 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 conjugation to the agent (eg, general reduction of possible conjugation sites or reduction at a specific conjugation site). 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.

[0179] The effective incubation time and temperature for treating the target binding protein with the reducing agent can be any time and temperature that at least partially reduces the target binding protein (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites) in a manner that allows the agent to conjugate with the target binding protein. In some embodiments, the incubation time and temperature for treating the target binding protein can be in the range of about 1 hour at 37° C. to about 12 hours at 37° C. (or any sub-range therein).

[0180] The effective pH of the reduction reaction for treating the target binding protein with the reducing agent can be any pH that at least partially reduces the target binding protein in a manner that allows conjugation of the target binding protein to the agent (e.g., general reduction of possible conjugation sites or reduction at a specific conjugation site).

[0181] When the partially reduced target binding protein is contacted with a thiol-containing agent, the agent can be conjugated to an interchain thiol in the target binding protein. The agent can be modified using a thiol-containing agent (e.g., cysteine ​​or N-acetylcysteine) to include a thiol. For example, the target binding protein can be partially reduced after incubation with a reducing agent (e.g., TEPC) at a desired ratio of reducing agent to target binding protein at about 37° C. for about 1 hour. The effective reducing agent to target binding protein ratio can be any ratio that partially reduces at least two interchain disulfide bonds located in the target binding protein in a manner that allows the thiol-containing agent to conjugate (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0182] In some embodiments, the target binding protein is reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds. In some embodiments, the target binding protein is reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0183] In some embodiments, the agent (e.g., an agent conjugated to a target binding protein) is a detectable portion, such as an image marker or other marker. For example, the agent can be or include radiolabeled amino acids, one or more biotinyl moieties that can be detected by labeled avidin (e.g., streptavidin containing fluorescent markers or enzyme activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable portion can 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 beta-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 tdimer2 (RFPtdimer2), HCRED or europium derivatives. In some embodiments, the luminescent label may include N-methyl acridine derivatives. In some embodiments, the label may include Alexa Fluor Tags, such as Alex 680 or Alexa 750. In some embodiments, the ligand-based label may comprise biotin, avidin, streptavidin, or one or more haptens.

[0184] Other examples of detectable labels 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, dichlorotriazinamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0185] In some embodiments, the agent can 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 can be conjugated to the target binding protein via a linker and / or a CM as described herein. In some embodiments, the agent can be conjugated to a cysteine ​​or lysine in the target binding protein. In some embodiments, the agent can be conjugated to another residue of the target binding protein (e.g., those residues disclosed herein).

[0186] In some embodiments, a variety of bifunctional protein coupling agents can be used to conjugate the agent to the target binding protein, including but not limited to N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl diimidoadipate hydrochloride), 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, carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelator can be used to conjugate radionucleotides to target binding proteins (see, e.g., WO94 / 11026).

[0187] Suitable conjugated 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 acetyl hydrazide derivatives coupled to target binding proteins via oligopeptides. In some embodiments, suitable conjugated moieties include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-disulfide)-toluene (Pierce Chem., Catalog No. (21558G)); (iii) SPDP (succinimidyl-6-[3-(2-pyridyldisulfide) propionamido] hexanoate (Pierce Chem., Catalog No. 21651G); (iv) sulfo-LC-SPDP (6-[3-(2-pyridyldisulfide)-propionamido] hexanoic acid sulfosuccinimidyl ester (Pierce Chem., Catalog No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.) conjugated to EDC. Chem., Cat. No. 24510). Additional exemplary conjugated moieties include SMCC, Sulfo-SMCC, SPDB, and Sulfo-SPDB.

[0188] The conjugated moieties described above may contain components with different properties, thus making the conjugates have different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. The solubility of the linker containing NHS esters is less than that of sulfo-NHS esters. In addition, SMPT contains a disulfide bond with steric hindrance and can form a conjugate with increased stability. In general, the stability of disulfide bonds is less than that of other bonds because disulfide bonds are cleaved in vitro, resulting in less available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. Compared to a single carbodiimide coupling reaction, carbodiimide coupling (e.g., EDC) forms an ester that is more resistant to hydrolysis when used in conjunction with sulfo-NHS.

[0189] Those of ordinary skill in the art will recognize that a variety of possible moieties can be coupled 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 binds the agent to the target binding protein while also allowing the agent and target binding protein to retain functionality.

[0190] Nucleic acids and vectors

[0191] In some aspects, the disclosure also provides nucleic acids comprising sequences encoding target binding proteins or their components or fragments. Nucleic acids may include coding sequences of heavy chain variable domains, light chain variable domains, TB, CM, MM, EM and joints in target binding proteins. In the case where the target binding protein comprises multiple peptides (e.g., multiple TBs on different peptides, or TB comprises multiple peptides), the nucleic acid may include coding sequences of multiple peptides. In some examples, the coding sequence of one of the peptides is located in one nucleic acid, and the coding sequence of another peptide is located in another nucleic acid. In some examples, the coding sequences of two or more peptides in multiple peptides are located in the same nucleic acid. The disclosure includes polynucleotides encoding proteins or parts thereof as described herein, and the use of such polynucleotides for producing 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 proteins as defined herein. The disclosure includes compositions comprising such polynucleotides. In some aspects, such compositions can be used therapeutically or prophylactically.

[0192] Unless otherwise specified, "protein encoding nucleic acid sequence" includes all nucleotide sequences that are degenerate forms of each other and therefore encode the same amino acid sequence. The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, or a combination thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides with similar binding properties to the reference nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses complementary sequences and sequences explicitly indicated. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid comprises a mixture or a heterocomplex of DNA and RNA.

[0193] 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, the term "N-terminal positioning" 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. 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, the term "C-terminal positioning" 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.

[0194] Modifications can be introduced into the nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with 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 amino acid families include: aliphatic-hydroxy amino acids (e.g., serine and threonine), amide family (e.g., asparagine and glutamine), aliphatic family (e.g., alanine, valine, leucine, and isoleucine), and aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0195] The present disclosure also provides vectors and vector groups comprising any nucleic acid described herein. Those skilled in the art will be able to select a vector or vector group (e.g., an expression vector) suitable for preparing any target binding protein described herein, and use the vector or vector group to express any target binding protein described herein. For example, when selecting a vector or vector group, a cell type can be selected so that one or more vectors may need to be able to be integrated into the chromosome of the cell and / or replicated in the cell. 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 recombinant protein (e.g., the first or second monomer) to be expressed in a cell (e.g., any cell described herein). "Vector" can deliver nucleic acids and fragments thereof to a host cell, and includes regulatory sequences (e.g., promoters, enhancers, polyadenylic acid (poly (A)) signals). Exogenous polynucleotides can be inserted into an expression vector for expression. The term "vector" also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0196] Methods for constructing suitable vectors comprising any nucleic acid described herein and suitable for transforming cells (e.g., mammalian cells) are well known in the art. See, e.g., Sambrook et al., ed., "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Press, 1989 and Ausubel et al., ed., "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0197] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector), as well as any Vector. The vector may, for example, include cis-acting elements sufficient to achieve expression; other elements for expression may be provided by the host mammalian cell or in an in vitro expression system. A skilled practitioner will be able to select vectors and mammalian cells suitable for preparing any target binding protein described herein.

[0198] In some embodiments, target binding proteins can be produced biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0199] cell

[0200] In some aspects, the disclosure provides host cells comprising any vector or nucleic acid described herein. Cells can be used to produce target binding proteins described herein. In some embodiments, cells can be animal cells, mammalian cells (e.g., human cells), rodent cells (e.g., mouse cells, rat cells, hamster cells, or guinea pig cells), non-human primate cells, insect cells, bacterial cells, fungal cells, or plant cells. In some embodiments, cells can be eukaryotic cells. As used herein, the term "eukaryotic cell" refers to a cell with a distinct, membrane-surrounded nucleus. Such cells may include, for example, mammals (e.g., rodents, non-human primates, or humans), insects, fungi, or plant cells. In some embodiments, eukaryotic cells are yeast cells, such as Saccharomyces cerevisiae. In some embodiments, eukaryotic cells are higher eukaryotic cells, such as mammals, birds, plants, or insect cells. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some embodiments, cells are prokaryotic cells.

