Compositions targeting prostate-specific membrane antigen (PSMA) and methods for making and using same

Abstract

Provided herein are, inter alia, antigen binding molecules with binding specificity for cluster of differentiation 3 T cell receptor (CD3), antigen binding molecules with binding specificity for prostate-specific membrane antigen (PSMA), cleavable linker sequences, and protease-activatable bispecific fusion proteins, such as protease-activatable T cell engagers, and methods of use and treatment.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application Nos. 63 / 444,839, filed February 10, 2023, and 63 / 499,031, filed April 28, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Prostate cancer is the second most common cancer in men, with one in nine men in the United States expected to develop the disease during their lifetime. Treatment of localized disease (as measured by Gleason score <6 and PSA <10 ng / mL) with radiation, radical prostatectomy, or active surveillance is successful in controlling early-stage disease; however, recurrence occurs in 20–50% of men. Patients who continue to progress despite first- and second-line androgen deprivation therapy (ADT) develop castration-resistant prostate cancer (CRPC), which often metastasizes to the bone, brain, liver, and lungs (metastatic castration-resistant prostate cancer, mCRPC). Chemotherapy, such as docetaxel and cabazitaxel, has shown improved survival in this population, but no cure for mCRPC exists.

[0003] Prostate-specific membrane antigen (PSMA; also known as folate hydrolase 1 and FOLH1) is an integral cell surface membrane protein that is frequently overexpressed in prostate cancer and is often associated with androgen-independent prostate cancer and secondary metastatic disease. PSMA is also expressed within the neovasculature of bladder, kidney, stomach, and colorectal cancers.

[0004] Immunotherapy has shown mixed success, including in prostate cancer. The first cell-based immunotherapy, sipuleucel-T (PROVENGE®), was approved for mCRPC in 2010, but checkpoint blockade targeting the immune inhibitory receptors PD-1 and CTLA-4 has rarely been shown to reduce response rates in prostate cancer compared with other solid tumor malignancies. It has been suggested that this may be due to the immunologically "cold" tumor microenvironment of primary prostate cancer tumors, characterized by low immune cell infiltration and a weak neoantigen load that has been shown to be required for response to checkpoint blockade inhibitors.

[0005] There is a long-standing unmet need for therapeutic intervention in tumors that express PSMA, including immunologically cold tumors. Summary of the Invention

[0006] The present disclosure provides, inter alia, antigen-binding molecules with binding specificity for PSMA, antigen-binding molecules with binding specificity for CD3, and bispecific antigen-binding molecules that bind both PSMA and CD3 for use in therapeutic settings where specific targeting and T cell-mediated killing of PSMA-expressing cells is desired. Embodiments disclosed herein address the long-standing unmet need for PSMA-targeted cancer therapeutics, including T cell engagers (TCEs) with increased therapeutic indices. Embodiments of the present disclosure also address the long-standing unmet need for therapeutic intervention of immunologically cold tumors, e.g., solid tumors, that express PSMA. Also included are, for example, protease-cleavable linkers, barcode fragments, antibody domain linkers, and activatable TCEs (including those that do not bind PSMA). Also included are fusion proteins, such as non-TCE fusion proteins, that target PSMA, CD3, and / or include the linkers and other components provided herein.

[0007] Certain aspects of the present disclosure include compounds, compositions, and methods for increasing a subject's therapeutic response to a checkpoint inhibitor (e.g., a PD-1 or CTLA-4 inhibitor, such as an anti-PD1 antibody or an anti-CTLA4 antibody). In some embodiments, the compounds provided herein recruit and activate effector T cells in a major histocompatibility complex-independent manner through engagement of CD3 on T cells. In some embodiments, the compound is a bispecific TCE that is administered in an inactive form and activated at and / or within the tumor site. In some embodiments, the bispecific TCE is administered before initiating checkpoint inhibitor therapy, concomitantly with checkpoint inhibitor therapy, or after checkpoint inhibitor therapy has been discontinued.

[0008] Certain aspects of the present disclosure are directed to chimeric polypeptides comprising a bispecific antibody domain, the bispecific antibody domain comprising a first antigen-binding domain that specifically binds prostate-specific membrane antigen (PSMA) and a second antigen-binding domain that binds cluster of differentiation 3 (CD3), wherein the first antigen-binding domain is a VHH or the second antigen-binding domain is a Fab or scFV, and the chimeric polypeptide further comprises a masking polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide is capable of reducing binding of the bispecific antibody domain to CD3 or PSMA, and the protease-cleavable release segment is cleavable by at least one protease present in a tumor.

[0009] Certain aspects of the present disclosure are directed to chimeric polypeptides comprising a bispecific antibody domain, the bispecific antibody domain comprising a first antigen-binding domain that specifically binds to a protease-specific membrane antigen (PSMA) and a second antigen-binding domain that binds to a cluster of differentiation 3 T-cell receptor (CD3), wherein the chimeric polypeptide further comprises a masking polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide is capable of reducing binding of the bispecific antibody domain to CD3 or PSMA, and wherein the protease-cleavable release segment is not cleavable by legumain in human plasma or legumain cleaves the protease-cleavable release segment in human plasma at a rate that is less than about 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain.

[0010] In some embodiments, the chimeric polypeptide comprises a structural arrangement defined from N-terminal to C-terminal as: (first antigen-binding domain)-(second antigen-binding domain)-(linker)-(mask polypeptide), (second antigen-binding domain)-(first antigen-binding domain)-(linker)-(mask polypeptide), (mask polypeptide)-(linker)-(first antigen-binding domain)-(second antigen-binding domain), or (mask polypeptide)-(linker)-(second antigen-binding domain)-(first antigen-binding domain), where each - is a covalent bond or polypeptide linker.

[0011] In some embodiments, the mask polypeptide is ELNN.

[0012] In some embodiments, the linker further comprises a spacer.

[0013] In some embodiments, the protease-cleavable release segment is fused to the bispecific antibody domain via a spacer.

[0014] In some embodiments, the spacer is characterized in that (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) comprises at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0015] In some embodiments, the spacer is 9 to 14 amino acids in length.

[0016] In some embodiments, the spacer comprises at least four amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the amino acids of the spacer consist of A, E, G, S, P, and / or T.

[0017] In some embodiments, the spacer is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0018] In some embodiments, the spacer comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 85% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 90% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 91% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 92% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 93% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 94% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 95% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 96% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 97% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 98% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 99% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 100% identity to a sequence listed in Table C.

[0019] In some embodiments, the spacer comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 85% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 90% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 91% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 92% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 93% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 94% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 94% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 95% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 96% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 97% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 98% identity to GTSESATPES or GTATPESGPG. In some embodiments, the spacer comprises an amino acid sequence having at least 99% identity to GTSESATPES or GTATPESGPG.In some embodiments, the spacer comprises an amino acid sequence having 100% identity to GTSESATPES or GTATPESGPG.

[0020] In some embodiments, the protease-cleavable release segment comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid except N. In some embodiments, X is S.

[0021] Certain aspects of the present disclosure relate to a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain having binding specificity for a cancer cell antigen and a second antigen-binding domain having binding specificity for an effector cell antigen expressed on an effector cell, wherein the chimeric polypeptide further comprises a first ELNN joined to the first antigen-binding domain via a first linker comprising a first protease-cleavable release segment (RS1) positioned between the first ELNN and the first antigen-binding domain, such that the first ELNN is capable of reducing binding of the first antigen-binding domain to the cancer cell antigen, and wherein the RS1 the chimeric polypeptide further comprises a second ELNN joined to the second antigen-binding domain via a second linker comprising a second protease-cleavable release segment (RS2) positioned between the second ELNN and the second antigen-binding domain, such that the second ELNN is capable of reducing binding of the first antigen-binding domain to an effector cell antigen, and RS2 is cleavable by at least one protease present in the tumor; the first ELNN has a shorter amino acid sequence than the second ELNN; and the cancer cell antigen is not HER2.

[0022] In some embodiments, the chimeric polypeptide comprises a structural arrangement defined from N- to C-terminus as: (ELNN1)-(linker1)-(first antigen-binding domain)-(second antigen-binding domain)-(linker2)-(ELNN2), (ELNN1)-(linker1)-(second antigen-binding domain)-(first antigen-binding domain)-(linker2)-(ELNN2), (ELNN2)-(linker2)-(first antigen-binding domain)-(second antigen-binding domain)-(linker1)-(ELNN1), or (ELNN2)-(linker2)-(second antigen-binding domain)-(first antigen-binding domain)-(linker1)-(ELNN1), where each - is individually a covalent bond or polypeptide linker.

[0023] In some embodiments, each - is a covalent bond. In some embodiments, each - is a peptide bond.

[0024] In some embodiments, Linker 1 further comprises a first spacer (Spacer 1). In some embodiments, Linker 2 further comprises a second spacer (Spacer 2).

[0025] In some embodiments, RS1 is fused to the bispecific antibody domain via spacer 1 and / or RS2 is fused to the bispecific antibody domain via spacer 2.

[0026] In some embodiments, the chimeric polypeptide comprises a structural arrangement defined, from N-terminus to C-terminus, as follows: (ELNN1)-(RS1)-(spacer 1)-(first antigen-binding domain)-(second antigen-binding domain)-(spacer 2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(spacer 1)-(second antigen-binding domain)-(first antigen-binding domain)-(spacer 2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(spacer 2)-(first antigen-binding domain)-(second antigen-binding domain)-(spacer 1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(spacer 2)-(second antigen-binding domain)-(first antigen-binding domain)-(spacer 1)-(RS1)-(ELNN1), where each - is, individually, a covalent bond or a polypeptide linker.

[0027] In some embodiments, each - is a covalent bond. In some embodiments, each - is a peptide bond.

[0028] In some embodiments, the chimeric polypeptide further comprises an antibody domain linker between the first antigen-binding domain and the second antigen-binding domain.

[0029] Certain aspects of the present disclosure relate to chimeric polypeptides comprising bispecific antibody domains, the chimeric polypeptides comprising, from N-terminal to C-terminal, a formula comprising Formula 1: (Mask 1)-(RS1)-(Spacer 1)-(First antigen-binding domain)-[Antibody domain linker]-(Second antigen-binding domain), Formula 2: (First antigen-binding domain)-[Antibody domain linker]-(Second antigen-binding domain)-(Spacer 2)-(RS2)-(Mask 2), or Formula 3: (Mask 1)-(RS1)-(Spacer 1)-(First antigen-binding domain)-[Antibody domain linker]-(Second antigen-binding domain)-(Spacer 2)-(RS2)-(Mask 2), wherein the first antigen-binding domain has binding specificity for a cancer cell antigen and the second antigen-binding domain has binding specificity for an effector cell expressed on an effector cell. and wherein the chimeric polypeptide has binding specificity for a cancer cell antigen, wherein each mask comprises, individually, a covalent bond or polypeptide linker; mask 1 is a polypeptide capable of reducing binding of the first antigen-binding domain to its target; mask 2 is a polypeptide capable of reducing binding of the second antigen-binding domain to its target; when the chimeric polypeptide comprises Formula 1, spacer 1 consists of A, E, G, S, P, and / or T residues; when the chimeric polypeptide comprises Formula 2, spacer 2 consists of A, E, G, S, P, and / or T residues; and when the chimeric polypeptide comprises Formula 3, spacer 1 and / or spacer 2 consist of A, E, G, S, P, and / or T residues; and the cancer cell antigen is not HER2.

[0030] In some embodiments, each -is individually a covalent connection. In some embodiments, each -is individually a covalent bond. In some embodiments, each -is a peptide bond. In some embodiments, each -is individually a polypeptide linker of 5 amino acids or less.

[0031] In some embodiments, the cancer cell antigen is human alpha 4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER3, HER4, PD-L1, prostate-specific membrane antigen (PSMA), CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3;9-O-acetyl-GD3, GM2, Globo H, fucosyl GM1, GD2, carbonic anhydrase IX, CD44v6, Sonic Hedgehog (Shh), Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, 6-transmembrane epithelial antigen of prostate (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (cancer antigen 125, CA-125, Mullerian inhibitory substance receptor type II (MISIIR), sialylated Tn antigen (TN), fibroblast activation antigen (FAP), endosialin (CD248), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen antigen), CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMPs, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, or EphA2. In some embodiments, the cancer cell antigen is PSMA.

[0032] In some embodiments, the effector cell antigen is cluster of differentiation 3 T cell receptor (CD3).

[0033] In some embodiments, the second antigen-binding domain has binding specificity for human CD3 and cynomolgus CD3.

[0034] In some embodiments, the second antigen-binding domain has binding specificity for human CD3.

[0035] In some embodiments, the effector cell antigen is CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.

[0036] In some embodiments, the effector cell antigen is CD3 epsilon.

[0037] In some embodiments, Mask 1 is a first ELNN and Mask 2 is a second ELNN.

[0038] In some embodiments, spacer 1 and / or spacer 2 are characterized in that (i) at least 90% of their amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) contain at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0039] In some embodiments, spacer 1 and / or spacer 2 are 9 to 14 amino acids in length.

[0040] In some embodiments, spacer 1 and / or spacer 2 comprise at least four amino acids selected from the group consisting of G, A, S, T, E, and P.

[0041] In some embodiments, the amino acids of spacer 1 and / or spacer 2 consist of A, E, G, S, P, and / or T.

[0042] In some embodiments, spacer 1 and / or spacer 2 are cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0043] 48. The chimeric polypeptide of any one of claims 42-47, wherein spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 85% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 90% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 91% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 92% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 93% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 94% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 95% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 96% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 97% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 98% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 99% identity to a sequence listed in Table C. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having 100% identity to a sequence listed in Table C.

[0044] In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 85% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 90% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 91% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 92% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 93% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 94% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 94% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 95% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 96% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 97% identity to GTSESATPES or GTATPESGPG.In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 98% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having at least 99% identity to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 comprise an amino acid sequence having 100% identity to GTSESATPES or GTATPESGPG.

[0045] In some embodiments, the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is about 294 amino acids in length and the amino acid sequence of the second ELNN is about 582 amino acids in length.

[0046] In some embodiments, the first antigen-binding domain comprises a first antibody or antigen-binding fragment thereof, and the second antigen-binding domain is a second antibody or antigen-binding fragment thereof.

[0047] In some embodiments, the first antigen-binding domain is a Fab, scFV, or ISVD. In some embodiments, the ISVD is a VHH domain. In some embodiments, the second antigen-binding domain is a Fab, scFV, or ISVD. In some embodiments, the ISVD is a VHH domain. In some embodiments, the first antigen-binding domain is a VHH domain. In some embodiments, the second antigen-binding domain is an scFV.

[0048] In some embodiments, there is an antibody domain linker between the first antigen binding domain and the second antigen binding domain.

[0049] In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 85% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 90% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 91% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 92% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 93% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 94% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 94% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 95% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 96% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 97% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 98% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 99% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence having 100% identity to a sequence listed in Table A or B.

[0050] In some embodiments, the antibody domain linker consists of G and S amino acid residues. In some embodiments, the antibody domain linker is about 9 residues in length. In some embodiments, the antibody domain linker comprises the amino acid sequence GGGGSGGGS.

[0051] In some embodiments, the scFv comprises a VL domain, a VH domain, and a linker between the VL and VH domains, wherein the linker consists of A, E, G, S, P, and / or T residues.

[0052] In some embodiments, the linker is characterized in that (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) comprises at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0053] In some embodiments, the linker between the VL and VH domains is 25 to 35 amino acids in length.

[0054] In some embodiments, the linker between the VL and VH domains comprises at least four amino acids selected from the group consisting of G, A, S, T, E, and P.

[0055] In some embodiments, the amino acids of the linker between the VL and VH domains consist of A, E, G, S, P, and / or T.