[0201] Methods for introducing nucleic acids and vectors (e.g., any vector or any vector group described herein) into cells are known in the art. Examples of methods that can be used to introduce nucleic acids into cells include: liposome transfection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0202] In some embodiments, the introducing step comprises introducing into the cell a vector (eg, any vector or set of vectors described herein) comprising a nucleic acid encoding a monomer that makes up any target binding protein described herein.

[0203] Compositions and kits

[0204] The present disclosure also provides compositions and kits comprising the target binding proteins described herein. The compositions and kits may also include one or more excipients, carriers, reagents, instructions required for using the target binding proteins.

[0205] In some embodiments, the composition can be a pharmaceutical composition comprising a target binding protein, its derivatives, fragments, analogs and homologues. The pharmaceutical composition may comprise a target binding protein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents compatible with drug administration, etc. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the art and 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. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, it is expected to be used in the composition. Supplementary active compounds may also be incorporated into the composition.

[0206] The pharmaceutical composition can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous applications may include one or more of the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates, and agents for adjusting tension, such as sodium chloride or dextrose. pH values ​​may be adjusted with acids or bases (e.g., hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic. In some, any target binding protein described herein is prepared with a carrier that prevents rapid elimination from the body, such as sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The preparation methods of such pharmaceutical compositions and formulations are obvious to those skilled in the art. For example, the target binding protein can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively) prepared by coacervation techniques or by interfacial polymerization, embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or embedded in coarse emulsions.

[0207] Sustained release preparations can be prepared.Sustained release preparations include semipermeable matrixes of solid hydrophobic polymers containing antibodies, and the matrix is ​​in the form of formed articles, such as films or microcapsules.Examples of sustained release matrixes include polyesters, hydrogels (such as poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides, copolymers of L-glutamic acid and L-glutamic acid γ ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate (leuprolide acetate)) and poly-D-(-)-3-hydroxybutyric acid.Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, the time for some hydrogels to release proteins is shorter.

[0208] In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where soluble in water) or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ), phosphate buffered saline (PBS), etc. The composition must be sterile and should be a fluid that is easy to inject. It must be stable under the conditions of manufacture and storage and must be preserved to prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained. Prevention of microbial effects can be achieved by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In some embodiments, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) and sodium chloride may be included in the composition. The extended absorption of the injectable composition can be achieved by including an agent (such as aluminum monostearate and gelatin) that delays absorption in the composition.

[0209] In some embodiments, the pharmaceutical composition may include a sterile injection solution. A sterile injection solution may be prepared by incorporating the required amount of active compound and a composition listed above or a combination thereof into an appropriate solvent as required, followed by filtration sterilization. Typically, dispersions may be prepared by incorporating the active compound into a sterile vehicle containing other components required in an alkaline dispersion medium and those components listed above. In the case of a sterile powder for the preparation of a sterile injection solution, the preparation method is vacuum drying and freeze drying to obtain a powder of the active ingredient plus any other required components from a previously sterile filtered solution.

[0210] In some embodiments, the pharmaceutical composition may include an oral composition. The oral composition may include an inert diluent or an edible carrier. They may be encapsulated in a gelatin capsule or compressed into a tablet. For the purpose of oral therapeutic administration, the active compound may be blended with an excipient and used in the form of a tablet, lozenge or capsule. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is taken orally and gargled, coughed out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: a binder, such as microcrystalline cellulose, tragacanth or gelatin; an excipient, such as starch or lactose; a disintegrant 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 mint, methyl salicylate or orange flavoring.

[0211] In one embodiment, the pharmaceutical compositions may be formulated for administration by inhalation. For example, the compounds may be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0212] In one embodiment, the pharmaceutical composition can be formulated for systemic administration. For example, systemic administration can be intravenous, or it can be transmucosal or transdermal. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound can be formulated into an ointment, salves, gels, or creams, as generally known in the art.

[0213] In some embodiments, the pharmaceutical compositions may be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0214] In one embodiment, the pharmaceutical composition can be prepared with a carrier that prevents rapid elimination from the body, such as sustained 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 formulations will be apparent to those skilled in the art.

[0215] Oral or parenteral compositions are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for treating individuals.

[0216] In some embodiments, a composition (eg, a pharmaceutical composition) may be included in a container, vial, syringe, pen, pack, or dispenser, optionally together with instructions for administration.

[0217] Also provided herein are kits comprising any target binding protein described herein, any composition comprising any target binding protein described herein, or any pharmaceutical composition comprising any target binding protein described herein. Also provided are kits comprising one or more second therapeutic agents in addition to the target binding protein described herein. The one or more second therapeutic agents can be provided in a dosage administration form separate from the target binding protein. Alternatively, the one or more second therapeutic agents can be formulated together with the target binding protein.

[0218] Any medicine box described herein may include instructions for using any composition (e.g., pharmaceutical composition) and / or any target binding protein described herein. In some embodiments, the medicine box may include instructions for performing any method described herein. In some embodiments, the medicine box may include at least one dose of any composition (e.g., pharmaceutical composition) described herein. In some embodiments, the medicine box may provide a syringe for administering any pharmaceutical composition described herein.

[0219] Also provided herein are target binding proteins produced by any of the methods described herein. Also provided 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.

[0220] Methods of producing target binding proteins

[0221] Provided herein are methods for producing any target binding protein described herein, the methods comprising: (a) culturing any recombinant host cell 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 cell and / or the liquid culture medium.

[0222] Methods of culturing cells are well known in the art. In some embodiments, cells can be maintained in vitro under conditions that are conducive to cell proliferation, cell differentiation, and cell growth. For example, cells can be cultured by contacting cells (e.g., any cells described herein) with a cell culture medium that includes necessary growth factors and supplements sufficient to support cell viability and growth.

[0223] 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, examples of separation methods include: separation using a protein purification tag (e.g., a 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.

[0224] 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 a target binding protein.

[0225] In some embodiments, the method further comprises formulating the separated target binding protein into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any separated 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).

[0226] Methods using target binding proteins

[0227] In some aspects, the disclosure also provides methods of using the target binding proteins herein. In some embodiments, the disclosure provides methods for treating a disease (e.g., cancer (e.g., any cancer described herein)) of a subject, the method comprising administering a therapeutically effective amount of any target binding protein described herein to the subject. In some embodiments, the disclosure provides methods for preventing a subject's disease, delaying the progression of a disease, treating a disease, alleviating the symptoms of a disease, or otherwise improving a disease, the method being performed by administering a therapeutically effective amount of a target binding protein described herein to a subject in need. The term "treatment" refers to improving at least one symptom of a condition. The condition may be cancer, an autoimmune disease, an infectious disease, chronic inflammation, or transplant rejection (e.g., a kidney, liver, or heart transplant). In some embodiments, the condition 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, Sjögren's syndrome ( syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease) or infectious diseases (e.g., chickenpox, common cold, diphtheria, Escherichia coli, Giardiasis, HIV / AIDS, infectious mononucleosis, influenza (flu), Lyme disease, malaria, measles, meningitis, mumps, poliomyelitis (polio), pneumonia, Rocky mountain spotted fever, rubella (German measles), Salmonella infections, severe acute respiratory syndrome (SARS), sexually transmitted diseases, shingles (herpes zoster), tetanus, toxic shock syndrome, tuberculosis, viral hepatitis, West Nile virus, whooping cough (whooping cough) cough) (pertussis) and the treatment is for the amelioration of at least one symptom of cancer, autoimmune disease, or infectious disease.

[0228] 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., a monkey (e.g., a baboon, a marmoset) or an 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 cancer described herein)).

[0229] In some embodiments, it is possible to identify a subject as having an increase in the expression and / or active HER2 gene mutation of HER2 in a mammalian cell (for example, any mammalian cell described herein).For example, the HER2 gene mutation that increases the expression and / or active HER2 in a mammalian cell can be gene repetition, and the mutation causes the HER2 expression with one or more amino acid substitutions (for example, 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 wild-type protein).See, for example, Weigelt and Reis-Filho, Cancer Discov.2013, 3 (2): 145-147.

[0230] Non-limiting examples of methods for detecting HER2-related diseases in a subject include: immunohistochemistry, fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH). See, for example, Yan et al., Cancer Metastasis Rev. 2015, 34: 157-164.

[0231] The therapeutically effective amount of target binding proteins disclosed herein generally relates to the amount required to achieve the therapeutic goal. As mentioned above, this may be a binding interaction between an antibody and its target antigen, which may interfere with the function of the target in some cases. The amount required for administration will also depend on the binding affinity of the target binding protein to its specific target, and will also depend on the rate at which the target binding protein administered is consumed from the free volume of other subjects to which it is administered. The common range of the therapeutically effective dose of target binding proteins disclosed herein may be (by way of 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 higher. The structure of target binding proteins disclosed herein makes it possible to reduce the dosage of the target binding proteins applied to a subject compared to conventional target binding proteins and compared to conventional antibodies. For example, the administered dose on a unit dose basis or the total dose during a dosing regimen may be reduced by 10%, 20%, 30%, 40% or 50% compared to the corresponding dose of a corresponding conventional target binding protein or a corresponding conventional antibody.

[0232] Typical dosing frequencies may range, for example, from once or twice daily, weekly, biweekly, or monthly.

[0233] The effectiveness of the treatment is determined in conjunction with any known method for diagnosing or treating a particular disease.Methods for screening target binding proteins with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immune-mediated techniques known in the art.

[0234] In another embodiment, target binding proteins for two or more targets are used in methods known in the art related to target localization and / or quantification (e.g., for measuring the level of one or more targets in an appropriate physiological sample, for diagnostic methods, for imaging proteins, etc.). In a given embodiment, target binding proteins for two or more targets, or derivatives, fragments, analogs or homologs thereof containing antibody-derived antigen binding domains, are used as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0235] The target binding proteins used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such target binding proteins can be administered to cancer patients at any stage, from early stage to metastatic. In some embodiments, the target binding proteins and formulations thereof can be administered to subjects suffering from or susceptible to a disease or condition associated with aberrant target expression and / or activity.

[0236] Any of a variety of methods known in the art can be used to identify subjects suffering from or susceptible to a disease or condition associated with abnormal target expression and / or activity. For example, any of a variety of clinical and / or laboratory tests (e.g., physical examination and blood, urine and / or stool analysis) can be used to assess health status to identify subjects suffering from cancer or other tumor disorders. For example, any of a variety of clinical and / or laboratory tests (e.g., physical examination and / or body fluid analysis, such as blood, urine and / or stool analysis) can be used to assess health status to identify subjects suffering from inflammation and / or inflammatory conditions.

[0237] In some embodiments, if any of a variety of laboratory or clinical goals are achieved, it is considered that the administration of the target binding protein to a patient suffering from a disease or condition associated with abnormal target expression and / or activity is successful. For example, if one or more symptoms associated with the disease or condition are alleviated, mitigated, suppressed, or do not progress to a further (i.e., worse) state, it is considered that the administration of the target binding protein to a patient suffering from a disease or condition associated with abnormal target expression and / or activity is successful. If the disease or condition enters remission or does not progress to a further (i.e., worse) state, it is considered that the administration of the target binding protein to a patient suffering from a disease or condition associated with abnormal target expression and / or activity is successful.

[0238] As used herein, the term "treating" 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 cancer described herein)) in a subject (e.g., any subject described herein). In some embodiments where the disease is cancer, treatment reduces cancer growth, inhibits cancer progression, inhibits cancer metastasis, or reduces the risk of cancer recurrence in a subject with cancer.

[0239] In some embodiments, the disease can be cancer. In some embodiments, the subject can have been identified or diagnosed as having cancer. Examples of cancer include: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas, sarcoma), osteosarcoma, B cell tumors, multiple myeloma, lymphoma (e.g., B cell lymphoma, B cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T cell lymphoma), leukemia (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL)), myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric cancer 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 a 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 cancer-ovarian cancer (BRCA1 or BRAC2 mutation) syndrome, etc. The disclosed method can also be used to treat non-solid cancers. Exemplary solid tumors include malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as lung, breast, lymph, gastrointestinal (e.g., colon) and urogenital (e.g., kidney, urothelial or testicular tumors) tracts, pharynx, prostate, and ovarian malignancies. Exemplary adenocarcinomas include colorectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small intestinal cancer. Other examples of cancers treatable by the compositions and methods herein include: acute lymphoblastic leukemia in adults; acute lymphoblastic leukemia in children; acute myeloid leukemia in adults; adrenocortical carcinoma; adrenocortical carcinoma in children; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma in children; cerebral astrocytoma in children; extrahepatic bile duct cancer; bladder cancer; bladder cancer in children; osteosarcoma / malignant fibrous histiocytoma bone cancer; brain stem glioma in children; brain tumors in adults; brain stem glioma brain tumors in children; cerebellar astrocytoma brain tumors in children; cerebral astrocytoma / malignant glioma brain tumors in children; ependymoma brain tumors in children; medulloblastoma brain tumors in children; supratentorial primitive neuroectodermal tumor brain tumors in children; optic pathway and hypothalamic glioma brain tumors in children; brain tumors in children (other); breast cancer; breast cancer and pregnancy; breast cancer in children; breast cancer in men; bronchial adenoma / carcinoid in children; carcinoid tumors in children; gastrointestinal carcinoid tumors;Adrenocortical carcinoma; Islet cell carcinoma; Carcinoma of unknown primary; Primary central nervous system lymphoma; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / glioblastoma, childhood; Cervical cancer; Childhood cancer; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorder; Clear cell sarcoma of the tendon sheath; Colon cancer; Childhood colorectal cancer; Cutaneous T-cell lymphoma; Endometrial carcinoma; Childhood ependymoma; Ovarian epithelial carcinoma; Esophageal cancer; Childhood esophageal cancer; Ewing family tumors; Childhood extracranial germ cell tumors; Extragonadal germ cell tumors tumors; extrahepatic bile duct cancer; intraocular melanoma eye cancer; retinoblastoma eye cancer; gallbladder cancer; stomach (gastric) cancer; childhood stomach (gastric) cancer; gastrointestinal carcinoid tumors; childhood extracranial germ cell tumors; extragonadal germ cell tumors; ovarian germ cell tumors; gestational trophoblastic tumors; childhood brain stem gliomas; childhood optic pathway and hypothalamic gliomas; hairy cell leukemia; head and neck cancer; adult (primary) hepatocellular (liver) cancer; childhood (primary) hepatocellular (liver) cancer; adult Hodgkin's lymphoma (Hodgkin's Lymphoma; Hodgkin's lymphoma in children; Hodgkin's lymphoma in pregnancy; Hypopharyngeal cancer; Hypothalamic and optic pathway gliomas in children; Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer; Laryngeal cancer; Laryngeal cancer in children; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children; Acute myeloid leukemia in adults; Acute myeloid leukemia in children; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cancer; Liver cancer (primary) in adults; Liver cancer (primary) in children; Non-small cell lung cancer; Small cell lung cancer; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children ; Chronic lymphocytic leukemia; AIDS-related lymphoma; Central nervous system (primary) lymphoma; Cutaneous T-cell lymphoma; Hodgkin's lymphoma, adult; Hodgkin's lymphoma, childhood; Hodgkin's lymphoma, pregnancy; Non-Hodgkin's lymphoma, adult; Non-Hodgkin's lymphoma, childhood; Non-Hodgkin's lymphoma, pregnancy; Primary central nervous system lymphoma; Macroglobulinemia, Waldenstrom's; Male breast cancer; Malignant mesothelioma, adult; Malignant mesothelioma, childhood; Malignant thymoma; Medulloblastoma, childhood; Melanoma; Intraocular melanoma; Merkel cell carcinoma CellCarcinoma); Malignant Mesothelioma; Metastatic Squamous Cell Carcinoma of the Neck with Occult Primary; Multiple Endocrine Neoplasia Syndrome in Children; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndrome; Chronic Myeloid Leukemia; Acute Myeloid Leukemia in Children; Multiple Myeloma; Chronic Myeloproliferative Disorders; Nasal and Paranasal Sinus Cancer; Nasopharyngeal Carcinoma; Nasopharyngeal Carcinoma in Children; Neuroblastoma; Non-Hodgkin's Lymphoma in Adults; Non-Hodgkin's Lymphoma in Children; Non-Hodgkin's Lymphoma in Pregnancy; Non-Small Cell Lung Cancer;Oral cancer, childhood; oral cavity and lip cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer, childhood; ovarian epithelial cancer; ovarian germ cell tumors; ovarian low-grade malignant potential tumors; pancreatic cancer; pancreatic cancer, childhood; islet cell pancreatic cancer; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; pineal and supratentorial primitive neuroectodermal tumors, childhood; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non Hodgkin lymphoma; primary central nervous system lymphoma; primary liver cancer, adults; primary liver cancer, children; prostate cancer; colorectal cancer; renal cell (kidney) cancer; renal cell cancer, children; transitional cell carcinoma of the renal pelvis and ureter; retinoblastoma; rhabdomyosarcoma, children; salivary gland cancer; salivary gland cancer, children; Ewing family tumor sarcoma; Kaposi sarcoma; sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; rhabdomyosarcoma, children; soft tissue sarcoma, adults; soft tissue sarcoma, children; Sezary syndrome Syndrome); skin cancer; childhood skin cancer; skin cancer (melanoma); Merkel cell skin cancer; small cell lung cancer; small intestinal cancer; adult soft tissue sarcoma; childhood soft tissue sarcoma; metastatic squamous cell carcinoma of the neck with occult primary site; stomach (gastric) cancer; childhood stomach (gastric) cancer; childhood supratentorial primitive neuroectodermal tumor; cutaneous T-cell lymphoma; testicular cancer; childhood thymoma; malignant thymoma; thyroid cancer; childhood thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic tumor; childhood cancer of unknown primary site; unusual childhood cancer; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; childhood optic pathway and hypothalamic glioma; vulvar cancer; Waldenstrom's macroglobulinemia; Wilms' Tumor; diffuse large B-cell lymphoma (DLBCL); and mantle cell lymphoma (MCL). Metastasis of the above-mentioned cancers can also be treated or prevented according to the methods described herein. ;