[0056] In some embodiments, the linker between the VL and VH domains is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0057] In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0058] In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 85% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 90% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 91% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 92% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 93% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 94% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 95% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 96% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 97% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 98% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 99% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having 100% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0059] In some embodiments, the first antigen-binding domain comprises a VHH domain comprising three VHH complementarity determining regions (CDRs), wherein the three VHH CDRs comprise CDR1, CDR2, and CDR3 of the VHH domain comprising the following amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0060] In some embodiments, the second antigen-binding domain comprises a VL domain comprising three VL CDRs, wherein the three VL CDRs comprise CDR1, CDR2, and CDR3 of the VL domain comprising the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSX1GAVTX2SNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX3YYCALWYX4NLWVFGGGTKLTVL, wherein X1 corresponds to T or N, X2 corresponds to T or S, X3 corresponds to E or V, and X4 corresponds to S or P.

[0061] In some embodiments, the second antigen-binding domain comprises a VL domain comprising three VL CDRs, wherein the three VL CDRs comprise CDR1, CDR2, and CDR3 of the VL domain comprising the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0062] In some embodiments, the second antigen-binding domain comprises a VH domain comprising three VH CDRs, wherein the three VH CDRs have the following amino acid sequence: EVQLX5ESGGGX6VQPGGSLX7LSCAASGFTFX8TYAMNWVRQAPGKGLEWVX9RIRX 10 KX 11 NNYATYYADSVKX 12 RFTISRDDSKNTX 13 YLQMNX 14 LKTEDTAVYYCVRHX 15 NFGNSYVSWFAX 16 X5 corresponds to V or L, X6 corresponds to I or L, X7 corresponds to R or K, X8 corresponds to S or N, X9 corresponds to G or A, and X 10 corresponds to T or S, and X 11 corresponds to R or Y, and X 12 corresponds to G or D, and X 13 corresponds to V or A, and X 14 corresponds to S or N, and X 15 corresponds to E or G, and X 16 corresponds to H or Y.

[0063] In some embodiments, the second antigen-binding domain comprises a VH domain comprising three VH CDRs, wherein the three VH CDRs comprise CDR1, CDR2, and CDR3 of the VH domain comprising the following amino acid sequence: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

[0064] In some embodiments, the second antigen-binding domain is 896 / 897, 902 / 903, 700 / 701, 702 / 703, 716 / 717, 718 / 719, 728 / 729, 736 / 737, 738 / 739, 740 / 741, 742 / 743, 744 / 745, 746 / 747, 748 / 749, 750 / 751, 752 / 753, 754 / 755, 756 / 757, 758 / 759, 760 / 761, 61, 762 / 763, 764 / 765, 766 / 767, 774 / 775, 776 / 777, 790 / 791, 792 / 793, 798 / 799, 800 / 801, 806 / 807, 808 / 809, 814 / 815, 816 / 817, 822 / 823, 824 / 825, or 826 / 867.

[0065] In some embodiments, (i) the first antigen-binding domain comprises a VHH CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs: GRTFGIYVWG; CDR2; and a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to AASNKEYGRTWYDFNESDY. and (ii) a VL domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs: RSSX1GAVTX2SNYAN (wherein X1 corresponds to T or N and X2 corresponds to T or S); a VL domain CDR2 comprising the sequence: ALWYX4NLWV (wherein X4 corresponds to S or P); a VL domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX4NLWV (wherein X4 corresponds to S or P); a VH domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX8TYAMN (wherein X8 corresponds to S or N); 10 KX 11 NNYATYYADSVKX 12 (In the formula, X 10 corresponds to T or S, and X 11 corresponds to R or Y, and X12 a VH domain CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a VH domain CDR3; 15 NFGNSYVSWFAX 16 (In the formula, X 15 corresponds to E or G, and X 16 and a VH domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a VH domain CDR3 (wherein

[0066] In some embodiments, (i) the first antigen-binding domain comprises a VHH CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs: GRTFGIYVWG; CDR2; and a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to AASNKEYGRTWYDFNESDY. (ii) a VHH comprising a second antigen-binding domain having the following CDRs: a VL domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN; a VL domain CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP; a VL domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN; a VH domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG;and a VH domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to HENFGNSYVSWFAH;

[0067] In some embodiments, the VHH is a VHH FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following framework regions (FR): QVQLVESGGGVVQPGRSLRLSCAAS; FR2; VHH FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDNSKNTLYLQMNSLRAEDTAVYYC; and VHH FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS.

[0068] In some embodiments, the second antigen-binding domain is a VL domain FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following FR: ELVVTQEPSLTVSPGGTVTLTC; a VL domain FR2 comprising the amino acid sequence GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL; a VL domain FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS; and a VH domain FR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG. a VH domain FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR; and a VH domain FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS.

[0069] In some embodiments, (i) the first antigen-binding domain comprises the following CDRs: a VHH CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG; a VHH CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK; and a VHH CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY. and (ii) a VL domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs: RSSX1GAVTX2SNYAN (wherein X1 corresponds to T or N and X2 corresponds to T or S); a VL domain CDR2 comprising the sequence: ALWYX4NLWV (wherein X4 corresponds to S or P); a VL domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX4NLWV (wherein X4 corresponds to S or P); a VH domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX8TYAMN (wherein X8 corresponds to S or N); 10 KX 11 NNYATYYADSVKX 12 (In the formula, X 10 corresponds to T or S, and X 11 corresponds to R or Y, and X12 a VH domain CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a VH domain CDR3; 15 NFGNSYVSWFAX 16 (In the formula, X 15 corresponds to E or G, and X 16 and a VH domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a VH domain CDR3 (wherein

[0070] In some embodiments, (i) the first antigen-binding domain comprises the following CDRs: a VHH CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG; a VHH CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK; and a VHH CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY. (ii) a VHH comprising a second antigen-binding domain having the following CDRs: a VL domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN; a VL domain CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP; a VL domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN; a VH domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG;and a VH domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to HENFGNSYVSWFAH;

[0071] In some embodiments, the VHH is a VHH FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following framework regions (FR): QVQLVESGGGVVQPGRSLRLSCAAS; FR2; VHH FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC; and VHH FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS.

[0072] In some embodiments, the second antigen-binding domain is a VL domain FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following FR: ELVVTQEPSLTVSPGGTVTLTC; a VL domain FR2 comprising the amino acid sequence GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL; a VL domain FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS; and a VH domain FR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG. a VH domain FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR; and a VH domain FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS.

[0073] In some embodiments, the second antigen-binding domain comprises a VL domain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to ELVVTQEPSLTVSPGGTVTLTCRSSX1GAVTX2SNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX3YYCALWYX4NLWVFGGGTKLTVL, wherein X1 corresponds to T or N, X2 corresponds to T or S, X3 corresponds to E or V, and X4 corresponds to S or P.

[0074] In some embodiments, the second antigen-binding domain comprises a VL domain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0075] In some embodiments, the second antigen-binding domain is EVQLX5ESGGGX6VQPGGSLX7LSCAASGFTFX8TYAMNWVRQAPGKGLEWVX9RIRX 10 KX 11 NNYATYYADSVKX 12 RFTISRDDSKNTX 13 YLQMNX 14 LKTEDTAVYYCVRHX 15 NFGNSYVSWFAX 16X5 corresponds to V or L, X6 corresponds to I or L, X7 corresponds to R or K, X8 corresponds to S or N, X9 corresponds to G or A, and X 10 corresponds to T or S, and X 11 corresponds to R or Y, and X 12 corresponds to G or D, and X 13 corresponds to V or A, and X 14 corresponds to S or N, and X 15 corresponds to E or G, and X 16 corresponds to H or Y.

[0076] In some embodiments, the second antigen-binding domain comprises a VH domain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

[0077] In some embodiments, the VL domain is N-terminal to the VH domain. In some embodiments, the VL domain is C-terminal to the VH domain.

[0078] In some embodiments, the second antigen-binding domain comprises an scFV comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS.

[0079] In some embodiments, the first antigen-binding domain comprises a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the amino acid sequence of PSMA.2, PSMA.3, PSMA.5, PSMA.6, PSMA.262, or PSMA.263.

[0080] In some embodiments, the first antigen-binding domain is QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVX 17 GWFRQAPGKEREFVGAX 18 SWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYX 19 CX 20 X 21 SNKX 22 YGRTWYDFNESDYWGQGTQVTVSS, wherein X is a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, 17 , X 18 , X 19 , X20 , X 21 and X6 each individually correspond to any naturally occurring amino acid. 17 corresponds to M or W, and X 18 corresponds to M or I, and X 19 corresponds to F or Y, and X 20 corresponds to A or G, and X 21 corresponds to A or G and / or X 22 corresponds to L, W, R, D, E, or G.

[0081] In some embodiments, the first antigen-binding domain comprises a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0082] In some embodiments, the RS comprises a protease cleavage site that is cleavable by at least one protease listed in Table 7.

[0083] In some embodiments, the RS comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 85% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 90% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 91% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 92% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 93% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 94% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 95% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 96% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 97% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 98% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 99% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having 100% identity to a sequence listed in Table 8a.

[0084] In some embodiments, the RS is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.

[0085] In some embodiments, RS is not cleavable by legumain. In some embodiments, RS is not cleavable by legumain in human blood, plasma, or serum. In some embodiments, RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, RS is not cleavable upon incubation with about 1 nM or less legumain in human blood, plasma, or serum for about 20 hours.

[0086] In some embodiments, legumain cleaves RS in human plasma at a rate that is less than about 50% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS in human plasma at a rate that is less than about 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS in human plasma at a rate that is less than about 10% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS in human plasma at a rate that is less than about 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS in human plasma at a rate that is less than about 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain.

[0087] In some embodiments, RS1 and / or RS2 comprise a protease cleavable by at least one protease listed in Table 7.

[0088] In some embodiments, RS1 and / or RS2 comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence having at least 85% identity to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence having at least 90% identity to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence having at least 91% identity to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence having at least 92% identity to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence having at least 93% identity to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence at least 94% identical to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence at least 95% identical to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence at least 96% identical to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence at least 97% identical to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence at least 98% identical to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence at least 99% identical to a sequence listed in Table 8a. In some embodiments, RS1 and / or RS2 comprise an amino acid sequence 100% identical to a sequence listed in Table 8a.

[0089] In some embodiments, RS1 and / or RS2 are cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.

[0090] In some embodiments, RS1 and / or RS2 are not cleavable by legumain. In some embodiments, RS1 and / or RS2 are not cleavable by legumain in human blood, plasma, or serum. In some embodiments, RS1 and / or RS2 are not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, RS1 and / or RS2 are not cleavable upon incubation with about 1 nM or less legumain in human blood, plasma, or serum for about 20 hours.

[0091] In some embodiments, legumain cleaves RS1 and / or RS2 in human plasma at a rate that is less than about 50% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS1 and / or RS2 in human plasma at a rate that is less than about 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS1 and / or RS2 in human plasma at a rate that is less than about 10% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS1 and / or RS2 in human plasma at a rate that is less than about 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves RS1 and / or RS2 in human plasma at a rate that is less than about 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain.

[0092] In some embodiments, RS1 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid other than N.

[0093] In some embodiments, RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid except N.

[0094] In some embodiments, RS1 and / or RS2 comprise a protease-cleavable amino acid sequence comprising the sequence: EAGRSASHTPAGLTGP (SEQ ID NO: 7628).

[0095] In some embodiments, RS1 and RS2 are the same. In some embodiments, RS1 and RS2 are different.

[0096] In some embodiments, the mask polypeptide is a first mask polypeptide, the protease-cleavable release segment is a first protease-cleavable release segment (RS1), and the chimeric polypeptide further comprises a second mask polypeptide and a second protease-cleavable release segment (RS2), wherein the second mask polypeptide is joined to the second antigen-binding domain via the second protease-cleavable release segment (RS2) located between the second mask polypeptide and the second antigen-binding domain, such that the second mask polypeptide reduces binding of the first antigen-binding domain to CD3, and RS2 is cleavable by at least one protease present in the tumor.

[0097] In some embodiments, a first masked polypeptide is attached to a first antigen-binding domain and a second masked polypeptide is attached to a second antigen-binding domain.

[0098] In some embodiments, the first masked polypeptide is a first ELNN and the second masked polypeptide is a second ELNN.

[0099] In some embodiments, the first ELNN and the second ELNN are each individually characterized in that (i) at least 90% of the amino acids of each of the first ELNN and the second ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) each contain at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0100] In some embodiments, the first ELNN and the second ELNN are each, individually, further characterized in that (i) each comprises at least 100 amino acid residues, and (ii) each comprises a plurality of non-overlapping sequence motifs that are 9 to 14 amino acids in length, wherein the plurality of non-overlapping sequence motifs comprises a set of non-overlapping sequence motifs, and each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least twice in the ELNN.

[0101] In some embodiments, the plurality of non-redundant sequence motifs comprises at least one non-redundant sequence motif that occurs only once within the ELNN. In some embodiments, the non-redundant sequence motifs comprise one or any combination of the sequence motifs listed in Table 1. In some embodiments, the non-redundant sequence motifs comprise at least two, three, or four of the sequence motifs listed in Table 1. In some embodiments, the non-redundant sequence motifs comprise any one or any combination of GTSTEPSEGSAP, GTSESATPESGP, GSGPGTSESATP, GSEPATSGSETP, GSPAGSPTSTEE, and GTSPSATPESGP.

[0102] In some embodiments, the first ELNN and the second ELNN each comprise at least four amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the amino acids of the first ELNN and the second ELNN each consist of A, E, G, S, P, and / or T.

[0103] In some embodiments, the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is about 294 amino acids in length and the amino acid sequence of the second ELNN is about 582 amino acids in length.

[0104] In some embodiments, the first ELNN and / or the second ELNN comprise an amino acid sequence that is at least 85% identical to an amino acid sequence listed in Table 3a or 3b.

[0105] In some embodiments, the first ELNN comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP (SEQ ID NO: 8021). In some embodiments, the first ELNN comprises an amino acid sequence having at least 85% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 91% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 92% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 93% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 94% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 95% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 96% identity to SEQ ID NO:8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8021.In some embodiments, the first ELNN comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence having 100% identity to SEQ ID NO: 8021.

[0106] In some embodiments, the second ELNN comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to (SEQ ID NO:8022). In some embodiments, the first ELNN comprises an amino acid sequence having at least 85% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 91% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 92% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 93% identity to SEQ ID NO:8022.In some embodiments, the first ELNN comprises an amino acid sequence having at least 94% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 95% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 96% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 97% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 98% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having at least 99% identity to SEQ ID NO:8022. In some embodiments, the first ELNN comprises an amino acid sequence having 100% identity to SEQ ID NO:8022.

[0107] In some embodiments, the chimeric polypeptide comprises one or more barcode fragments. In some embodiments, the chimeric polypeptide comprises two or more barcode fragments. In some embodiments, each barcode fragment is different from every other barcode fragment.

[0108] In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptide upon complete digestion of the chimeric polypeptide by a non-mammalian protease.