[0240] In some embodiments, the methods herein may result in a decrease in the number, severity, or frequency of one or more symptoms of cancer in a subject (eg, compared to the number, severity, or frequency of one or more symptoms of cancer in the subject prior to treatment).

[0241] The method may also include administering one or more additional agents to the subject. In some embodiments, one or more additional agents are chemotherapeutic agents, such as chemotherapeutic agents selected from the group consisting of: docetaxel, paclitaxel, abraxane (ie, albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan and gemcitabine. In some embodiments, one or more additional agents may be checkpoint inhibitors, kinase inhibitors, agents targeting inhibitors in tumor microenvironment and / or T cells or NK agonists. In some embodiments, one or more additional agents are radiotherapy alone or in combination with one or more additional agents such as chemotherapeutic agents or anti-tumor agents. In some embodiments, one or more additional agents may be vaccines, tumor viruses and / or DC activators, such as toll-like receptor (TLR) agonists and / or α-CD40 by way of non-limiting examples. In some embodiments, one or more additional agents may be tumor-targeting antibodies designed to kill tumors via ADCC or via direct conjugation to a toxin (eg, an antibody drug conjugate (ADC)).

[0242] In some embodiments, the checkpoint inhibitor can be an inhibitor of proteins 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 an IDO inhibitor, an α-CSF1R inhibitor, an α-CCR4 inhibitor, TGF-β, myeloid-derived suppressor cells or T regulatory cells. In some embodiments, the agonist can be Ox40, GITR, CD137, ICOS, CD27 or HVEM.

[0243] In some embodiments, the target binding protein can be co-administered with one or more additional agents during and / or after treatment. In some embodiments, the target binding protein can be formulated into a single therapeutic composition, and the target binding protein and one or more additional agents can be administered simultaneously. Alternatively, the target binding protein and one or more additional agents can be separated from each other, for example, each formulated into a separate therapeutic composition, and the target binding protein and additional agent are administered simultaneously, or the target binding protein and additional agent are administered at different times during the treatment regimen. For example, the target binding protein can be administered before administering an additional agent, after administering an additional agent, or in an alternating manner. The target binding protein and one or more additional agents can be administered in a single dose or multiple doses.

[0244] One or more target binding proteins herein can be co-formulated and / or co-administered with one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzyme inhibitors. Examples of drugs or inhibitors that can be used include one or more of the following: nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids, such as prednisolone; cytokine inhibitory anti-inflammatory drugs (CSAIDs); ID); nucleotide biosynthesis inhibitors, such as purine biosynthesis inhibitors, folate antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamate); and pyrimidine biosynthesis inhibitors, such as dihydroorotate dehydrogenase (DHODH) inhibitors. Therapeutic agents suitable for use in combination with the antibodies of the present disclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide) and folate antagonists (e.g., methotrexate). Additional inhibitors Examples of agents include one or more of the following: corticosteroids (oral, inhaled, and locally injected); immunosuppressants, such as cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, such as sirolimus (rapamycin or a rapamycin derivative, such as a soluble rapamycin derivative (e.g., an ester rapamycin derivative, such as CCI-779); agents that interfere with the signaling of proinflammatory cytokines such as TNFα or IL-1 (e.g., such as IRAK, NIK, IKK, p38 or MAP kinase inhibitors); COX2 inhibitors, such as celecoxib, rofecoxib and variants thereof; phosphodiesterase inhibitors, such as R973401 (a type IV phosphodiesterase inhibitor); phospholipase inhibitors, such as cytoplasmic phospholipase 2 (cPLA2) inhibitors (e.g., trifluoromethylketone analogs); vascular endothelial growth factor or growth factor receptor inhibitors, such as VEGF inhibitors and / or VEGF-R inhibitors; and angiogenesis inhibitors.Therapeutic agents suitable for use in combination with the antibodies of the present disclosure are immunosuppressants, such as cyclosporine, tacrolimus (FK-506); mTOR inhibitors, such as sirolimus (rapamycin) or rapamycin derivatives, such as soluble rapamycin derivatives (e.g., ester rapamycin derivatives, such as CCI-779); COX2 inhibitors, such as celecoxib and variants thereof; and phospholipase inhibitors, such as cytosolic phospholipase 2 (cPLA2) inhibitors, such as trifluoromethyl ketone analogs. 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 sulfasalazine; mesalazine; olsalazine; chloroquine / hydroxychloroquine; penicillamine; aurothiourea (intramuscular and oral); azathioprine; colchicine; beta-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeteral); xanthines (theophylline, aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

[0245] The present 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 a subject or biological sample with a target binding protein, wherein the target binding protein includes a detectable label located on a portion of the target binding protein that is released after cleavage of a CM substrate; and (ii) measuring the level of activated target binding protein in the subject or biological sample, wherein a detectable level of activated target binding protein in the subject or biological sample indicates that a cleavage agent, a target, or both a cleavage agent and a target are not present and / or insufficiently present 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 wherein a decrease in the detectable level of activated target binding protein in the subject or biological sample indicates that a cleavage agent and a target are present in the subject or biological sample.