[0109] In some embodiments, the non-mammalian protease is Glu-C. In some embodiments, the chimeric polypeptide comprises a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0110] In some embodiments, the chimeric polypeptide has the following amino acid sequence: SGPE.SGPGX n SGPE.SGPG, SGPE.SGPGX nATPE.SGPG、SGPE.SGPGX n GTSE.SATP、SGPE.SGPGX n TTPE.SGPG、SGPE.SGPGX n STPE.SGPG、SGPE.SGPGX n GTPE.SGPG、SGPE.SGPGX n GTPE.TPGS、SGPE.SGPGX n GTPE.TPGS、SGPE.SGPGX n SGSE.TGTP、SGPE.SGPGX n GTPE.GSAP、SGPE.SGPGX n EPSE.SATP、ATPE.SGPGX n SGPE.SGPG、ATPE.SGPGX n ATPE.SGPG、ATPE.SGPGX n GTSE.SATP、ATPE.SGPGX n TTPE.SGPG、ATPE.SGPGX n STPE.SGPG、ATPE.SGPGX n GTPE.SGPG、ATPE.SGPGX n GTPE.TPGS、ATPE.SGPGX n SGSE.TGTP、ATPE.SGPGX n GTPE.GSAP、ATPE.SGPGX n EPSE.SATP、GTSE.SATPX n SGPE.SGPG、GTSE.SATPX n ATPE.SGPG、GTSE.SATPX n GTSE.SATP、GTSE.SATPX n TTPE.SGPG、GTSE.SATPX n STPE.SGPG、GTSE.SATPX n GTPE.SGPG、GTSE.SATPX n GTPE.TPGS、GTSE.SATPX n SGSE.TGTP、GTSE.SATPX n GTPE.GSAP、GTSE.SATPX n EPSE.SATP、TTPE.SGPGX nSGPE.SGPG、TTPE.SGPGX n ATPE.SGPG、TTPE.SGPGX n GTSE.SATP、TTPE.SGPGX n TTPE.SGPG、TTPE.SGPGX n STPE.SGPG、TTPE.SGPGX n GTPE.SGPG、TTPE.SGPGX n GTPE.TPGS、TTPE.SGPGX n SGSE.TGTP、TTPE.SGPGX n GTPE.GSAP、TTPE.SGPGX n EPSE.SATP、STPE.SGPGX n SGPE.SGPG、STPE.SGPGX n ATPE.SGPG、STPE.SGPGX n GTSE.SATP、STPE.SGPGX n TTPE.SGPG、STPE.SGPGX n STPE.SGPG、STPE.SGPGX n GTPE.SGPG、STPE.SGPGX n GTPE.TPGS、STPE.SGPGX n SGSE.TGTP、STPE.SGPGX n GTPE.GSAP、STPE.SGPGX n EPSE.SATP、GTPE.SGPGX n SGPE.SGPG、GTPE.SGPGX n ATPE.SGPG、GTPE.SGPGX n GTSE.SATP、GTPE.SGPGX n TTPE.SGPG、GTPE.SGPGX n STPE.SGPG、GTPE.SGPGX n GTPE.SGPG、GTPE.SGPGX n GTPE.TPGS、GTPE.SGPGX n SGSE.TGTP、GTPE.SGPGX n GTPE.GSAP、GTPE.SGPGX n EPSE.SATP、GTPE.TPGSX nSGPE.SGPG、GTPE.TPGSX n ATPE.SGPG、GTPE.TPGSX n GTSE.SATP、GTPE.TPGSX n TTPE.SGPG、GTPE.TPGSX n STPE.SGPG、GTPE.TPGSX n GTPE.SGPG、GTPE.TPGSX n GTPE.TPGS、GTPE.TPGSX n SGSE.TGTP、GTPE.TPGSX n GTPE.GSAP、GTPE.TPGSX n EPSE.SATP、SGSE.TGTPX n SGPE.SGPG、SGSE.TGTPX n ATPE.SGPG、SGSE.TGTPX n GTSE.SATP、SGSE.TGTPX n TTPE.SGPG、SGSE.TGTPX n STPE.SGPG、SGSE.TGTPX n GTPE.SGPG、SGSE.TGTPX n GTPE.TPGS、SGSE.TGTPX n SGSE.TGTP、SGSE.TGTPX n GTPE.GSAP、SGSE.TGTPX n EPSE.SATP、GTPE.GSAPX n SGPE.SGPG、GTPE.GSAPX n ATPE.SGPG、GTPE.GSAPX n GTSE.SATP、GTPE.GSAPX n TTPE.SGPG、GTPE.GSAPX n STPE.SGPG、GTPE.GSAPX n GTPE.SGPG、GTPE.GSAPX n GTPE.TPGS、GTPE.GSAPX n SGSE.TGTP、GTPE.GSAPX n GTPE.GSAP、GTPE.GSAPX n EPSE.SATP、EPSE.SATPX nSGPE.SGPG, EPSE.SATPX n ATPE.SGPG, EPSE.SATPX n GTSE.SATP, EPSE.SATPX n TTPE.SGPG, EPSE.SATPX n STPE.SGPG, EPSE.SATPX n GTPE.SGPG, EPSE.SATPX n GTPE.TPGS, EPSE.SATPX n SGSE.TGTP, EPSE.SATPX n GTPE.GSAP or EPSE.SATPX n EPSE.SATP, where each "." is a Glu-C cleavage site and n is any integer between 0 and 50. In some embodiments, the chimeric polypeptide has the following amino acid sequence: SGPE.SGPGX n ATPE.SGPG, ATPE.SGPGX n GTSE.SATP, ATPE.SGPGX n TTPE.SGPG, ATPE.SGPGX n STPE.SGPG, ATPE.SGPGX n ATPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n ATPE.SGPG, GTPE.SGPGX n GTPE.SGPG, GTPE.SGPGX n STPE.SGPG, GTPE.SGPGX n TTPE.SGPG, GTPE.SGPGX n STPE.SGPG, GTPE.TPGSX n SGSE.TGTP, GTPE.GSAPX n EPSE.SATP, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n ATPE.SGPG, ATPE.SGPGXn GTPE.SGPG, TTPE.SGPGX n TTPE.SGPG or STPE.SGPGX n STPE.SGPG, In the formula, each "." is a Glu-C cleavage site, and n is any integer from 0 to 30.

[0111] In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4.

[0112] In some embodiments, X n is PGTGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTAGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST.

[0113] In some embodiments, X n is TGTS, SGP, TTPG, TPPT, TSPS, TGSA, TGGA, TSPG, TSGS, TSSA, TGAG, TGST, EPAT, GTST, TAGS, TSSG, TAGP, TPGT, TGGP, or TGSG.

[0114] In some embodiments, neither the N-terminal nor the C-terminal amino acid of the chimeric polypeptide is included in the barcode fragment.

[0115] In some embodiments, the chimeric polypeptide comprises an ELNN comprising a non-overlapping sequence motif that occurs only once within the ELNN, and the ELNN further comprises a barcode fragment that comprises at least a portion of the non-overlapping sequence motif that occurs only once within the ELNN.

[0116] In some embodiments, the chimeric polypeptide comprises a first ELNN comprising a first barcode fragment and a second ELNN comprising a second barcode fragment, wherein neither the first nor the second barcode fragment comprises a glutamic acid immediately adjacent to another glutamic acid (if present) in the ELNN containing the barcode fragment.

[0117] In some embodiments, at least one of the barcode fragments comprises a glutamic acid at its C-terminus.

[0118] In some embodiments, at least one of the barcode fragments has an N-terminal amino acid in the chimeric polypeptide that is immediately preceded by a glutamic acid.

[0119] In some embodiments, the glutamic acid preceding the N-terminal amino acid of the barcode fragment is not immediately adjacent to another glutamic acid.

[0120] In some embodiments, at least one of the barcode fragments does not include a second glutamic acid at a position other than the C-terminus of the barcode fragment, unless the second glutamic acid is immediately followed by a proline.

[0121] In some embodiments, the chimeric polypeptide comprises a single polypeptide chain, wherein the chimeric polypeptide comprises a barcode fragment located within the polypeptide chain that is 10-200 amino acids or 10-125 amino acids from the N-terminus or C-terminus of the chimeric polypeptide. In some embodiments, a first ELNN is N-terminal to the bispecific antibody domain, and the first barcode fragment is located within 200, 150, 100, or 50 amino acids of the N-terminus of the chimeric polypeptide. In some embodiments, a second ELNN is C-terminal to the bispecific antibody domain, and the second barcode fragment is located within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.

[0122] In some embodiments, at least one of the barcode fragments is at least four amino acids in length.

[0123] In some embodiments, at least one of the barcode fragments is between 4 and 20, 5 and 15, 6 and 12, or 7 and 10 amino acids in length.

[0124] In some embodiments, each masked polypeptide comprises one barcode fragment listed in Table 2 or disclosed in Table 3a.

[0125] In some embodiments, the chimeric polypeptide comprises a barcode fragment comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SGPGSGPGTSE or SGPGTSPSATPE.

[0126] In some embodiments, the chimeric polypeptide comprises one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGSGPGTSE and one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGTSPSATPE.

[0127] In some embodiments, the barcode fragment consists of A, E, G, S, P, and / or T residues.

[0128] In some embodiments, the barcode fragment is part of a mask peptide.

[0129] In some embodiments, the mask peptide is a first ELNN or a second ELNN.

[0130] In some embodiments, a chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table D (SEQ ID NOS: 1000-1009). In some embodiments, a chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1000. In some embodiments, a chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1001. In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1002. In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1003. In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1004. In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1005. In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1006.In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1007. In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1008. In some embodiments, the chimeric polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1009.

[0131]

[0132]

[0133] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising a chimeric polypeptide described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0134] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0135] Certain aspects of the present disclosure are directed to polynucleotide sequences that encode the chimeric polypeptides described herein.

[0136] Certain aspects of the present disclosure are directed to expression vectors that include the polynucleotides described herein.

[0137] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0138] Certain aspects of the present disclosure are directed to methods of producing the chimeric polypeptides described herein. In some embodiments, the methods further comprise isolating the chimeric polypeptide from the host cell.

[0139] Certain aspects of the present disclosure are directed to methods of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a chimeric polypeptide described herein.

[0140] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is metastatic prostate cancer. In some embodiments, the prostate cancer is androgen-independent. In some embodiments, the prostate cancer is non-metastatic castration-resistant prostate cancer (nmCRPC). In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

[0141] In some embodiments, the method further comprises administering docetaxel to the subject.

[0142] In some embodiments, the method further comprises administering a checkpoint inhibitor to the subject. In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the checkpoint inhibitor is pembrolizumab or cemiplimab.

[0143] Certain aspects of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 85% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 90% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 91% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 92% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 93% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 94% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 95% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 96% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 97% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 98% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having at least 99% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain embodiments of the present disclosure are directed to linker polypeptides comprising an amino acid sequence having 100% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0144] In some embodiments, the linker polypeptide is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0145] In some embodiments, a linker polypeptide connects the first polypeptide moiety to the second polypeptide moiety, hi some embodiments, the first polypeptide moiety is a VL domain and the second polypeptide moiety is a VH domain.

[0146] Certain aspects of the present disclosure are directed to antigen-binding polypeptides comprising a VL domain and a VH domain, wherein the VL domain is linked to the VH domain by a linker polypeptide comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0147] In some embodiments, the linker polypeptide is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0148] In some embodiments, the antigen-binding polypeptide is an scFv.

[0149] In some embodiments, the antigen is CD3. In some embodiments, the antigen is CD3 epsilon.

[0150] In some embodiments, the VL domain is N-terminal to the VH domain. In some embodiments, the VH domain is N-terminal to the VL domain.

[0151] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising a linker polypeptide described herein or an antigen-binding polypeptide described herein and at least one pharmaceutically acceptable excipient.

[0152] In some embodiments, the pharmaceutical composition is in liquid form or is frozen, hi some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0153] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0154] Certain aspects of the present disclosure are directed to polynucleotide sequences that encode a linker as described herein or an antigen-binding polypeptide as described herein.

[0155] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0156] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0157] Certain aspects of the present disclosure are directed to methods of producing a linker described herein or an antigen-binding polypeptide described herein. In some embodiments, the method further comprises isolating the linker or antigen-binding polypeptide from the host cell.

[0158] Certain aspects of the present disclosure are directed to isolated polypeptides comprising a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid except N. In some embodiments, X is S.

[0159] In some embodiments, the isolated polypeptide is not cleavable by legumain. In some embodiments, the isolated polypeptide is not cleavable by legumain in human blood, plasma, or serum. In some embodiments, the isolated polypeptide is not cleavable upon incubation with about 1 nM or less of legumain for about 20 hours. In some embodiments, the isolated polypeptide is not cleavable upon incubation with about 1 nM or less of legumain in human blood, plasma, or serum for about 20 hours.

[0160] In some embodiments, legumain cleaves a polypeptide isolated in human plasma at a rate that is less than about 50% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves a polypeptide isolated in human plasma at a rate that is less than about 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves a polypeptide isolated in human plasma at a rate that is less than about 10% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves a polypeptide isolated in human plasma at a rate that is less than about 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves the polypeptide isolated in human plasma at a rate that is less than about 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain.

[0161] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising an isolated polypeptide described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0162] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0163] Certain aspects of the present disclosure are directed to polynucleotide sequences that encode the isolated polypeptides described herein.

[0164] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0165] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0166] Certain aspects of the present disclosure are directed to methods of producing the isolated polypeptides described herein. In some embodiments, the methods further comprise isolating the isolated polypeptide from the host cell.

[0167] Certain aspects of the present disclosure are directed to fusion proteins comprising a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid except N, and the protease-cleavable amino acid sequence links a first polypeptide moiety to a second polypeptide moiety. In some embodiments, X is S.

[0168] In some embodiments, the fusion protein is not cleavable by legumain. In some embodiments, the fusion protein is not cleavable by legumain in human blood, plasma, or serum. In some embodiments, the fusion protein is not cleavable upon incubation with about 1 nM or less of legumain for about 20 hours. In some embodiments, the fusion protein is not cleavable upon incubation with about 1 nM or less of legumain in human blood, plasma, or serum for about 20 hours.

[0169] In some embodiments, legumain cleaves protease-cleavable amino acid sequences in human plasma at a rate that is less than about 50% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves protease-cleavable amino acid sequences in human plasma at a rate that is less than about 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves protease-cleavable amino acid sequences in human plasma at a rate that is less than about 10% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves protease-cleavable amino acid sequences in human plasma at a rate that is less than about 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain. In some embodiments, legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legumain.

[0170] In some embodiments, the first polypeptide portion comprises an antigen-binding domain and the second polypeptide portion comprises a masking polypeptide.

[0171] In some embodiments, the first polypeptide moiety comprises an antigen-binding domain and the second polypeptide moiety is a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin.

[0172] In some embodiments, the first polypeptide moiety is a cytokine, enzyme, hormone, growth factor, chemotherapeutic polypeptide, antiviral polypeptide, or toxin, and the second polypeptide moiety is a masking polypeptide.

[0173] In some embodiments, the masking polypeptide comprises ELNN.

[0174] In some embodiments, the fusion protein comprises a single polypeptide chain comprising, from N-terminal to C-terminal, a first polypeptide, followed by a protease-cleavable amino acid sequence, followed by a second polypeptide portion. In some embodiments, the fusion protein comprises a single polypeptide chain comprising, from N-terminal to C-terminal, a second polypeptide, followed by a protease-cleavable amino acid sequence, followed by the first polypeptide portion.

[0175] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising a fusion protein described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0176] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0177] Certain aspects of the present disclosure are directed to polynucleotide sequences that encode the fusion proteins described herein.

[0178] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0179] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0180] Certain aspects of the present disclosure are directed to methods of producing the fusion proteins described herein. In some embodiments, the methods further comprise isolating the fusion protein from the host cell.

[0181] Certain aspects of the present disclosure are directed to an ELNN polypeptide comprising the following amino acid sequence: ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP.

[0182] Certain embodiments of the present disclosure are directed to an ELNN polypeptide comprising the following amino acid sequence:

[0183] Certain aspects of the present disclosure are directed to fusion proteins comprising the ELNN polypeptides described herein.

[0184] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising an ELNN polypeptide described herein or a fusion protein described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted before administration.

[0185] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0186] Certain aspects of the present disclosure are directed to polynucleotide sequences that encode the ELNN polypeptides described herein or the fusion proteins described herein.

[0187] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0188] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0189] Certain aspects of the present disclosure are directed to methods of producing an ELNN polypeptide described herein or a fusion protein described herein.

[0190] 289. The method of Claim 288, in some embodiments further comprising isolating the ELNN polypeptide or fusion protein from the host cell.

[0191] Certain aspects of the present disclosure are directed to barcode fragments that include an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SGPGTGTSATPE, SGPGSGPGTSE, SGPGTTPGTTPE, SGPGTPPTSTPE, SGPGTSPSATPE, SGPGTGSAGTPE, SGPGTGGAGTPE, SGPGTSPGATPE, SGPGTSGSGTPE, SGPGTSSASTPE, SGPGTGAGTTPE, SGPGTGSTSTPE, TPGSEPATSGSE, GSAPGTSTEPSE, SGPGTAGSGTPE, SGPGTSSGGTPE, SGPGTAGPATPE, SGPGTPGTGTPE, SGPGTGGPTTPE, or SGPGTGSGSTPE.