[0246] The reduction in the level of the detectable marker can be, for example, a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or substantially a reduction of 100%. In some embodiments, the detectable marker can be conjugated to a component (e.g., TB) of the target binding protein. In some embodiments, a secondary reagent specifically bound to an activating antibody can be used to measure the level of the target binding protein in a subject or sample, wherein the reagent comprises a detectable marker. The secondary reagent can be an antibody comprising a detectable marker.

[0247] In some embodiments, the target binding protein can also be used to detect the target in the patient sample and thus can be used as a diagnostic agent. For example, the target binding protein can be used in an in vitro assay, such as an ELISA, to detect the target level in the patient sample. For example, the target binding protein can be fixed on a solid support (e.g., a well of a microtiter plate). The fixed target binding protein can be used as a capture antibody for any target that may be present in the test sample. Before contacting the fixed antibody with the patient sample, the solid support can be rinsed and treated with a blocking agent (e.g., milk protein or albumin) to prevent nonspecific adsorption of the analyte.

[0248] In some embodiments, based on the results obtained in in vitro diagnostic assays using the target binding protein, the disease of the subject can be staged based on the expression level of the target antigen. For a given disease, blood samples are drawn from subjects diagnosed as being at various stages of disease progression and / or at various time points of disease therapeutic treatment. Using a sample population that provides statistically significant results for each stage of progression or treatment, a range of antigen concentrations that can be considered as characteristic of each stage is specified.

[0249] The target binding proteins herein can also be used in diagnostic and / or imaging methods. In some embodiments, such methods can be in vitro methods. In some embodiments, such methods can be in vivo methods. In some embodiments, such methods can be in situ methods. In some embodiments, such methods can be ex vivo methods. For example, a target binding protein with a CM can be used to detect the presence or absence of an enzyme capable of cleaving the CM. Such target binding proteins can be used in diagnosis, which may include detecting (e.g., qualitatively or quantitatively) enzyme activity in vivo (or in some embodiments, an environment with an increased reduction potential, such as an environment that can provide disulfide bond reduction) by measuring the accumulation of activated antibodies (i.e., antibodies produced by cleavage of the target binding protein) in a given cell or tissue of a given host organism. This accumulation of activated antibodies not only indicates that the tissue expresses the enzyme activity (or an increased reduction potential depending on the nature of the CM substrate), but also indicates that the tissue expresses the target to which the activated antibody binds.

[0250] For example, CM can be selected as a protease substrate for a protease found at a tumor site, a viral or bacterial infection site at a biologically restricted site (e.g., such as an abscess, an organ, etc.). The TB can be a TB that binds to a target antigen. Using methods familiar 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 discussed in the context of the above screening methods, and additional specific examples are provided below. Using a TB specific for a protein or peptide of a disease state and a protease with increased activity in the disease tissue of interest, the target binding protein will exhibit an increased binding rate to the disease tissue relative to tissues in which the CM-specific enzyme is not present at a detectable level or is present at a level lower than that in the disease tissue or is inactive (e.g., in a zymogen form or in a complex with an inhibitor). Because the kidney filtration system rapidly clears small proteins and polypeptides from the blood, and because the enzyme specific for CM is not present at a detectable level (or is present at a lower level in non-disease tissue or in an inactive conformation), the accumulation of activated antibodies in the disease tissue relative to non-disease tissue is increased.

[0251] In some embodiments, the target binding protein can be used for in vivo imaging, wherein the detection of a fluorescent signal in a subject (e.g., a mammal, including a human) indicates that the disease site contains the target and contains a protease specific for the CM of the target binding protein. In vivo imaging can be used to identify or otherwise refine 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 the protease that cleaves the substrate in the CM of the target binding protein being tested (e.g., activated antibodies accumulate at the disease site) is identified as a candidate suitable for treatment with such a target binding protein comprising such CM. Similarly, a patient who tests negative can be identified as a suitable candidate for another therapy (i.e., not suitable for treatment with the target binding protein being tested). In some embodiments, such patients who test negative for the first target binding protein can be tested with other target binding proteins containing different CMs until a target binding protein suitable for treatment is identified (e.g., a target binding protein containing CM cleaved by the patient at the disease site).

[0252] In some embodiments, in situ imaging can be used to identify methods for patients to be treated. For example, in situ imaging, target binding proteins can be used to screen patient samples to identify those patients with appropriate proteases and targets at appropriate locations, such as tumor sites. In some embodiments, in situ imaging is used to identify or otherwise refine patient populations suitable for treatment with target binding proteins of the present disclosure. For example, patients who test positive for both the target and the protease of the substrate in the CM of the target binding protein being tested for cleavage (e.g., activated antibodies accumulate at the disease site) are identified as candidates suitable for treatment with such target binding proteins comprising such CM. Similarly, patients who test negative for one or both of the proteases of the target and the substrate in the CM substrate of the target binding protein being tested using these methods are identified as suitable candidates for another therapy (i.e., not suitable for treatment with the tested target binding protein). In some embodiments, such patients who test negative for the first target binding protein may be tested with additional target binding proteins comprising different CMs until a target binding protein suitable for treatment is identified (e.g., an activatable target binding protein comprising a CM that is cleaved by the patient at the disease site).

[0253] The present disclosure includes any combination of the following numbered items:

[0254] 1. A target binding protein, comprising:

[0255] A heavy chain variable domain, the heavy chain variable domain comprising

[0256] a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the sequence of TYAMN (SEQ ID NO: 3),

[0257] a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the sequence of RIRSKYNNYATYY ADSVKD (SEQ ID NO: 5), and

[0258] a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the sequence of HGNFGNSYVSWX1AY (SEQ ID NO:6), wherein X1 is W or F; and

[0259] A light chain variable domain, the light chain variable domain comprising

[0260] a variable light chain complementarity determining region 1 (VL CDR1) comprising a sequence of X2SSTGAVTTSNYX3N (SEQ ID NO: 10), wherein X2 is R or G; and X3 is P or V,

[0261] a variable light chain complementarity determining region 2 (VL CDR2) comprising the sequence of GTNCRAP (SEQ ID NO: 14), and

[0262] 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,

[0263] wherein the heavy chain variable domain and the light chain variable domain are located in one or more polypeptides,

[0264] Wherein the target binding protein specifically binds to CD3ε.

[0265] 2. The target binding protein of item 1, wherein:

[0266] a. the VH CDR1 comprises the sequence of TYAMN (SEQ ID NO: 3);

[0267] b. the VH CDR2 comprises a sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO: 5);

[0268] c. the VH CDR3 comprises the sequence of HGNFGNSYVSWWAY (SEQ ID NO: 7) or HGNFGNSYVSWFAY (SEQ ID NO: 8);

[0269] d. the VL CDR1 comprises the sequence of RSSTGAVTTSNYPN (SEQ ID NO: 11), RSSTGAVTTSNYVN (SEQ ID NO: 12) or GSSTGAVTTSNYVN (SEQ ID NO: 13);

[0270] e. the VLCDR2 comprises the sequence of GTNCRAP (SEQ ID NO: 14); and

[0271] f. The VL CDR3 comprises the sequence of VLWYSNRWV (SEQ ID NO: 16), VLWYSNLWV (SEQ ID NO: 17) or ILWYSNRWV (SEQ ID NO: 18).