[0192] Certain aspects of the present disclosure are directed to barcode fragments that include an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SGPGSGPGTSE or SGPGTSSATPE.

[0193] In some embodiments, the barcode fragment comprises the amino acid sequence: SGPGSGPGTSEE. In some embodiments, the barcode fragment comprises the amino acid sequence: SGPGTSPSATPE.

[0194] Certain aspects of the present disclosure are directed to fusion proteins comprising the barcode fragments described herein.

[0195] Certain aspects of the present disclosure are directed to fusion proteins that include a Glu-C cleavage site that includes one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0196] Certain embodiments of the present disclosure include the following amino acid sequences: SGPE.SGPGX n SGPE.SGPG, SGPE.SGPGX nATPE.SGPG、SGPE.SGPGX n GTSE.SATP、SGPE.SGPGX n TTPE.SGPG、SGPE.SGPGX n STPE.SGPG、SGPE.SGPGX n GTPE.SGPG、SGPE.SGPGX n GTPE.TPGS、SGPE.SGPGX n GTPE.TPGS、SGPE.SGPGX n SGSE.TGTP、SGPE.SGPGX n GTPE.GSAP、SGPE.SGPGX n EPSE.SATP、ATPE.SGPGX n SGPE.SGPG、ATPE.SGPGX n ATPE.SGPG、ATPE.SGPGX n GTSE.SATP、ATPE.SGPGX n TTPE.SGPG、ATPE.SGPGX n STPE.SGPG、ATPE.SGPGX n GTPE.SGPG、ATPE.SGPGX n GTPE.TPGS、ATPE.SGPGX n SGSE.TGTP、ATPE.SGPGX n GTPE.GSAP、ATPE.SGPGX n EPSE.SATP、GTSE.SATPX n SGPE.SGPG、GTSE.SATPX n ATPE.SGPG、GTSE.SATPX n GTSE.SATP、GTSE.SATPX n TTPE.SGPG、GTSE.SATPX n STPE.SGPG、GTSE.SATPX n GTPE.SGPG、GTSE.SATPX n GTPE.TPGS、GTSE.SATPX n SGSE.TGTP、GTSE.SATPX n GTPE.GSAP、GTSE.SATPX n EPSE.SATP、TTPE.SGPGX nSGPE.SGPG、TTPE.SGPGX n ATPE.SGPG、TTPE.SGPGX n GTSE.SATP、TTPE.SGPGX n TTPE.SGPG、TTPE.SGPGX n STPE.SGPG、TTPE.SGPGX n GTPE.SGPG、TTPE.SGPGX n GTPE.TPGS、TTPE.SGPGX n SGSE.TGTP、TTPE.SGPGX n GTPE.GSAP、TTPE.SGPGX n EPSE.SATP、STPE.SGPGX n SGPE.SGPG、STPE.SGPGX n ATPE.SGPG、STPE.SGPGX n GTSE.SATP、STPE.SGPGX n TTPE.SGPG、STPE.SGPGX n STPE.SGPG、STPE.SGPGX n GTPE.SGPG、STPE.SGPGX n GTPE.TPGS、STPE.SGPGX n SGSE.TGTP、STPE.SGPGX n GTPE.GSAP、STPE.SGPGX n EPSE.SATP、GTPE.SGPGX n SGPE.SGPG、GTPE.SGPGX n ATPE.SGPG、GTPE.SGPGX n GTSE.SATP、GTPE.SGPGX n TTPE.SGPG、GTPE.SGPGX n STPE.SGPG、GTPE.SGPGX n GTPE.SGPG、GTPE.SGPGX n GTPE.TPGS、GTPE.SGPGX n SGSE.TGTP、GTPE.SGPGX n GTPE.GSAP、GTPE.SGPGX n EPSE.SATP、GTPE.TPGSX nSGPE.SGPG、GTPE.TPGSX n ATPE.SGPG、GTPE.TPGSX n GTSE.SATP、GTPE.TPGSX n TTPE.SGPG、GTPE.TPGSX n STPE.SGPG、GTPE.TPGSX n GTPE.SGPG、GTPE.TPGSX n GTPE.TPGS、GTPE.TPGSX n SGSE.TGTP、GTPE.TPGSX n GTPE.GSAP、GTPE.TPGSX n EPSE.SATP、SGSE.TGTPX n SGPE.SGPG、SGSE.TGTPX n ATPE.SGPG、SGSE.TGTPX n GTSE.SATP、SGSE.TGTPX n TTPE.SGPG、SGSE.TGTPX n STPE.SGPG、SGSE.TGTPX n GTPE.SGPG、SGSE.TGTPX n GTPE.TPGS、SGSE.TGTPX n SGSE.TGTP、SGSE.TGTPX n GTPE.GSAP、SGSE.TGTPX n EPSE.SATP、GTPE.GSAPX n SGPE.SGPG、GTPE.GSAPX n ATPE.SGPG、GTPE.GSAPX n GTSE.SATP、GTPE.GSAPX n TTPE.SGPG、GTPE.GSAPX n STPE.SGPG、GTPE.GSAPX n GTPE.SGPG、GTPE.GSAPX n GTPE.TPGS、GTPE.GSAPX n SGSE.TGTP、GTPE.GSAPX n GTPE.GSAP、GTPE.GSAPX n EPSE.SATP、EPSE.SATPX nSGPE.SGPG, EPSE.SATPX n ATPE.SGPG, EPSE.SATPX n GTSE.SATP, EPSE.SATPX n TTPE.SGPG, EPSE.SATPX n STPE.SGPG, EPSE.SATPX n GTPE.SGPG, EPSE.SATPX n GTPE.TPGS, EPSE.SATPX n SGSE.TGTP, EPSE.SATPX n GTPE.GSAP or EPSE.SATPX n The present invention relates to a fusion protein comprising at least one of EPSE.SATP, wherein each "." is a Glu-c cleavage site and n is any integer between 0 and 50.

[0197] In some embodiments, the fusion protein has the following amino acid sequence: SGPE.SGPGX n ATPE.SGPG, ATPE.SGPGX n GTSE.SATP, ATPE.SGPGX n TTPE.SGPG, ATPE.SGPGX n STPE.SGPG, ATPE.SGPGX n ATPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n ATPE.SGPG, GTPE.SGPGX n GTPE.SGPG, GTPE.SGPGX n STPE.SGPG, GTPE.SGPGX n TTPE.SGPG, GTPE.SGPGX n STPE.SGPG, GTPE.TPGSX n SGSE.TGTP, GTPE.GSAPX n EPSE.SATP, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGXn ATPE.SGPG, ATPE.SGPGX n GTPE.SGPG, TTPE.SGPGX n TTPE.SGPG or STPE.SGPGX n STPE.SGPG, where each "." is a Glu-C cleavage site and n is any integer from 0 to 30.

[0198] In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4.

[0199] In some embodiments, X n is PGTGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTAGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST.

[0200] In some embodiments, X n is TGTS, SGP, TTPG, TPPT, TSPS, TGSA, TGGA, TSPG, TSGS, TSSA, TGAG, TGST, EPAT, GTST, TAGS, TSSG, TAGP, TPGT, TGGP, or TGSG.

[0201] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising a barcode fragment described herein or a fusion protein described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0202] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0203] Certain aspects of the present disclosure are directed to polynucleotide sequences that encode the barcode fragments described herein or the fusion proteins described herein.

[0204] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0205] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0206] Certain aspects of the present disclosure are directed to methods of producing a barcode fragment described herein or a fusion protein described herein. In some embodiments, the method further comprises isolating the barcode fragment or fusion protein from the host cell.

[0207] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment thereof that specifically binds to PSMA, comprising a VHH domain or fragment thereof comprising three VHH CDRs, wherein the three VHH CDRs comprise CDR1, CDR2, and CDR3 from the following amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0208] Certain embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to PSMA, the antibody or antigen-binding fragment thereof comprising a VHH CDR1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs: GRTFGIYVWG; and a VHH CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to AASNKEYGRTWYDFNESDY.

[0209] In some embodiments, the antibody or fragment is a VHH FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following FR: QVQLVESGGGVVQPGRSLRLSCAAS; a VHH FR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG FR2; VHH FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDNSKNTLYLQMNSLRAEDTAVYYC; and VHH FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS.

[0210] Certain embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to PSMA, comprising the following CDRs: a VHH CDR1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG; a VHH CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK; and a VHH CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY. The present invention relates to an antibody or antigen-binding fragment thereof, comprising the CDR3.

[0211] In some embodiments, the antibody or fragment is a VHH FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following FR: QVQLVESGGGVVQPGRSLRLSCAAS; a VHH FR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG FR2; VHH FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC; and VHH FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS.

[0212] Certain aspects of the present disclosure are directed to antibodies or antigen-binding fragments thereof that specifically bind to PSMA, comprising a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0213] In some embodiments, the antibody or fragment is an isolated antibody or fragment thereof.

[0214] Certain aspects of the present disclosure are directed to antibodies or antigen-binding fragments thereof that specifically bind to PSMA, comprising a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to PSMA.2, PSMA.3, PSMA.5, PSMA.6, PSMA.262, or PSMA.263.

[0215] Certain aspects of the present disclosure include an antibody or antigen-binding fragment thereof that specifically binds to PSMA, comprising: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVX 17 GWFRQAPGKEREFVGAX 18 SWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYX 19 CX 20 X 21 SNKX 22 YGRTWYDFNESDYWGQGTQVTVSS, wherein X is a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, 17 , X 18 , X 19 , X 20 , X 21 and X6 each individually corresponds to any naturally occurring amino acid. 17 corresponds to M or W, and X 18 corresponds to M or I, and X 19 corresponds to F or Y, and X 20 corresponds to A or G, and X 21 corresponds to A or G and / or X 22 corresponds to L, W, R, D, E, or G.

[0216] In some embodiments, the PSMA comprises the following amino acid sequence: (SEQ ID NO: 1044).

[0217] Certain aspects of the present disclosure are directed to an antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising a VL domain and a VH domain, wherein (i) the VL domain comprises the VL CDRs of the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), or (ii) the VH domain comprises the VH CDRs of the amino acid sequence of EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

[0218] Certain embodiments of the present disclosure relate to an anti-CD3 antibody or antigen-binding fragment thereof, comprising a VL domain CDR1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to RSSNGAVTSSNYAN, or 100% identity to GTNKRAP; a VL domain CDR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV; a VL domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV; a VH domain CDR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to and / or a VH domain CDR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to HENFGNSYVSWFAH.

[0219] In some embodiments, the antibody or fragment comprises a VL domain FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following FR: ELVVTQEPSLTVSPGGTVTLTC; a VL domain FR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC; a VL domain FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL; R4; a VH domain FR1 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS; a VH domain FR2 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG; RFTISRDDS a VH domain FR3 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to KNTVYLQMNSLKTEDTAVYYCVR; and / or a VH domain FR4 comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS.

[0220] In some embodiments, the antibody or fragment comprises a VL domain.

[0221] In some embodiments, the VL domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0222] In some embodiments, the antibody or fragment comprises a VH domain.

[0223] In some embodiments, the VH domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

[0224] Certain aspects of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising a VL domain and a VH domain, wherein the pair of VL domain amino acid sequence SEQ ID NO / VH domain amino acid sequence SEQ ID NO is 896 / 897, 902 / 903, 700 / 701, 702 / 703, 716 / 717, 718 / 719, 728 / 729, 736 / 737, 738 / 739, 740 / 741, 742 / 743, 744 / 745, 746 / 747, 748 / 749 , 750 / 751, 752 / 753, 754 / 755, 756 / 757, 758 / 759, 760 / 761, 762 / 763, 764 / 765, 766 / 767, 774 / 775, 776 / 777, 790 / 791, 792 / 793, 798 / 799, 800 / 801, 806 / 807, 808 / 809, 814 / 815, 816 / 817, 822 / 823, 824 / 825, or 826 / 867.

[0225] In some embodiments, the antibody or fragment thereof is an isolated antibody or fragment thereof.

[0226] In some embodiments, the antibody or fragment thereof is an antibody.

[0227] In some embodiments, the antibody or fragment thereof is a Fab, scFv, or monoclonal antibody.

[0228] In some embodiments, the antibody or fragment thereof is an scFv.

[0229] In some embodiments, the VL domain is N-terminal to the VH domain in an scFv.

[0230] In some embodiments, the VL domain is C-terminal to the VH domain in an scFv.

[0231] In some embodiments, the scFv comprises a linker between the VL and VH domains, wherein the linker consists of A, E, G, S, P, and / or T residues.

[0232] In some embodiments, the linker is an ELNN.

[0233] In some embodiments, the ELNN is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0234] In some embodiments, the ELNN comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0235] In some embodiments, the scFv comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS.

[0236] In some embodiments, the CD3 is CD3 epsilon.

[0237] In some embodiments, CD3 epsilon comprises the following amino acid sequence: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 1043).

[0238] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof described herein and at least one pharmaceutically acceptable excipient.

[0239] In some embodiments, the pharmaceutical composition is in liquid form or is frozen, hi some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0240] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0241] Certain aspects of the present disclosure are directed to polynucleotide sequences encoding the antibodies or antigen-binding fragments thereof described herein.

[0242] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0243] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0244] Certain aspects of the present disclosure are directed to methods of producing the antibodies or antigen-binding fragments thereof described herein. In some embodiments, the methods further comprise isolating the antibodies or antigen-binding fragments thereof from the host cell.

[0245] Certain aspects of the present disclosure are directed to multispecific antibodies comprising an anti-PSMA antibody domain comprising an antibody or antibody fragment described herein, and / or an anti-CD3 antibody domain comprising an antibody or antibody fragment described herein.

[0246] Certain aspects of the present disclosure are directed to multispecific antibodies comprising an anti-PSMA antibody domain comprising an antibody or antibody fragment described herein, and an anti-CD3 antibody domain comprising an antibody or antibody fragment described herein.

[0247] In some embodiments, the affinity of the anti-PSMA antibody domain for PSMA is higher than the affinity of the anti-CD3 antibody domain for CD3. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody is a T cell engager.

[0248] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising a multispecific antibody described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0249] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0250] Certain aspects of the present disclosure are directed to polynucleotide sequences encoding the multispecific antibodies described herein.

[0251] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0252] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0253] Certain aspects of the present disclosure are directed to methods of producing the multispecific antibodies described herein. In some embodiments, the method further comprises isolating the multispecific antibody from the host cell.

[0254] Certain aspects of the present disclosure relate to a T cell engager comprising a first antigen-binding domain that binds to prostate-specific membrane antigen (PSMA) and a second antigen-binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the first antigen-binding domain comprises a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to QLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549); and the second antigen-binding domain comprises a VHH comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549). a VL domain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to NKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), and and a VH domain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to LEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

[0255] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising a T cell engager described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0256] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0257] Certain aspects of the present disclosure are directed to polynucleotide sequences encoding the T cell engagers described herein.

[0258] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0259] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0260] Certain aspects of the present disclosure are directed to methods of producing the T cell engagers described herein. In some embodiments, the methods further comprise isolating the T cell engager from the host cell.

[0261] Certain aspects of the present disclosure are directed to a protease-activatable T cell engager (paTCE) comprising a T cell engager (TCE) described herein in the form of a single polypeptide chain, wherein the N-terminus of the TCE is fused to a first masking polypeptide by a first protease-cleavable linker and the C-terminus of the TCE is fused to a second masking polypeptide by a second protease-cleavable linker.

[0262] In some embodiments, the first masking polypeptide is a first ELNN. In some embodiments, the second masking polypeptide is a second ELNN.