[0272] 3. The target binding protein of item 1, wherein

[0273] 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16);

[0274] 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16);

[0275] 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16);

[0276] 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises ILWYSNR WV (SEQ ID NO:18);

[0277] 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNL WV (SEQ ID NO:17); or

[0278] 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16).

[0279] 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0280] 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.

[0281] 6. The target binding protein of 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.

[0282] 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0283] 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.

[0284] 9. The target binding protein of 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.

[0285] 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), and 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0286] 11. The target binding protein of claim 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.

[0287] 12. The target binding protein of 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.

[0288] 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 RIRSKYNNYA TYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNS YVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises: the VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO: 12), the VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising ILWYSNRWV (SEQ ID NO: 18).

[0289] 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.

[0290] 15. The target binding protein of 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.

[0291] 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 RIRSKYNNYA TYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNS YVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises: the VL CDR1 comprising GSSTGAVTTSNYVN (SEQ ID NO: 13), the VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNLWV (SEQ ID NO: 17).

[0292] 17. The target binding protein of claim 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.

[0293] 18. The target binding protein of 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.

[0294] 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 RIRSKYNNYA TYYADSVKD (SEQ ID NO: 5), the VH CDR3 comprising HGNFGNS YVSWWAY (SEQ ID NO: 7), and the light chain variable domain comprises: the VL CDR1 comprising RSSTGAVTTSNYVN (SEQ ID NO: 12), the VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

[0295] 20. The target binding protein of claim 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.

[0296] 21. The target binding protein of 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.

[0297] 22. A target binding protein, comprising:

[0298] a heavy chain variable domain comprising the sequence EVQLVESGGG LVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSK YNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWX1AYWGX6GTLVTVSS (SEQ ID NO: 20); and

[0299] a light chain variable domain comprising the sequence QTVVTQEPSL TVSPGGTVTLTCX2SSTGAVTTSNYX3NWVQQKPGX7APRGLIGGT NKRAPGTPARFSGSLJGGKAALTLSGX9QPEDEAEYYCX4LWYSNX5WVFGGGTKLTVL (SEQ ID NO: 21),

[0300] in:

[0301] X1 is W or F; X2 is R or G; X3 is P or V; X4 is V or I; X5 is R or L,

[0302] X6 is Q and X7 is Q; or X6 is C and X7 is C,

[0303] When X9 is V, J is L; or when X9 is A, J is I, and

[0304] The target binding protein specifically binds to CD3ε.

[0305] 23. The target binding protein of claim 22, wherein X6 is Q and X7 is Q.

[0306] 24. The target binding protein of claim 22, wherein X6 is C and X7 is C.

[0307] 25. The target binding protein of item 22, wherein

[0308] 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;

[0309] 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;

[0310] 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;

[0311] 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;

[0312] 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

[0313] 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.

[0314] 26. The target binding protein of any one or combination of items 1-25, wherein the heavy chain variable domain and the light chain variable domain are located in the same polypeptide.

[0315] 27. The target binding protein of item 26, wherein the heavy chain variable domain and the light chain variable domain are coupled via a linker.

[0316] 28. The target binding protein of claim 27, wherein the linker is 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 in length.

[0317] 29. The target binding protein of item 26, which comprises the sequence of SEQ ID NO: 50, 28, 114, 99, 108 or 32.

[0318] 30. The target binding protein of any one or combination of items 26-29, wherein the target binding protein comprises a single-chain variable fragment (scFv).

[0319] 31. The target binding protein of any one or combination of items 1-30, wherein the target binding protein comprises BiTE, (scFv)2, Nanobodies - HSAVHH-scAb, VHH-Fab, double scFab, F(ab')2, double-chain antibodies, DAF (two-in-one), DAE (four-in-one), DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, 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 TM , DVI-IgG, diabody-CH3, tribody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KI'H, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, L'HH-Fc KiH, Fab-VHH-Fc, intracellular antibody, docking and locking, (monoclonal TCR (T cell receptor) for immune mobilization against cancer), IgG-IgG conjugate, Cov-X-Body, scFvl-PEG-scFv2, Adnectin, Fibronectin, IgG, IgM, IgA, IgE, IgD or DEP conjugates, TMEAbody TM , Or SHIELD antibodies.

[0320] 32. The target binding protein of any one or combination of items 1-30, wherein the target binding protein comprises an IgG, IgM, IgA, IgE or IgD antibody or a fragment thereof.

[0321] 33. The target binding protein of item 32, wherein the target binding protein comprises an IgG1, IgG2, IgG3 or IgG4 antibody.

[0322] 34. The target binding protein of any one or combination of items 1-33, wherein the target binding protein is humanized.

[0323] 35. The target binding protein of any one or combination of items 1-34, further comprising a masking portion that inhibits the binding of the target binding protein to CD3 in an inactive state.

[0324] 36. The target binding protein of item 35, wherein the masking portion is coupled to the target binding protein via a cleavable portion, and the cleavable portion is a substrate of a protease.

[0325] 37. A target binding protein of any one or a combination of items 36, wherein 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, apoptotic protease, apoptotic protease 1, apoptotic protease 2, apoptotic protease 3, apoptotic protease 4 , apoptosis proteinase 5, apoptosis proteinase 6, apoptosis proteinase 7, apoptosis proteinase 8, apoptosis proteinase 9, apoptosis proteinase 10, apoptosis proteinase 14, cysteine ​​cathepsins, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteinases, cruzi proteinase, legumin, Otubain-2, KLKs, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK1 3. KLK14, metalloproteinases, transmembrane peptidases, enkephalinases, PSMA, BMP-1, MMPs, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine proteases, activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor proteases, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, granzyme B, guanidinobenzoic acidase, HtrA1, human neutrophil elastase, lactoferrin, malathion, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSPs, DESC1, DPP-4, FAP, amberlin, interstitial proteinase-2, MT-SP1 / interstitial proteinase, TMPRSS2, TMPRSS3 or TMPRSS4.

[0326] 38. A target binding protein of any one or a combination of items 1-37, wherein the heavy chain variable domain and / or the light chain variable domain is conjugated to a toxin, a radioactive isotope, a small molecule, a diagnostic agent, a therapeutic macromolecule, a targeting moiety or a detectable moiety via a conjugated moiety.

[0327] 39. The target binding protein of claim 38, wherein the conjugated portion can be cleaved by a protease.

[0328] 40. The target binding protein of claim 38, wherein the conjugated portion cannot be cleaved by a protease.

[0329] 41. A composition comprising the target binding protein of any one or a combination of items 1-40 and a carrier.

[0330] 42. The composition of claim 41, wherein the composition is a pharmaceutical composition, and wherein the carrier is a pharmaceutically acceptable carrier.

[0331] 43. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition of item 41 or 42.

[0332] 44. A nucleic acid comprising a sequence encoding a target binding protein according to any one or a combination of items 1-40.

[0333] 45. A vector comprising the nucleic acid of item 44.

[0334] 46. ​​A cell comprising the nucleic acid of item 44 or the vector of item 45.

[0335] 47. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a target binding protein of any one or combination of items 1-40 or a composition of item 41 or 42.

[0336] 48. The method of claim 47, wherein the subject has been identified or diagnosed as having cancer, an inflammatory disorder, condition or disease, or an autoimmune disorder, condition or disease.

[0337] 49. A method for producing a target binding protein, the method comprising:

[0338] Cultivating the cell of item 46 in a culture medium under conditions sufficient to produce the target binding protein; and

[0339] The target binding protein is recovered from the cells or the culture medium.

[0340] 50. The method of item 49, which also includes isolating the target binding protein recovered from the cells or the liquid culture medium.

[0341] 51. The method of item 50, which also includes formulating the target binding protein into a pharmaceutical composition.