[0263] In some embodiments, the TCE comprises an anti-PSMA VHH comprising the following amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0264] In some embodiments, the TCE comprises an anti-CD3 scFv comprising a VH domain having the following amino acid sequence: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311), and a VL domain having the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0265] Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising the paTCE described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted prior to administration.

[0266] Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

[0267] Certain aspects of the present disclosure are directed to polynucleotide sequences encoding the paTCEs described herein.

[0268] Certain aspects of the present disclosure are directed to expression vectors comprising the polynucleotide sequences described herein.

[0269] Certain aspects of the present disclosure are directed to host cells comprising the expression vectors described herein.

[0270] Certain aspects of the present disclosure are directed to methods of producing the paTCE described herein. In some embodiments, the method further comprises isolating the paTCE from the host cell.

[0271] Certain aspects of the present disclosure are directed to a chimeric polypeptide, an isolated polypeptide, a fusion protein, an antigen-binding polypeptide, an antibody or antigen-binding fragment thereof that specifically binds to PSMA, an antibody or antigen-binding fragment thereof that specifically binds to CD3, a multispecific antibody, a T cell engager, or a paTCE produced by the methods described herein.

[0272] Certain aspects of the present disclosure are directed to polynucleotide sequences that encode the amino acid sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is an amino acid sequence other than N.

[0273] In some embodiments, the polynucleotide is a vector.

[0274] In some embodiments, the polynucleotide is an isolated polynucleotide.

[0275] Certain aspects of the present disclosure are directed to cell lines that express an exogenous polynucleotide comprising the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is an amino acid sequence other than N.

[0276] In some embodiments, the exogenous polypeptide is a fusion protein described herein.

[0277] In some embodiments, the cell lines are cultured or frozen in glass or plastic containers.

[0278] In some embodiments, the cell line is in a bioreactor.

[0279] In some embodiments, the cells are stable cell lines.

[0280] In some embodiments, the cell is a mammalian cell.

[0281] In some embodiments, the cell line is a CHO cell or a HEK293 cell.

[0282] In some embodiments, the cell line is a prokaryotic cell.

[0283] In some embodiments, the cell line is an Escherichia coli cell.

[0284] Certain aspects of the present disclosure are directed to non-human animals comprising an exogenous polypeptide comprising the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid except N. In some embodiments, X is D, E, or Q. In some embodiments, X is G, A, V, L, or I. In some embodiments, X is P. In some embodiments, X is F, Y, or W. In some embodiments, X is H, K, or R. In some embodiments, X is S, C, U, T, or M. In some embodiments, X is S.

[0285] Certain aspects of the present disclosure are directed to fusion proteins comprising an anti-PSMA antibody or fragment described herein and a biologically active protein.

[0286] Certain aspects of the present disclosure are directed to fusion proteins comprising an anti-CD3 antibody or fragment described herein and a biologically active protein.

[0287] In some embodiments, the biologically active protein comprises a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin.

[0288] Certain aspects of the present disclosure are directed to immunoconjugates comprising an anti-PSMA antibody or fragment described herein and a compound.

[0289] Certain aspects of the present disclosure are directed to immunoconjugates comprising an anti-CD3 antibody or fragment described herein and a compound.

[0290] In some embodiments, the compound comprises a chemotherapeutic agent.

[0291] In some embodiments, the compound comprises a diagnostic agent.

[0292] In some embodiments, the compound comprises a toxin, a radioactive molecule, an imaging agent, or a drug.

[0293] The present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising a heavy chain variable region (VH) comprising three heavy chain CDRs and a light chain variable region (VL) comprising three light chain CDRs, wherein the three heavy chain CDRs are selected from the group consisting of: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311). and the three light chain CDRs comprise CDR1, CDR2, and CDR3 from ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), wherein the CDRs are identified by the Kabat, Chothia, AbM, IMGT, or contact definition. In some embodiments, the antibody is an scFv.

[0294] Included herein is an antigen binding protein comprising: (i) a light chain variable domain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to a light chain variable domain sequence comprising CDR sequences selected from the group consisting of RSSNGAVTSSNYAN, GTNKRAP, and ALWYPNLWV; and (ii) a heavy chain variable domain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to a heavy chain variable domain sequence comprising CDR sequences selected from the group consisting of SEQ ID NOs:GFTFSTYAMN, RIRTKRNNYATYYADSVKG, and HENFGNSYVSWFAH, wherein the antigen binding protein specifically binds to CD3. In some embodiments, the antibody is an scFv.

[0295] Disclosed herein is an antibody or antigen-binding fragment thereof that specifically binds to CD3, wherein the antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (CDR) sequences of SEQ ID NOs: RSSNGAVTSSNYAN, GTNKRAP, and ALWYPNLWV, and three heavy chain complementarity determining region (CDR) sequences of SEQ ID NOs: GFTFSTYAMN, RIRTKRNNYATYYADSVKG, and HENFGNSYVSWFAH. In some embodiments, the antibody is an scFv.

[0296] Included herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising the amino acid sequence of DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 1043), and comprising a heavy chain variable region comprising three heavy chain CDRs and a light chain variable region comprising three light chain CDRs, wherein the three heavy chain CDRs are selected from the group consisting of EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIR An isolated antibody or antigen-binding fragment thereof comprising CDR1, CDR2 and CDR3 from TKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (sequence number 311), and the three light chain CDRs comprising CDR1, CDR2 and CDR3 from ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (sequence number 361). In some embodiments, the heavy chain CDR1 comprises GFTFSTYAMN, the heavy chain CDR2 comprises RIRTKRNNYATYYADSVKG, the heavy chain CDR3 comprises HENFGNSYVSWFAH, the light chain CDR1 comprises RSSNGAVTSSNYAN, the light chain CDR2 comprises GTNKRAP, and the light chain CDR3 comprises ALWYPNLWV. In some embodiments, the heavy chain variable region comprises EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).In some embodiments, the light chain variable region comprises ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361). In some embodiments, the heavy chain variable region comprises EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311) and the light chain variable region comprises ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0297] In some embodiments, included herein are compounds having a K of about 300 nM or less, e.g., as measured by surface plasmon resonance. D and specifically binds to CD3 (e.g., a protein having a heavy chain variable region amino acid sequence of SEQ ID NO: 311 and a light chain variable region amino acid sequence of SEQ ID NO: 361). In some embodiments, the antibody or antigen-binding portion thereof has a K of about 200 nM or less, about 150 or less, about 100 nM or less, or about 75 nM or less. D It presents.

[0298] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD3 (e.g., a protein having the heavy chain variable region amino acid sequence of SEQ ID NO: 311 and the light chain variable region amino acid sequence of SEQ ID NO: 361), comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment thereof has (a) an affinity for CD3 (K) of about 100 nM or less. D(b) a heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 311; and (c) a light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 361; (d) an affinity for CD3 (K) of about 300 nM or less. D and (c) a heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 311; and a light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 361; or (d) an affinity for CD3 (K) of about 75 nM or less. Dand a light chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 361), characterized in that: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS; and a light chain variable region having an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 361), characterized in that:

[0299] The present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to PSMA, comprising a VHH domain comprising three VHH CDRs, wherein the three VHH CDRs comprise CDR1, CDR2, and CDR3 from QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549), and the CDRs are identified by the Kabat definition, the Chothia definition, the AbM definition, the IMGT definition, or the contact definition.

[0300] Included herein are antigen binding proteins comprising: (i) a VHH domain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or 100% identical, to a VHH CDR sequence selected from the group consisting of GRTFGIYVWG, AMSWSGSNRK, and AASNKEYGRTWYDFNESDY, wherein the antigen binding protein specifically binds to PSMA.

[0301] Included herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to PSMA, comprising the amino acid sequence of (SEQ ID NO: 1044), which includes a VHH region comprising three VHH CDRs from QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).In some embodiments, VHH CDR1 comprises GRTFGIYVWG, VHH CDR2 comprises AMSWSGSNRK, and VHH CDR3 comprises AASNKEYGRTWYDFNESDY. In some embodiments, the VHH region comprises QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0302] In some embodiments, included herein are compounds having a K of about 300 nM or less, e.g., as measured by surface plasmon resonance. D and specifically binds to PSMA (e.g., a protein having the amino acid sequence of SEQ ID NO: 549). In some embodiments, the antibody or antigen-binding portion thereof has a K of about 200 nM or less, about 150 or less, about 100 nM or less, or about 50 nM or less. D It presents.

[0303] Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to PSMA (e.g., proteins comprising the amino acid sequence of SEQ ID NO: 549), and that have (a) an affinity for PSMA (K) of about 100 nM or less. D (b) a VHH region having an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549), characterized in that: (a) an affinity for PSMA (K) of about 300 nM or less; DQVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549), characterized by: (c) an affinity for PSMA (K) of about 50 nM or less. D and a VHH region having an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 549, wherein the amino acid sequence is QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS.

[0304] The present disclosure includes a bispecific T cell engager comprising: (i) an antibody or antigen-binding fragment thereof that specifically binds to human CD3 provided herein (a protein having a heavy chain variable region amino acid sequence of SEQ ID NO: 311 and a light chain variable region amino acid sequence of SEQ ID NO: 361), and (ii) an antibody or antigen-binding fragment thereof that specifically binds to human PSMA provided herein (SEQ ID NO: 549).

[0305] Various features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0306] [Figure 1A] 1 shows a non-limiting schematic diagram of an exemplary paTCE. [Figure 1B]1A shows a schematic diagram of the completely unmasked paTCE (unmasked paTCE, uTCE) and singly masked metabolites paTCE(1x-N) and paTCE(1x-C) from the exemplary paTCE shown in FIG. 1A. [Figure 2A] Biophysical characterization data for PMSA.2 variant antibodies are shown. Figure 2A shows the concentration of PMSA.2 variant antibodies. Figure 2B shows the relative binding of PMSA.2 variant antibodies to PSMA. Figure 2C shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 65°C. The AC clone numbers of the uTCEs tested are indicated in the figures. uTCEs were used in these experiments, not paTCEs. [Figure 2B] Biophysical characterization data for PMSA.2 variant antibodies are shown. Figure 2A shows the concentration of PMSA.2 variant antibodies. Figure 2B shows the relative binding of PMSA.2 variant antibodies to PSMA. Figure 2C shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 65°C. The AC clone numbers of the uTCEs tested are indicated in the figures. uTCEs were used in these experiments, not paTCEs. [Figure 2C] Biophysical characterization data for PMSA.2 variant antibodies are shown. Figure 2A shows the concentration of PMSA.2 variant antibodies. Figure 2B shows the relative binding of PMSA.2 variant antibodies to PSMA. Figure 2C shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 65°C. The AC clone numbers of the uTCEs tested are indicated in the figures. uTCEs were used in these experiments, not paTCEs. [Figure 2D]Biophysical characterization data for PMSA.2 variant antibodies are shown. Figure 2A shows the concentration of PMSA.2 variant antibodies. Figure 2B shows the relative binding of PMSA.2 variant antibodies to PSMA. Figure 2C shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 65°C. The AC clone numbers of the uTCEs tested are indicated in the figures. uTCEs were used in these experiments, not paTCEs. [Figure 3A] Biophysical characterization data for PMSA.3 variant antibodies are shown. Figure 3A shows the relative binding of PMSA.3 variant antibodies to PSMA. Figures 3B and 3C show the thermal stability of PMSA.3 variant antibodies as measured by monomer concentration (Figure 3B) or aggregate concentration (Figure 3C) (pM) at 63.5°C. The AC clone numbers of the uTCEs tested are indicated in the figures. uTCE, rather than paTCE, was used in these experiments. [Figure 3B] Biophysical characterization data for PMSA.3 variant antibodies are shown. Figure 3A shows the relative binding of PMSA.3 variant antibodies to PSMA. Figures 3B and 3C show the thermal stability of PMSA.3 variant antibodies as measured by monomer concentration (Figure 3B) or aggregate concentration (Figure 3C) (pM) at 63.5°C. The AC clone numbers of the uTCEs tested are indicated in the figures. uTCE, rather than paTCE, was used in these experiments. [Figure 3C] Biophysical characterization data for PMSA.3 variant antibodies are shown. Figure 3A shows the relative binding of PMSA.3 variant antibodies to PSMA. Figures 3B and 3C show the thermal stability of PMSA.3 variant antibodies as measured by monomer concentration (Figure 3B) or aggregate concentration (Figure 3C) (pM) at 63.5°C. The AC clone numbers of the uTCEs tested are indicated in the figures. uTCE, rather than paTCE, was used in these experiments. [Figure 4]The PTE scores of representative PSMA and CD3 variants are shown. The graph shows molecules containing known anti-drug antibodies (ADAs) and their corresponding PTE scores. Higher scores indicate a higher likelihood of harboring a putative T cell epitope. [Figure 5-1] Figure 1 shows T cell proliferation in an EpiScreen™ DC:T cell immunogenicity assay. PSMA.350 and the positive control KLH were tested. For each donor data point, each bar represents, from left to right, days 9, 10, 11, and 12. [Figure 5-2] Figure 1 shows T cell proliferation in an EpiScreen™ DC:T cell immunogenicity assay. PSMA.350 and the positive control KLH were tested. For each donor data point, each bar represents, from left to right, days 9, 10, 11, and 12. [Figure 6A] Figure 1 shows PTE score assessment using internal PTE algorithm v22 for anti-CD3 pool 2 antibodies. [Figure 6B] The percent of antibody remaining after the thermostability assay is shown. [Figure 7A] 1 shows an alignment of the RSR-2295 and RSR-3213 amino acid sequences and the proteases capable of cleaving them. [Figure 7B] 1 shows in vitro protease digestion of paTCE with RSR-2295 or RSR-3213. The RSR-3213 sequence has been modified to substantially reduce cleavage by legumain. [Figure 8A] Figure 8 shows the relative plasma stability of paTCE using RSR-2295 or RSR-3213 measured on days 0 and 7. In Figure 8A, RSR-2295 used the SCy5.5 fluorophore, and RSR-3213 used the SCy7.5 fluorophore. In Figure 8B, RSR-2295 used the SCy7.5 fluorophore, and RSR-3213 used the SCy5.5 fluorophore. [Figure 8B]Figure 8 shows the relative plasma stability of paTCE using RSR-2295 or RSR-3213 measured on days 0 and 7. In Figure 8A, RSR-2295 used the SCy5.5 fluorophore, and RSR-3213 used the SCy7.5 fluorophore. In Figure 8B, RSR-2295 used the SCy7.5 fluorophore, and RSR-3213 used the SCy5.5 fluorophore. [Figure 8C] The figures show the in vivo cleavage activity observed from tumor homogenates from three different mouse tumor models. For each set of bar graphs (i.e., 1x-C%, 1x-N%, uTCE%), each bar represents, from left to right, B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent four different mice from the first tumor model (NCI-N87). A1-A4 represent four different mice from the second tumor model (HT-29). 43-1-43-4 represent four different mice from the third tumor model (HT-55). [Figure 8D] The % of the sum of the three metabolites plus paTCE (paTCE, 1x-N, 1x-C, and uTCE) is shown when using RSR-2295 or RSR-3213. [Figure 9] Figure 1 shows the relative tumor uptake of paTCE with RSR-2295 or RSR-3213. Plasma:tumor ratios were calculated in three different mouse tumor models (four mice per tumor model). There is a "Mouse 1" for each of the three different tumor models, a "Mouse 2" for each of the three different tumor models, a "Mouse 3" for each of the three different tumor models, and a "Mouse 4" for each of the three different tumor models. [Figure 10] This graph shows the binding activity of AC3092 (AMX-500-P1) and AC3896 (AMX-500-P4, also referred to herein as simply AMX-500) to human PSMA or cyno PSMA on PSMA-transfected CHO cells. Surface binding was detected with a labeled secondary antibody specific for anti-CD3 scFv. [Figure 11A]Dose-response curves of relative in vitro cytotoxicity of LNCaP PSMAhigh cells (FIG. 11A) and 22Rv1 PSMAlow cells (FIG. 11B) are shown. [Figure 11B] Dose-response curves of relative in vitro cytotoxicity of LNCaP PSMAhigh cells (FIG. 11A) and 22Rv1 PSMAlow cells (FIG. 11B) are shown. [Figure 12A] Dose-response curves for the relative in vitro cytotoxicity of LNCaP PSMAhigh cells (FIGS. 12A and 12B) and 22Rv1 PSMAlow cells (FIG. 12C) using three different donor human PBMC samples are shown. [Figure 12B] Dose-response curves for the relative in vitro cytotoxicity of LNCaP PSMAhigh cells (FIGS. 12A and 12B) and 22Rv1 PSMAlow cells (FIG. 12C) using three different donor human PBMC samples are shown. [Figure 12C] Dose-response curves for the relative in vitro cytotoxicity of LNCaP PSMAhigh cells (FIGS. 12A and 12B) and 22Rv1 PSMAlow cells (FIG. 12C) using three different donor human PBMC samples are shown. [Figure 13A] Figure 13A shows graphs of the relative in vitro cytotoxicity of LNCaP PSMAhigh cells from donor 1 incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Figure 13A shows assay results from donor 1. Figure 13B shows similar results from donors 2-5. [Figure 13B] Figure 13A shows graphs of the relative in vitro cytotoxicity of LNCaP PSMAhigh cells from donor 1 incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Figure 13A shows assay results from donor 1. Figure 13B shows similar results from donors 2-5. [Figure 14A]Figure 14A shows graphs of the relative in vitro cytotoxicity of 22Rv1 PSMAlow cells from donor 1 incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Figure 14A shows the assay results from donor 1. Figure 14B shows similar results from donors 2 and 3. [Figure 14B] Figure 14A shows graphs of the relative in vitro cytotoxicity of 22Rv1 PSMAlow cells from donor 1 incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Figure 14A shows the assay results from donor 1. Figure 14B shows similar results from donors 2 and 3. [Figure 15-1] This graph shows in vitro cytokine release from LNCaP PSMAhigh cells incubated with various concentrations of AMX-500 (uTCE), AMX-500, AMX-500 (1x-N), AMX-500 (1x-C), and AMX-500 (NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMC to LNCaP cells. The levels of cytokines INF-γ, TNF-α, IL-6, IL-10, GM-CSF, IL-1β, IL-2, IL-4, and MCP-1 are shown. [Figure 15-2] This graph shows in vitro cytokine release from LNCaP PSMAhigh cells incubated with various concentrations of AMX-500 (uTCE), AMX-500, AMX-500 (1x-N), AMX-500 (1x-C), and AMX-500 (NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMC to LNCaP cells. The levels of cytokines INF-γ, TNF-α, IL-6, IL-10, GM-CSF, IL-1β, IL-2, IL-4, and MCP-1 are shown. [Figure 15-3]This graph shows in vitro cytokine release from LNCaP PSMAhigh cells incubated with various concentrations of AMX-500 (uTCE), AMX-500, AMX-500 (1x-N), AMX-500 (1x-C), and AMX-500 (NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMC to LNCaP cells. The levels of cytokines INF-γ, TNF-α, IL-6, IL-10, GM-CSF, IL-1β, IL-2, IL-4, and MCP-1 are shown. [Figure 15-4] This graph shows in vitro cytokine release from LNCaP PSMAhigh cells incubated with various concentrations of AMX-500 (uTCE), AMX-500, AMX-500 (1x-N), AMX-500 (1x-C), and AMX-500 (NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMC to LNCaP cells. The levels of cytokines INF-γ, TNF-α, IL-6, IL-10, GM-CSF, IL-1β, IL-2, IL-4, and MCP-1 are shown. [Figure 15-5] This graph shows in vitro cytokine release from LNCaP PSMAhigh cells incubated with various concentrations of AMX-500 (uTCE), AMX-500, AMX-500 (1x-N), AMX-500 (1x-C), and AMX-500 (NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMC to LNCaP cells. The levels of cytokines INF-γ, TNF-α, IL-6, IL-10, GM-CSF, IL-1β, IL-2, IL-4, and MCP-1 are shown. [Figure 16] This figure shows graphs of CD69, CD25, and PD-1 expression on CD4+ T cells from LNCaP PSMAhigh / PBMC cocultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMCs to LNCaP cells. PBMCs were collected from donor 1. [Figure 17] This figure shows graphs of CD69, CD25, and PD-1 expression on CD8+ T cells from LNCaP PSMAhigh / PBMC cocultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMCs to LNCaP cells. PBMCs were collected from donor 1. [Figure 18-1] This figure shows graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC cocultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMCs to LNCaP cells. PBMCs were collected from donor 2. [Figure 18-2] This figure shows graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC cocultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMCs to LNCaP cells. PBMCs were collected from donor 2. [Figure 19-1] This figure shows graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC cocultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMCs to LNCaP cells. PBMCs were collected from donor 3. [Figure 19-2]This figure shows graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC cocultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). Cells were co-incubated with PBMCs at a 10:1 ratio of PBMCs to LNCaP cells. PBMCs were collected from donor 3. [Figure 20A] The relative target binding of AMX-500 (FIG. 20A) and AMX-500-P7 (AC3934, FIG. 20B) to approximately 6,000 different HEK293T membrane proteins is shown. [Figure 20B] The relative target binding of AMX-500 (FIG. 20A) and AMX-500-P7 (AC3934, FIG. 20B) to approximately 6,000 different HEK293T membrane proteins is shown. [Figure 21] This figure shows a graph of tumor volume from human prostate tumor mouse models. Tumor mouse models were generated using 22Rv1 PSMA-low cells, LNCaP PSMA-high cells, or C4-2 PSMA-high cells. For the 22Rv1 model, AMX-500paTCE was administered at 2 mg / kg, 16 nmol / kg, and AMX-500 unmasked TCE (uTCE) was administered at 0.35 mg / kg, 7.6 nmol / kg. For the LNCaP model, AMX-500paTCE was administered at 3 mg / kg, 24 nmol / kg, and AMX-500uTCE was administered at 0.35 mg / kg, 7.6 nmol / kg. For the C4-2 model, Dose A was 7.5 mg / kg, 59 nmol / kg, BIW, and Dose B was 3.5 mg / kg, 27 nmol / kg, BIW. [Figure 22] 1 shows a graph of tumor volume from a LNCaP PSMAhigh cell human prostate tumor mouse model. [Figure 23] 1 shows a graph of tumor volume from a human prostate tumor mouse model of 22Rv1 PSMAlow cells. [Figure 24]1 shows a graph of tumor volume from a mouse model of human prostate tumor administered AMX-500, the anti-PD-1 antibody pembrolizumab, or a combination of AMX-500 and pembrolizumab. [Figure 25] 1 shows the tissue distribution of AMX-500 in a mouse tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0307] There is a significant unmet need in cancer treatment for PSMA-targeted bispecific therapeutic modalities that are effective against solid tumors, particularly those residing in immunologically cold microenvironments. While TCEs have been shown to be effective in inducing remission in certain cancers, their extreme potency and on-target, off-tumor toxicity in healthy tissues have prevented their widespread development as therapeutics.

[0308] Without being bound by any scientific theory, TCE forms a bridge between T cells and tumor cells, activating T cell-mediated tumor cell killing and initiating a cytokine amplification cascade. The cytokine amplification cascade can promote further tumor cell killing and potentially provide long-term immunity. T cells activated by TCE release soluble perforin / granzymes in a manner independent of antigen-MHC recognition. This creates a two-fold response: direct tumor cell death and amplification of tumor killing through the initiation of a potent cytokine response from tumor cells. Direct tumor cell death results in the release of tumor antigens. The cytokine response can include, among others, an increase in interferon-g, which stimulates CD8 T cell activity and antigen presentation by APCs; an increase in IL2, which leads to increased proliferation of activated T cells; and an increase in CXCL9 and 10 responses, which increase T cell recruitment. Together, the release of tumor antigens and the initiation of a cytokine response result in the activation of endogenous T cell responses, which potentially leads to epitope spreading and induces long-term immunity.

[0309] One toxicity challenge with TCEs arises from the fact that many tumor targets are also expressed to some degree in healthy tissues, and normal cells can also generate cytokine responses resulting in cytokine release syndrome (CRS). These two potent responses of healthy tissues to T cell activation by TCEs often result in an overall lack of an acceptable therapeutic index for these agents.

[0310] The present disclosure addresses an unmet need and provides protease-activatable TCEs (paTCEs) that are superior in one or more aspects compared to conventional antibody or bispecific therapeutics that are active upon injection, including improved terminal half-life, targeted delivery, and / or improved therapeutic ratio with reduced toxicity to healthy tissue.

[0311] Included herein are compounds, compositions, and methods that overcome the shortcomings in existing TCEs by providing a paTCE that targets PSMA (referred to herein as PSMA-paTCE and exemplified as AMX-500).

[0312] AMX-500 comprises the amino acid sequence set forth as SEQ ID NO: 1000. Without being bound by any scientific theory, it is understood that the paTCE described herein exploits the dysregulated protease activity present in tumors versus healthy tissue, allowing for an expanded therapeutic index. The paTCE core comprises antigen-binding domains, one of which targets CD3 and the other targets PSMA. The two antigen-binding domains, in exemplary embodiments, can be two different antibody formats (e.g., single chain antibody fragment (scFv) and VHH, etc.) or the same antibody format (e.g., scFv, etc.). Many different antibody fragments or formats can be used.

[0313] In some embodiments, the PSMA-targeting paTCE comprises a first portion that is a VHH that binds PSMA and a second portion that is an scFv that binds CD3. One or more (e.g., two) unstructured polypeptide masks are attached to the core. In some embodiments, these unstructured polypeptide masks sterically reduce target engagement of either the tumor target and / or CD3 and also extend protein half-life. In some embodiments, the unstructured polypeptide mask is an extended length non-natural polypeptide (ELNN).

[0314] In some embodiments, the properties of ELNNs also minimize immunogenicity potential, as the lack of a stable tertiary structure is unfavorable to antibody binding, and the absence of hydrophobic, aromatic, positively charged residues that serve as anchor residues for peptide MHC II binding reduces T cell epitope potential.

[0315] In some embodiments, the base of the ELNN or the protease cleavage site of the ELNN allows proteolytic activation of paTCE in the tumor microenvironment, unleashing a smaller, highly potent TCE that can redirect cytotoxic T cells to kill target-expressing tumor cells. In some embodiments, in healthy tissues where protease activity is tightly regulated, paTCE remains largely inactive, thus extending its therapeutic index compared to unmasked TCE.

[0316] In some embodiments, the short half-life of the unmasked TCE form, in addition to localized activation, further broadens the therapeutic index while providing the potency of T cell immunity to improve solid tumor eradication. In some embodiments, the release moiety used in paTCE can be cleaved across a wide range of tumors by proteases collectively involved in all cancer hallmarks (growth; survival and mortality; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, by taking advantage of enhanced protease activity that is upregulated in all stages of cancer and tumor development but tightly regulated in healthy tissues, the TCE activity of paTCE is localized to the tumor.

[0317] term As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0318] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof, unless the context clearly dictates otherwise.

[0319] Furthermore, as used herein, "and / or" is to be interpreted as a specific disclosure of each of two specified features or components with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: "A, B, and C," "A, B, or C," "A or C," "A or B," "B or C," "A and C," "A and B," "B and C," "A" (alone), "B" (alone), and "C" (alone).

[0320] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0321] Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0322] The term "about" is used herein to mean approximately, roughly, around, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above or below (higher or lower) the stated value, for example, by a variance of 10 percent above or below. In some embodiments, the term indicates a deviation from the stated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the stated numerical value. In some embodiments, "about" indicates a ±4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" refers to a deviation of ±0.01% from the indicated numerical value.

[0323] With respect to a naturally occurring compound, the term "isolated" refers to a compound (i.e., a polypeptide or polynucleotide) that is not in its natural state (e.g., free from, to varying degrees including, components that naturally accompany the compound in nature). No particular level of purification is required. For example, an isolated polypeptide can be simply removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this disclosure, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. "Isolate" and "isolated" can also refer to the degree of separation from the original source or surroundings, depending on the context.

[0324] The term "polypeptide" refers to any polymer of two or more amino acids. Thus, the terms peptide, dipeptide, tripeptide, oligopeptide, protein, amino acid chain, or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide." The term "polypeptide" also encompasses amino acid polymers that have been modified by any other manipulation (e.g., post-translational modification), such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. Depending on the context, the term "polypeptide" can also be used to refer to a protein comprising two or more polymers of two or more amino acids.

[0325] A "host cell" includes an individual cell (e.g., in culture) that contains an exogenous polynucleotide. A host cell may include the progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genome of total DNA complement) to the original parent cell due to naturally occurring or genetically engineered mutations.

[0326] A "fusion" or "chimeric" polypeptide or protein comprises a first polypeptide portion linked to a second polypeptide portion that is not naturally linked in nature. In some embodiments, these portions may normally be present in separate proteins and are combined in the fusion polypeptide, or they may normally be present in the same protein but are arranged in a new arrangement in the fusion polypeptide, or the portions may be combined from different sources. In some embodiments, a fusion or chimeric protein comprises two or more portions that do not exist in nature (e.g., human-created, designed, or generated, such as binding domains, masks, linkers, barcodes, and other polypeptides provided herein). Chimeric proteins can be created, for example, by chemical synthesis or by recombinant expression (e.g., creating and translating a polynucleotide in which peptide regions are encoded in the desired relationship).

[0327] "Conjugated," "linked," "fused," and "fusion" may be used interchangeably herein, depending on the context. These terms may refer to the covalent joining of two or more chemical (e.g., polypeptide) elements or components by any means, including chemical conjugation or recombinant means.

[0328] As is known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. Similarly, "sequence identity" between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide with the sequence of a second polynucleotide. The terms "% identical," "% identity," or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids (where applicable) that are identical in optimal alignment between the compared sequences. The percentage may be purely statistical, in which the differences between the two sequences may, but need not, be randomly distributed over the entire length of the compared sequences. Comparison of two sequences is usually performed by comparing the sequences over a segment or "window of comparison" after optimal alignment to identify local regions of corresponding sequences. For example, optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the search for identity algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available at the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) gap costs set to linear; and (vi) a filter for low-complexity regions is used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) expectation threshold set to 10; (ii) word size set to 3; (iii) maximum match within query range set to 0; (iv) matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) conditional composition score matrix adjustment. As discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the percentage of identity is, of course, calculated over the entire length of the reference polypeptide sequence, with parameters set to allow gaps in homology of up to 5% of the total number of amino acids in the reference sequence.

[0329] As used herein, the terms "mask polypeptide," "mask," and "masking moiety," in the context of the fusion proteins (such as chimeric polypeptides) provided herein, refer to a polypeptide that is capable of reducing binding of an antigen-binding domain (e.g., an antibody) to a target antigen. Exemplary mask polypeptides include, but are not limited to, the ELNN polypeptides described herein. Additional mask polypeptides include albumin, polypeptides consisting of proline, serine, and alanine, coiled-coil domains, albumin-binding domains, Fc domains, and binding domains with specificity for conserved regions of antibody variable domains. Mask polypeptides are described in further detail in Lucchi et al. (ACS Cent Sci. 2021 May 26;7(5):724-738).

[0330] As used herein, the terms "ELNN polypeptide" and "ELNN" are synonymous and refer to an extended-length polypeptide comprising a non-naturally occurring, substantially non-repetitive sequence (e.g., a polypeptide motif) composed primarily of small, hydrophilic amino acids, which has little or no secondary or tertiary structure under physiological conditions. ELNN polypeptides include unstructured hydrophilic polypeptides comprising a repeating motif of the six naturally occurring amino acids (G, A, P, E, S, and / or T). In some embodiments, ELNN polypeptides comprise multiple motifs of the six naturally occurring amino acids (G, A, P, E, S, T), where the motifs comprise a combination of the same or different motifs. In some embodiments, ELNN polypeptides, when linked to proteins, including T cell engagers disclosed herein, can confer certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties. Such desirable properties can include, but are not limited to, improved pharmacokinetic parameters and solubility characteristics, as well as an improved therapeutic index. ELNN polypeptides are known in the art, and non-limiting descriptions of and examples of ELNN polypeptides known as XTEN® polypeptides are available in Schellenberger et al., (2009) Nat Biotechnol 27(12):1186-90, Brandl et al., (2020) Journal of Controlled Release 327:186-197, and Radon et al., (2021) Advanced Functional Materials 31, 2101633 (pages 1-33), the entire contents of each of which are incorporated herein by reference.