[0342] Example

[0343] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0344] Example 1: Generation of monovalent bispecific antibodies comprising anti-CD3 scFv

[0345] This embodiment shows an exemplary method for producing a monovalent bispecific antibody comprising an anti-CD3 scFv and a Her2 binding domain. The monovalent bispecific antibody is prepared by a recombinant method. The host cell is transformed with an expression vector comprising a polynucleotide encoding sequence of an anti-CD3 scFv of SEQ ID NO: 50, 28, 114, 32, 99, 108 or 164 and other components of the monovalent bispecific antibody, and then the resulting recombinant host cell is cultured under conditions suitable for producing a monovalent bispecific antibody comprising an anti-CD3 scFv to prepare the protein. The supernatant was harvested on the 5th day after transformation, and the protein A binding material was titrated using the Octet assay (Table 7), followed by affinity chromatography (protein A and / or CH1) and size exclusion chromatography to purify the monovalent bispecific antibody. Higher protein A Octet titers are generally associated with desirable higher yields, and several CD3 variants exhibit this desirable property (Table 7).

[0346] Example 2: Characterization of monovalent bispecific antibodies comprising anti-CD3 scFv

[0347] The CD3 binding of the monovalent bispecific antibodies produced in Example 1 was determined using the Octet assay. The binding kinetics of anti-CD3 scFv to human CD3ε was analyzed using the biolayer interferometry on the Octet system (Sartorius, Octet RED96). All proteins were diluted in kinetic buffer (PBS, 2% BSA, 0.1% Tween-20). Super streptavidin (SSA)-coated biosensors (Sartorius, 18-5057) were balanced in kinetic buffer at room temperature for 10 minutes before data acquisition, and the experiment was carried out at 30°C with 1000rpm stirring. The initial baseline level lasted for 60 seconds ("s"). 10nM recombinant his-tagged and biotinylated CD3ε protein (AcroBiosystems, CDE-H8223) was loaded onto the SSA biosensor for 120s. Before analyte association, another baseline level was established for 60s. In the off-rate screen, the loaded sensor was associated with 200 nM monovalent CD3 module and entered into a concentration range of 200 nM to 3.125 nM in 150 s in the complete kinetic assay, followed by a 300 s dissociation step in kinetic buffer. Each off-rate screening assay included a reference molecule containing v12_LH (SEQ ID NO: 164). Prior to fitting, a reference sensor loaded with ligand but without analyte was subtracted from the data. The data were fitted using a mass transport model. Analysis was performed using ForteBio DataAnalysis 10.0 software. The CD3 monovalent bispecific antibodies exhibited specific binding to CD3 as shown in Table 7 below. d and K off As shown in the data.

[0348] The monomer content of the monovalent bispecific antibody was analyzed using analytical size exclusion chromatography (SEC). The analysis was performed using an Agilent 1260 Infinity HPLC system with UV detection at 280 nm absorbance. A protein aliquot (approximately 25 μg) of the affinity chromatography (protein A or CH1) purified material was injected into a 7.8 mm × 15 cm TSKgel QC-PAK GFC300 column (TOSOH Biosciences, King of Prussia, PA) with 0.1 M Sodium phosphate , 0.1 M sodium sulfate pH 6.8 equilibration, flow rate 1.0 mL / min. A low monomer content of the affinity purified material (before the size exclusion polishing step) is generally undesirable because it will negatively affect the overall yield of the polishing step. The CD3 variants described in Table 7 all have acceptable ≥ 50% monomer content in the affinity purification step.

[0349] Thermal denaturation was studied using Nanotemper Prometheus NT.48 (NanoTemper Technologies, Munich). Sample concentrations were between 0.8 mg / ml-1 mg / ml and the heating rate was 1°C / min. Stability data were recorded using the change in tryptophan fluorescence with temperature at emission wavelengths of 330 nm and 350 nm. The intrinsic fluorescence of tryptophan and tyrosine (Trp / Try) was measured at wavelengths of 330 and 350 nm, and a graph of the intrinsic fluorescence versus temperature from 15 to 95°C during the unfolding process was plotted at a heating rate of 1°C / min. The unfolding / denaturation causes a change in the polarity of the microenvironment around the tryptophan residues, which results in a change in fluorescence, which is reflected in the melting curve. The graph of the fluorescence ratio F350 / F330 versus temperature produces a clear melting transition that can be used to analyze the melting temperature. The melting temperature was determined by detecting the maximum value of the first derivative of the fluorescence ratio (F350 / F330). The melting temperature (Tm) was calculated from the first derivative of the F350 / 330 graph. The higher the Tm value, the more stable the protein. All CD3 variants described in Table 7 (except for the reference v12_LH) have ideal Tm, above 55°C, and significantly higher than the reference v12_LH. The monovalent bispecific antibodies are identified by the CD3 binding domains in Table 7 below. Koff data are reported as relative measurements, where "+++" indicates that the Koff rate of CD3 binding is about the same as or better than the reference monovalent bispecific antibody comprising v12_LH (SEQ ID NO: 164) as the anti-CD3 scFv.

[0350] Table 7

[0351]

[0352] *Both numbers are from two independent transformations and represent the variation in transformation / expression.

[0353] Example 3 Cytotoxicity of a monovalent bispecific antibody comprising an anti-CD3 scFv and a Her2 binding domain

[0354] 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 plated together for co-culture at a target cell to effector cell ratio of 1:10 in RPMI medium (Gibco catalog number 22400071) supplemented with 5% human serum (MP Bio catalog number 2930949). To this co-culture was added a titration of the monovalent bispecific antibody produced in Example 1 and the plates were incubated at 37°C and 5% CO2 for approximately 48 hours. After incubation, the ONE-Glo TMCytotoxicity was assessed using a luciferase assay system (Promega catalog number E6130) and luminescence was measured on a plate reader (TECAN). Percent cytotoxicity was calculated as follows: (1-(experimental RLU / average RLU of untreated)) × 100. Percent cytotoxicity data were plotted using GraphPad PRISM and EC was calculated. 50 Values. The results are shown in Table 8 below as relative values ​​of EC50 compared to the EC50 of an internal control monovalent bispecific antibody comprising v12-LH as an 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 errors are about 2-3 times, so a ratio of about 0.5 to about 2 indicates that the tested monovalent bispecific antibody has a potency similar to that of a reference monovalent bispecific antibody comprising v12-LH as an anti-CD3 binding protein. A ratio of less than about 0.5 indicates that the tested monovalent bispecific antibody is more effective than the reference monovalent bispecific antibody comprising v12-LH anti-CD3 scFv. The monovalent bispecific antibodies are recognized by their CD3 binding domains. The EC50 ratios of most CD3 variants in Table 8 (except v619_HLp2) indicate that their potency is superior to v12-LH, within the limits of assay variation.

[0355] Table 8. Cytotoxicity of monovalent bispecific antibodies comprising anti-CD3 scFv.

[0356]

[0357] Examples of sequences are listed below in Tables 9A and 9B.

[0358] Table 9A Exemplary sequences

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365] Table 9B Exemplary sequences of heavy chain variable domains, light chain variable domains, and related molecules.

[0366] The CDRs in the heavy chain variable domain respectively comprise the amino acid sequences at positions 31-35, 50-68 and 101-114 of the heavy chain variable domain. The CDRs in the light chain variable domain respectively comprise the amino acid sequences at positions 23-36, 52-58 and 91-99 of the light chain variable domain.

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392] Other Implementations

[0393] It will be appreciated that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0394] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail. The materials, methods, and examples are illustrative only and are not intended to be limiting.