[0331] In some embodiments, the repetitiveness of an ELNN sequence refers to its trimeric repetitiveness, which can be measured by a computer program or algorithm or other means known in the art. In some embodiments, the trimeric repetitiveness of an ELNN can be assessed by determining the number of occurrences of overlapping trimeric sequences within a polypeptide. For example, a 200-amino acid residue polypeptide has 198 overlapping three-amino acid sequences (trimers), with the number of unique trimeric sequences depending on the amount of repetition within the sequence. In some embodiments, a score (hereinafter, "subsequence score") can be generated that reflects the degree of trimeric repetitiveness within the entire polypeptide sequence. In this context, "subsequence score" refers to the sum of the occurrences of each unique trimeric frame across the 200 consecutive amino acid sequence of the polypeptide, divided by the absolute number of unique trimeric subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repetitive and non-repetitive polypeptides are provided in Example 73 of International Patent Application Publication No. 2010 / 091122(A1), which is incorporated by reference in its entirety.

[0332] In some embodiments, in the context of ELNN, a "substantially non-repetitive sequence" refers to an ELNN sequence in which (1) there are few or no instances of four identical amino acids in the string of ELNN sequences, and (2) the ELNN has a subsequence score (as defined in the preceding paragraphs herein) of 12 or 10 or less, or there is no pattern in the N- to C-terminal ordering of sequence motifs that make up the polypeptide sequence.

[0333] A "vector" is a nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. In some embodiments, a vector is autonomously replicating in a suitable host. The term includes vectors that function primarily for the insertion of DNA or RNA into a cell, replicating vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be used to transcribe mRNA that is translated into a polypeptide. In some embodiments, an "expression system" is a suitable host cell containing an expression vector that can function to produce a desired expression product. The terms "treatment" or "treating" and "ameliorating" may be used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit. "Therapeutic benefit" refers to eradication or amelioration of the underlying disorder being treated. In some embodiments, the therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disease state, such that improvement is observed in a subject, even though the subject may still be afflicted with the underlying disorder. In some embodiments, the therapeutic benefit comprises slowing or halting the growth of one or more tumors. In some embodiments, the therapeutic benefit comprises reducing the size of one or more tumors. In some embodiments, the therapeutic benefit comprises eradicating one or more tumors from a subject. In some embodiments, the therapeutic benefit comprises causing the death of cancer cells.

[0334] As used herein, the term "therapeutically effective amount" refers to an amount of a biologically active agent (such as, for example, a fusion protein provided herein as part of a pharmaceutical composition) that, when administered to a subject in one or multiple doses, is capable of having any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition. Such an effect need not be absolute to be beneficial. A disease state can refer to a disorder or disease, for example, cancer or a symptom of cancer.

[0335] Antigen-binding domains, cleavage sequences, barcode fragments, and fusion polypeptides The present disclosure provides, inter alia, new and useful anti-PSMA antibodies, new and useful anti-CD3 antibodies, cleavage sequences, barcode fragments, and fusion proteins comprising them. Included herein is a fusion polypeptide comprising (i) one or more mask polypeptides (such as ELNNs), (ii) a bispecific antibody (BsAb, e.g., TCE) linked to the mask polypeptide, and (iii) one or more protease-cleavable release segments (RSs), wherein the RSs are positioned between the mask polypeptides and the BsAbs.

[0336] In some embodiments, the anti-PSMA antibodies provided herein have the sequence: It comprises a VHH domain comprising the CDRs of a VHH domain comprising QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (sequence number 549).

[0337] In some embodiments, the anti-CD3 antibodies provided herein have the sequence: A VH domain comprising the CDRs of a VH domain comprising EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311) and / or sequences: It comprises a VL domain comprising the CDRs of the VL domain comprising ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0338] Also provided are BsAbs, including, for example, anti-PSMA antibodies and / or anti-CD3 antibodies disclosed herein. In some embodiments, a bispecific antibody comprises an anti-PSMA VHH region disclosed herein. In some embodiments, a BsAb comprises the VH and VL regions of an anti-CD3 antibody disclosed herein. In some embodiments, a BsAb comprises an anti-PSMA VHH region described herein and an anti-CD3 scFV comprising a VH and VL pair disclosed herein. In some embodiments, the BsAb is a TCE.

[0339] In some embodiments, the fusion polypeptide comprises a first ELNN (such as an ELNN described herein). In some embodiments, the polypeptide further comprises a second ELNN (such as an ELNN described herein). In some embodiments, the polypeptide comprises an ELNN at or near its N-terminus (an "N-terminal ELNN"). In some embodiments, the polypeptide comprises an ELNN at or near its C-terminus (a "C-terminal ELNN"). In some embodiments, the polypeptide comprises both an N-terminal ELNN and a C-terminal ELNN.

[0340] In some embodiments, the fusion polypeptide comprises a BsAb, wherein a first ELNN is attached to the N-terminus of the BsAb by a first RS, and a second ELNN is attached to the C-terminus of the BsAb by a second RS. In some embodiments, each RS is cleavable by a protease as described herein. In some embodiments, each RS comprises an RS sequence as disclosed herein. In some embodiments, the fusion polypeptide is paTCE.

[0341] Included herein are polypeptide sequences that can be used, for example, to link one polypeptide moiety to another within a fusion protein. For example, useful linkers are provided that are cleaved by multiple proteases but not by legumain. In some embodiments, such linkers can be used outside the context of antibodies, such as those described herein.

[0342] In some embodiments, a fusion polypeptide (e.g., one or more ELNNs of paTCE, and / or another portion of the fusion polypeptide, such as a linker or spacer sequence) can include one or more barcode fragments (e.g., as described herein) that are capable of releasing (e.g., configured to be released) the fusion polypeptide upon cleavage or digestion of the fusion polypeptide (e.g., paTCE) by a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, each barcode fragment differs in sequence and molecular weight from all other peptide fragments (including all other barcode fragments, if present) that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease, thereby making it unique and its presence detectable through techniques such as mass spectrometry.

[0343] Extended recombinant polypeptide (ELNN) Chain length and amino acid composition In some embodiments, the ELNN comprises at least 100 or at least 150 amino acids. In some embodiments, the ELNN is 100 to 3,000 or 150 to 3,000 amino acids in length. In some embodiments, the ELNN is 100 to 1,000 or 150 to 1,000 amino acids in length. In some embodiments, the ELNN is at least (about) 100, at least (about) 150, at least (about) 200, at least (about) 250, at least (about) 300, at least (about) 350, at least (about) 400, at least (about) 450, at least (about) 500, at least (about) 550, at least (about) 600, at least (about) 650, at least (about) 700, at least (about) 750, at least (about) 800, at least at least (about) 850, at least (about) 900, at least (about) 950, at least (about) 1,000, at least (about) 1,100, at least (about) 1,200, at least (about) 1,300, at least (about) 1,400, at least (about) 1,500, at least (about) 1,600, at least (about) 1,700, at least (about) 1,800, at least (about) 1,900, or at least (about) 2,000 amino acids in length. In some embodiments, the ELNNs are at most (about) 100, at most (about) 150, at most (about) 200, at most (about) 250, at most (about) 300, at most (about) 350, at most (about) 400, at most (about) 450, at most (about) 500, at most (about) 550, at most (about) 600, at most (about) 650, at most (about) 700, at most (about) 750, at most (about) 800, at most (about) at most (about) 850, at most (about) 900, at most (about) 950, at most (about) 1,000, at most (about) 1,100, at most (about) 1,200, at most (about) 1,300, at most (about) 1,400, at most (about) 1,500, at most (about) 1,600, at most (about) 1,700, at most (about) 1,800, at most (about) 1,900, or at most (about) 2,000 amino acids in length.In some embodiments, the ELNNs are (about) 100, (about) 150, (about) 200, (about) 250, (about) 300, (about) 350, (about) 400, (about) 450, (about) 500, (about) 550, (about) 600, (about) 650, (about) 700, (about) 750, (about) 800, (about) 850, (about) 900, (about) In some embodiments, the ELNN has a length of 950, (about) 1,000, (about) 1,100, (about) 1,200, (about) 1,300, (about) 1,400, (about) 1,500, (about) 1,600, (about) 1,700, (about) 1,800, (about) 1,900, or (about) 2,000 amino acids, or a range between any two of the foregoing. In some embodiments, at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in an ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, an ELNN comprises at least three different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN comprises at least four different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN comprises at least five different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN consists of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN comprises the amino acids G, A, S, T, E, and P. In some embodiments, an ELNN (e.g., ELNN1, ELNN2, etc.) is characterized in that it (i) contains at least 100 or at least 150 amino acids, (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P), and (iii) contains at least four different types of amino acids from G, A, S, T, E, or P.As used herein, the term "glutamate" is synonymous with "glutamic acid" and refers to a glutamic acid residue, whether or not the side chain carboxyl is deprotonated. In some embodiments, an ELNN-containing fusion polypeptide comprises a first ELNN and a second ELNN. In some embodiments, the sum of the total number of amino acids of the first ELNN and the total number of amino acids of the second ELNN is at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids.

[0344] Non-overlapping sequence motifs In some embodiments, an ELNN comprises or is formed from multiple non-overlapping sequence motifs. In some embodiments, at least one of the non-overlapping sequence motifs is repetitive (or is repeated at least twice in the ELNN). In some embodiments, the ELNN comprises at least one other non-overlapping sequence motif that is non-repetitive (or is found only once in the ELNN). In some embodiments, the multiple non-overlapping sequence motifs comprise (a) a set of (repetitive) non-overlapping sequence motifs, where each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least twice in the ELNN, and (b) a non-overlapping (non-repetitive) sequence motif that occurs (or is found) only once in the ELNN. In some embodiments, each non-overlapping sequence motif is 9-14 (or 10-14, or 11-13) amino acids in length. In some embodiments, each non-overlapping sequence motif is 12 amino acids in length. In some embodiments, the plurality of non-overlapping sequence motifs comprises a set of non-overlapping (repeated) sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs (1) is repeated at least twice in the ELNN and (2) is 9 to 14 amino acids in length. In some embodiments, the set of (repeated) non-overlapping sequence motifs comprises the 12-mer sequence motifs identified herein by SEQ ID NOs: 179-200 and 1715-1722 in Table 1. In some embodiments, the set of (repeated) non-overlapping sequence motifs comprises the 12-mer sequence motifs identified herein by SEQ ID NOs: 186-189 in Table 1. In some embodiments, the set of (repeated) non-overlapping sequence motifs comprises at least two, at least three, or all four of the 12-mer sequence motifs of SEQ ID NOs: 186-189 in Table 1. In some embodiments, the ELNN further comprises sequences other than the 12-mer sequence motifs shown in Table 1. In some embodiments, the ELNN comprises a sequence not found in Table 1, such as ASSATPESGP, GSGPGTSESATP, or GTSESATP. In some embodiments, the ELNN comprises a sequence not found in Table 1, such as ATPESGP, GTSPSATPESGP, or GTSESAGEPEA. In some embodiments, the ELNN comprises a barcode sequence.

[0345] [Table 1] * indicates individual motif sequences that, when used together in various permutations, give rise to a "family of sequences"

[0346] Identification of unstructured polypeptides In various embodiments, the ELNN component (or components) of the fusion protein have an unstructured conformation under physiological conditions, regardless of the length of the polymer (e.g., extended length). For example, ELNNs are characterized by a large conformational freedom of the peptide backbone. In some embodiments, ELNNs are characterized by a lack of long-range interactions as determined by NMR. In some embodiments, the present disclosure provides ELNNs that resemble the structure of a denatured sequence that is largely devoid of secondary structure under physiological conditions. In some embodiments, an ELNN may be substantially devoid of secondary structure under physiological conditions. As used in this context, "substantially devoid" means that less than 50% of the ELNN amino acid residues of the ELNN contribute to secondary structure, as measured or determined by the means described herein. As used in this context, "substantially devoid" means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the ELNN amino acid residues of the ELNN sequence do not contribute to secondary structure, as measured or determined by the means described herein.

[0347] A variety of methods have been established in the art for identifying the presence or absence of secondary and tertiary structure in a given polypeptide. In some embodiments, the secondary structure of ELNNs can be measured spectrophotometrically, for example, by circular dichroism spectroscopy in the "far-ultraviolet" spectral region (190-250 nm). Secondary structural elements such as alpha-helices and beta-sheets each give rise to characteristic shapes and sizes in the CD spectrum. Secondary structure can also be predicted for polypeptide sequences via certain computer programs or well-known algorithms such as the Chou-Fasman algorithm (Chou, PY, et al. (1974) Biochemistry, 13:222-45) and the Garnier-Osguthorpe-Robson ("GOR") algorithm (Garnier J, Gibrat JF, Robson B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553, as described in U.S. Patent Application Publication No. 20030228309(A1), the entire contents of which are incorporated herein by reference). For a given sequence, the algorithm can predict the presence of some or no secondary structure, expressed, for example, as the total and / or percentage of residues in the sequence that form alpha-helices or beta-sheets, or the percentage of residues in the sequence that are predicted to result in random coil formation (lacking secondary structure).

[0348] In some embodiments, the ELNN used in the fusion protein composition may have an alpha-helix percentage ranging from 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition may have a beta-sheet percentage ranging from 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition may have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition may have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. In some embodiments, the ELNN of the fusion protein composition may have a high degree of random coil percentage, as determined by the GOR algorithm. In some embodiments, ELNNs may have at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil as determined by the GOR algorithm.

[0349] Net Charge In some embodiments, ELNN polypeptides can have unstructured characteristics imparted by incorporating amino acid residues with a net charge and / or reducing the proportion of hydrophobic amino acids in the ELNN sequence. The overall net charge and net charge density can be controlled, for example, by modifying the content of charged amino acids in the ELNN. In some embodiments, the net charge density of the ELNNs of the composition can be greater than +0.1 or less than -0.1 charges / residue. In some embodiments, the net charge of the ELNNs can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more.

[0350] Because most tissues and surfaces in humans or animals have a net negative charge, ELNNs can optionally be designed to have a net negative charge to minimize nonspecific interactions between ELNN-containing compositions and various surfaces, such as blood vessels, healthy tissues, or various receptors. Without being bound by any particular theory, ELNNs individually carry a high net negative charge and may adopt an open conformation due to electrostatic repulsion between the individual amino acids of the ELNN polypeptide distributed throughout the sequence of the ELNN polypeptide. Such distribution of net negative charges over the extended sequence length of the ELNN may result in an unstructured conformation, which in turn may result in an effective increase in the hydrodynamic radius. Thus, in some embodiments, the ELNN contains glutamine such that glutamine constitutes about 8, 10, 15, 20, 25, or even about 30% of the amino acids in the sequence. The ELNNs of the compositions of the present disclosure generally have no or a low content of positively charged amino acids. In some embodiments, ELNNs may have less than about 10% positively charged amino acid residues, or less than about 7%, or less than about 5%, or less than about 2% positively charged amino acid residues. However, the present disclosure contemplates polypeptides in which a limited number of positively charged amino acids, such as lysine, may be incorporated into the ELNN to allow, for example, conjugation between the epsilon amine of lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule conjugated to the ELNN backbone.

[0351] In some embodiments, ELNNs can contain charged residues separated by other residues, such as serine or glycine, which can result in better expression or purification behavior. Based on net charge, ELNNs of the subject compositions can have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In some embodiments, ELNNs have an isoelectric point between 1.5 and 4.5. In some embodiments, ELNNs incorporated into paTCE fusion proteins carry a net negative charge under physiological conditions, contributing to an unstructured conformation and reduced binding of the ELNN component to mammalian proteins and tissues.