Claims

1. A target binding protein, comprising: A heavy chain variable domain, the 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 RIRSKYNNYATYY ADSVKD (SEQ ID NO: 5), and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the sequence of HGNFGNSYVSWX1AY (SEQ ID NO:6), wherein X1 is W or F; and A light chain variable domain, the light chain variable domain comprising a variable light chain complementarity determining region 1 (VL CDR1) comprising a 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 of GTNCRAP (SEQ ID NO: 14), and 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, wherein the heavy chain variable domain and the light chain variable domain are located in one or more polypeptides, Wherein the target binding protein specifically binds to CD3ε.

2. The target binding protein of claim 1, wherein: a. the VH CDR1 comprises the sequence of TYAMN (SEQ ID NO: 3); b. the VH CDR2 comprises a sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO: 5); c. the VH CDR3 comprises the sequence of HGNFGNSYVSWWAY (SEQ ID NO: 7) or HGNFGNSYVSWFAY (SEQ ID NO: 8); d. the VL CDR1 comprises the sequence of RSSTGAVTTSNYPN (SEQ ID NO: 11), RSSTGAVTTSNYVN (SEQ ID NO: 12) or GSSTGAVTTSNYVN (SEQ ID NO: 13); e. the VLCDR2 comprises the sequence of GTNCRAP (SEQ ID NO: 14); and f. The VL CDR3 comprises the sequence of VLWYSNRWV (SEQ ID NO: 16), VLWYSNLWV (SEQ ID NO: 17) or ILWYSNRWV (SEQ ID NO: 18).

3. 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 RSSTGAVTTSNYPN (SEQ ID NO:11), the VL CDR2 comprises GTNKRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16); 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16); 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 VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16); 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises ILWYSNRWV (SEQ ID NO:18); 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNLWV (SEQ ID NO:17); 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 GTNCRAP (SEQ ID NO:14), and the VLCDR3 comprises VLWYSNR WV (SEQ ID NO:16).

4. The target binding protein of claim 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

5. The target binding protein of claim 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.

6. The target binding protein of claim 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.

7. The target binding protein of claim 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

8. The target binding protein of claim 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.

9. The target binding protein of claim 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.

10. The target binding protein of claim 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

11. The target binding protein of claim 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.

12. The target binding protein of claim 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.

13. The target binding protein of claim 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising ILWYSNRWV (SEQ ID NO: 18).

14. The target binding protein of claim 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.

15. The target binding protein of claim 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.

16. The target binding protein of claim 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNLWV (SEQ ID NO: 17).

17. A target binding protein as described in claim 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.

18. The target binding protein of claim 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.

19. The target binding protein of claim 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 VLCDR2 comprising GTNCRAP (SEQ ID NO: 14), and the VL CDR3 comprising VLWYSNRWV (SEQ ID NO: 16).

20. The target binding protein of claim 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.

21. The target binding protein of claim 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.

22. A target binding protein, comprising: a heavy chain variable domain comprising the sequence EVQLVESGGG LVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSK YNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWX1AYWGX6GTLVTVSS (SEQ ID NO: 20); and a light chain variable domain comprising the sequence QTVVTQEPSL TVSPGGTVTLTCX2SSTGAVTTSNYX3NWVQQKPGX7APRGLIGGT NKRAPGTPARFSGSLJGGKAALTLSGX9QPEDEAEYYCX4LWYSNX5WVFGGGTKLTVL (SEQ ID NO: 21), in: X1 is W or F; X2 is R or G; X3 is P or V; X4 is V or I; X5 is R or L, X6 is Q and X7 is Q; or X6 is C and X7 is C, When X9 is V, J is L; or when X9 is A, J is I, and The target binding protein specifically binds to CD3ε.

23. The target binding protein of claim 22, wherein X6 is Q and X7 is Q.

24. The target binding protein of claim 22, wherein X6 is C and X7 is C.

25. The target binding protein of claim 22, 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:49; 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; 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; 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; 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.

26. The target binding protein of any one of claims 1-25, wherein the heavy chain variable domain and the light chain variable domain are located within the same polypeptide.

27. The target binding protein of claim 26, wherein the heavy chain variable domain and the light chain variable domain are coupled via a linker.

28. The target binding protein of claim 27, wherein the linker is 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 in length.

29. The target binding protein of claim 26, comprising the sequence of SEQ ID NO: 50, 28, 114, 99, 108 or 32.

30. The target binding protein of any one of claims 26-29, wherein the target binding protein comprises a single chain variable fragment (scFv).

31. The target binding protein of any one of claims 1-30, wherein the target binding protein comprises BiTE, (scFv)2, Nanobodies - HSA VH H-scAb, VHH-Fab, double scFab, F(ab')2, double-chain antibody, DAF (two-in-one), DAE (four-in-one), DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, 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 TM , DVI-IgG, diabody-CH3, tribody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KI′H, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, L'HH-Fc KiH, Fab-VHH-Fc, intracellular antibody, docking and locking, (monoclonal TCR (T cell receptor) for immune mobilization against cancer), IgG-IgG conjugate, Cov-X-Body, scFvl-PEG-scFv2, Adnectin, Fibronectin, IgG, IgM, IgA, IgE, IgD or DEP conjugates, TMEAbody TM , Or SHIELD antibodies.

32. The target binding protein of any one of claims 1-30, wherein the target binding protein comprises an IgG, IgM, IgA, IgE or IgD antibody or a fragment thereof.

33. The target binding protein of claim 32, wherein the target binding protein comprises an IgG1, IgG2, IgG3, or IgG4 antibody.

34. The target binding protein of any one of claims 1-33, wherein the target binding protein is humanized.

35. The target binding protein of any one of claims 1-34, further comprising a masking moiety that inhibits binding of the target binding protein to CD3 in an inactive state.

36. The target binding protein of claim 35, wherein the masking moiety is coupled to the target binding protein via a cleavable moiety, and the cleavable moiety is a substrate for a protease.

37. The target binding protein of any one of claims 36, wherein 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, apoptotic protease, apoptotic protease 1, apoptotic protease 2, apoptotic protease 3, apoptotic protein Cathepsin 4, Cathepsin 5, Cathepsin 6, Cathepsin 7, Cathepsin 8, Cathepsin 9, Cathepsin 10, Cathepsin 14, Cysteine ​​cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Cysteine ​​proteases, Cruzi proteases, Pod protein, Otubain-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KL K13, KLK14, metalloproteinases, transmembrane peptidases, enkephalinases, 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 proteases, activated protein C, cathepsin A, cathepsin G, chymotrypsin, coagulation factor protease , FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, granzyme B, guanidinobenzoic acidase, HtrA1, human neutrophil elastase, lactoferrin, malacin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSP, DESC1, DPP-4, FAP, amber, interstitial proteinase-2, MT-SP1 / interstitial proteinase, TMPRSS2, TMPRSS3 or TMPRSS4.

38. The target binding protein of any one of claims 1-37, 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 conjugated moiety.

39. The target binding protein of claim 38, wherein the conjugated moiety is cleavable by a protease.

40. The target binding protein of claim 38, wherein the conjugated moiety is not cleavable by a protease.

41. A composition comprising the target binding protein of any one of claims 1-40 and a carrier.

42. The composition of claim 41, wherein the composition is a pharmaceutical composition, and wherein the carrier is a pharmaceutically acceptable carrier.

43. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition of claim 41 or 42.

44. A nucleic acid comprising a sequence encoding the target binding protein of any one of claims 1-40.

45. A vector comprising the nucleic acid of claim 44.

46. ​​A cell comprising the nucleic acid of claim 44 or the vector of claim 45.

47. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a target binding protein as described in any one of claims 1-40 or a composition as described in claim 41 or 42.

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

49. A method for producing a target binding protein, the method comprising: Culturing the cell of claim 46 in a culture medium under conditions sufficient to produce the target binding protein; as well as The target binding protein is recovered from the cells or the culture medium.

50. The method of claim 49, further comprising isolating the target binding protein recovered from the cells or the liquid culture medium.

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

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