[0352] Because hydrophobic amino acids can impart structure to a polypeptide, in some embodiments, the hydrophobic amino acid content of an ELNN is less than 5%, or less than 2%, or less than 1%. In some embodiments, an ELNN has no hydrophobic amino acids. In some embodiments, the methionine and tryptophan amino acid content of the ELNN component of a paTCE fusion protein is less than 5%, or less than 2%, and most preferably less than 1%. In some embodiments, an ELNN has a sequence with less than 10% positively charged amino acid residues, or less than about 7%, or less than about 5%, or less than about 2% positively charged amino acid residues, the sum of methionine and tryptophan residues is less than 2% of the total ELNN sequence, and the sum of asparagine and glutamine residues is less than 10% of the total ELNN sequence. In some embodiments, an ELNN has no methionine or tryptophan residues.

[0353] Increased hydrodynamic radius In some embodiments, ELNNs can have a high hydrodynamic radius, conferring a corresponding increased apparent molecular weight to paTCE fusion proteins incorporating the ELNN. Linking an ELNN to a BsAb (e.g., TCE) sequence can result in a paTCE composition that can have an increased hydrodynamic radius, increased apparent molecular weight, and increased apparent molecular weight factor compared to a BsAb (e.g., TCE) that is not linked to an ELNN. For example, in some therapeutic applications where extended half-life is desirable, one or more ELNNs with a high hydrodynamic radius can be incorporated into a fusion protein containing a BsAb (e.g., TCE), effectively expanding the hydrodynamic radius of the fusion protein beyond the glomerular pore size of approximately 3-5 nm (corresponding to an apparent molecular weight of approximately 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv. Drug Deliv. Rev. 55:1261-1277), resulting in reduced renal clearance of circulating proteins. In some embodiments, the hydrodynamic radius of a protein is determined by its molecular weight and by its structure, including shape and compactness. Without being bound by any particular theory, ELNNs may adopt an open conformation due to electrostatic repulsion between individual charges in the peptide or inherent flexibility conferred by certain amino acids in the sequence that lack the potential to confer secondary structure. In some embodiments, the open, extended, unstructured conformation of an ELNN polypeptide has a proportionally larger hydrodynamic radius compared to a polypeptide of comparable sequence length and / or molecular weight that has secondary structure and / or tertiary structure, such as a typical globular protein. Methods for determining hydrodynamic radius, such as by using size exclusion chromatography (SEC), as described in U.S. Patent Nos. 6,406,632 and 7,294,513, are well known in the art.In some embodiments, the addition of increasing ELNN length results in a proportional increase in the parameters of hydrodynamic radius, apparent molecular weight, and apparent molecular weight factor, allowing for tuning of the paTCE to a desired characteristic cutoff apparent molecular weight or hydrodynamic radius. Thus, in some embodiments, a paTCE fusion protein can be composed of ELNNs such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In some embodiments, the large hydrodynamic radius conferred by the ELNNs in the paTCE fusion protein can result in reduced renal clearance of the resulting fusion protein, resulting in a corresponding increase in terminal half-life, increased mean residence time, and / or decreased renal clearance rate.

[0354] In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of a selected length and sequence can be selectively incorporated into a paTCE to produce a fusion protein having an apparent molecular weight under physiological conditions of at least about 150 kDa, or at least about 300 kDa, or at least about 400 kDa, or at least about 500 kDa, or at least about 600 kDa, or at least about 700 kDa, or at least about 800 kDa, or at least about 900 kDa, or at least about 1000 kDa, or at least about 1200 kDa, or at least about 1500 kDa, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or more. In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of selected length and sequence can be selectively linked to a BsAb (e.g., TCE) to produce a paTCE fusion protein that has, under physiological conditions, an apparent molecular weight factor of at least 3, alternatively at least 4, alternatively at least 5, alternatively at least 6, alternatively at least 7, alternatively at least 8, alternatively at least 9, alternatively at least 10, alternatively at least 15, or an apparent molecular weight factor of at least 20 or more. In some embodiments, the paTCE fusion protein has, under physiological conditions, an apparent molecular weight factor that is about 4 to about 20, or about 6 to about 15, or about 8 to about 12, or about 9 to about 10 relative to the actual molecular weight of the fusion protein. In some embodiments, the fusion polypeptide exhibits an apparent molecular weight factor that is greater than about 6 under physiological conditions.

[0355] Increased terminal half-life In some embodiments, a fusion polypeptide comprising an ELNN (such as paTCE) has a terminal half-life that is at least 2-fold longer, or at least 3-fold longer, or at least 4-fold longer, or at least 5-fold longer than a corresponding biologically active polypeptide that is not linked to an ELNN. In some embodiments, the (fusion) polypeptide has a terminal half-life that is at least 2-fold longer than a biologically active polypeptide that is not linked to an ELNN.

[0356] In some embodiments, administering a therapeutically effective amount of a paTCE fusion protein to a subject in need thereof results in at least a two-fold, or at least a three-fold, or at least a four-fold, or at least a five-fold or greater increase in the time that the fusion protein remains within the therapeutic window compared to a corresponding BsAb (e.g., TCE) that is not linked to an ELNN when administered at an equivalent dose to a subject.

[0357] In some embodiments, the TCE released from paTCE upon protease cleavage comprises one or more short polypeptides (e.g., about 30, 25, 20, 15, 14, 13, 12, 11, 10, or less amino acids in length) that have no amino acids other than G, A, P, E, S, and / or T. For example, a short polypeptide that has no amino acids other than G, A, P, E, S, and / or T can be incorporated into one or more spacer or linker sequences of the TCE and / or into a portion of one or more spacers or linkers that remain part of the TCE after cleavage. In some embodiments, the TCE released from paTCE comprises GTSESATPES N-terminal to the TCE (e.g., the closest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid position of, or the sequence includes, the N-terminus). In some embodiments, the TCE released from the paTCE has a C-terminal sequence of GTATPESGPG (e.g., the nearest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid position of the N-terminal amino acid, or the sequence includes the N-terminus). In some embodiments, the TCE comprises an internal linker (e.g., between the VL and VH regions of an scFV) that includes a polypeptide sequence that contains no amino acids other than G, A, P, E, S, and / or T, such as SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0358] Low immunogenicity In some embodiments, the present disclosure provides compositions in which ELNNs have a low degree of immunogenicity or are substantially non-immunogenic. Several factors may contribute to the low immunogenicity of ELNNs, such as a substantially non-repetitive sequence, an unstructured conformation, a high degree of solubility, a low degree or lack of self-aggregation, a low degree or lack of proteolytic sites within the sequence, and a low degree or lack of epitopes in the ELNNs.

[0359] Those skilled in the art will generally understand that polypeptides having highly repetitive short amino acid sequences (e.g., a 200 amino acid long sequence contains a limited set of trimers or tetramers with an average of 20 or more repeats) and / or polypeptides having consecutive repeated amino acid residues (e.g., a pentameric or hexameric sequence with identical amino acid residues) have a tendency to aggregate or form higher order structures or form contacts resulting in crystalline or pseudocrystalline structures.

[0360] In some embodiments, the ELNN sequence is substantially non-repetitive such that (1) the ELNN sequence does not have three consecutive amino acids that are the same type of amino acid unless the amino acid is serine, in which case no more than three consecutive amino acids may be serine residues, and (2) the ELNN does not contain a three amino acid sequence (trimer) that occurs more than 16 times, more than 14 times, more than 12 times, or more than 10 times within a sequence of at least 200 amino acids in length of the ELNN (e.g., the entire span of the ELNN that is at least amino acids in length). Without being bound by any scientific theory, such substantially non-repetitive sequences have a reduced tendency to aggregate, thus allowing for the design of ELNNs of longer sequences with a relatively low frequency of charged amino acids that are more likely to aggregate when the sequence or amino acid residues are more repetitive.

[0361] Conformational epitopes can be formed by regions on the protein surface composed of multiple, noncontiguous amino acid sequences of a protein antigen. Without being bound by any scientific theory, precise protein folding places these sequences in well-defined, stable spatial configurations or epitopes that can be recognized as "foreign" by the host's humoral immune system, resulting in the production of antibodies against the protein and / or eliciting a cell-mediated immune response. In the latter case, an individual's immune response to a protein is largely influenced by T cell epitope recognition, which correlates with the peptide-binding specificity of that individual's HLA-DR allotype. Engagement of an MHC class II peptide complex by a cognate T cell receptor on the T cell surface, along with cross-linking of certain other coreceptors, such as CD4 molecules, can induce an activated state within the T cell. Activation can result in the release of cytokines, which can further activate other lymphocytes, such as B cells, to produce antibodies or activate T killer cells as part of a complete cellular immune response.

[0362] Without being bound by any scientific theory, the ability of a peptide to bind to a given MHC class II molecule for presentation on the surface of an antigen-presenting cell (APC) may depend on several factors, most notably its primary sequence. In some embodiments, a lower degree of immunogenicity can be achieved by engineering ELNNs to resist antibody processing in antigen-presenting cells and / or by selecting sequences that do not bind sufficiently to MHC receptors. In some embodiments, ELNN-containing fusion proteins have substantially non-repetitive ELNN polypeptides designed to reduce binding to MHC II receptors and to avoid the formation of epitopes for T cell receptor or antibody binding, resulting in a lower degree of immunogenicity. Without being bound by any scientific theory, the avoidance of immunogenicity is, in part, a direct result of the conformational flexibility of ELNNs, i.e., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular interest are sequences that have a low tendency to adopt a compactly folded conformation in aqueous solution or under physiological conditions that may give rise to conformational epitopes. Administration of fusion proteins containing ELNNs using conventional therapeutic practices and doses generally does not result in the formation of neutralizing antibodies to ELNNs and may reduce the immunogenicity of BsAb (e.g., TCE) fusion partners in paTCE compositions.

[0363] In some embodiments, the ELNNs utilized in the subject fusion proteins may be substantially free of epitopes recognized by human T cells. Eliminating such epitopes to generate less immunogenic proteins has been previously disclosed; see, for example, International Publication Nos. 98 / 52976, 02 / 079232, and 00 / 3317, which are incorporated herein by reference. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281:95-108). Of particular interest are peptide sequences that can oligomerize without generating T cell epitopes or non-human sequences. This can be achieved by testing direct repeats of these sequences for the presence of T cell epitopes and the occurrence of non-human 6- to 15-mer, and particularly 9-mer, sequences, and then modifying the design of the ELNN sequence to eliminate or destroy the epitope sequences. In some embodiments, ELNNs are rendered substantially non-immunogenic by limiting the number of epitopes of ELNN predicted to bind to MHC receptors. With a reduction in the number of epitopes capable of binding to MHC receptors, there is a concomitant reduction in the potential for T cell activation and T cell helper function, reduced B cell activation or upregulation, and reduced antibody production. The low level of predicted T cell epitopes can be determined, for example, by epitope prediction algorithms such as TEPITOPE (Sturniolo, T., et al. 1999) Nat Biotechnol, 17:555-61), as shown in Example 74 of International Patent Application Publication No. 2010 / 144502(A2), the entire contents of which are incorporated by reference. Aspects of the TEPITOPE score for a given peptide frame within a protein are disclosed in Sturniolo, T., et al. (1999) Nature Biotechnology 17:555. The score should be at least 20 log above, between about 10 and about -10 (10e 10 K D ~10e -10 K D(corresponding to the binding constraints of 10-15), and can be reduced by avoiding hydrophobic amino acids that can function as anchor residues during peptide display on MHC, such as M, I, L, V, or F. In some embodiments, the ELNN components incorporated into paTCE do not have predicted T cell epitopes with a TEPITOPE score of about -5 or higher, or -6 or higher, or -7 or higher, or -8 or higher, or a TEPITOPE score of -9 or higher. As used herein, a score of "-9 or higher" encompasses TEPITOPE scores between 10 and -9 (inclusive), but does not encompass a score of -10, since -10 is less than -9.

[0364] In some embodiments, ELNNs, including those incorporated into a subject paTCE fusion protein, can be rendered substantially non-immunogenic by restricting known proteolytic sites from the sequence of the ELNN, reducing processing of the ELNN into small peptides capable of binding to MHC II receptors. In some embodiments, ELNN sequences can be rendered substantially non-immunogenic by using sequences that are substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Thus, reducing the TEPITOPE score and eliminating known proteolytic sites from the ELNN can render an ELNN composition comprising an ELNN in a paTCE fusion protein composition substantially unable to be bound by mammalian receptors, including receptors of the immune system. In some embodiments, the ELNN in the paTCE fusion protein has a K of greater than 100 nM for mammalian receptors. D Binding or K >500 nM to mammalian cell surface or circulating polypeptide receptors D or a K greater than 1 μM D may have:

[0365] Additionally, the substantially non-repetitive sequence and lack of corresponding epitopes of such embodiments of ELNNs may limit the ability of B cells to bind to or be activated by the ELNN. In some embodiments, an ELNN may contact many different B cells over its extended sequence, but each individual B cell may only contact one or a few individual ELNNs. As a result, ELNNs may typically have a much lower tendency to stimulate B cell proliferation and thus an immune response. In some embodiments, paTCE may have reduced immunogenicity compared to a corresponding BsAb (e.g., TCE) that is not fused to a masked polypeptide such as an ELNN. In some embodiments, administration of up to three parenteral doses of paTCE to a mammal may result in detectable anti-paTCE IgG at a serum dilution of 1:100, but not at a dilution of 1:1000. In some embodiments, administration of up to three parenteral doses of paTCE to a mammal can result in detectable anti-BsAb (e.g., TCE) IgG at a serum dilution of 1:100, but may not result in a dilution of 1:1000. In some embodiments, administration of up to three parenteral doses of paTCE to a mammal can result in detectable anti-ELNN IgG at a serum dilution of 1:100, but may not result in a dilution of 1:1000. In some embodiments, the mammal can be, for example, a mouse, rat, rabbit, cynomolgus monkey, or human. In some embodiments, the mammal is a human.

[0366] An additional feature of certain ELNNs having substantially non-repetitive sequences compared to these less non-repetitive sequences (such as those with three consecutive amino acids that are identical) is that the non-repetitive ELNNs may form weaker contacts (e.g., monovalent interactions) with antibodies, thereby resulting in a lower likelihood of immune clearance, such that the paTCE composition may remain in the circulation for an increased period of time.

[0367] In some embodiments, biologically active polypeptides (such as BsAbs, e.g., TCEs) comprising ELNNs are less immunogenic than fusion polypeptides that are not linked to any ELNN, as confirmed by measuring the production of IgG antibodies that selectively bind to the biologically active polypeptides after administration of equivalent doses to subjects.

[0368] Barcode fragment In some embodiments, a polypeptide (e.g., a fusion polypeptide, or a portion thereof, such as an ELNN) comprises one or more barcode fragments (e.g., a first, second, or third barcode fragment) that are releasable from the polypeptide upon digestion with a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, the protease is a prokaryotic protease. As used herein, the term "barcode fragment" (or "barcode" or "barcode sequence") can refer to either a portion of a polypeptide that is cleavably fused within the polypeptide or the resulting peptide fragment released from the polypeptide.

[0369] In some embodiments, a barcode fragment can be a portion of an ELNN that (1) contains at least a portion of a (non-repeated, non-overlapping) sequence motif that occurs (or is found) only once within the ELNN, and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete cleavage or digestion of the polypeptide by a protease.

[0370] In some embodiments, the barcode fragment does not include the N-terminal or C-terminal amino acid of the fusion polypeptide. As described herein, in some embodiments, the barcode fragment is releasable (e.g., configured to be released) upon Glu-C digestion of the fusion polypeptide. In some embodiments, the barcode fragment is in an ELNN and does not include a glutamic acid immediately adjacent to another glutamic acid (if present) in the ELNN. In some embodiments, the barcode fragment has a glutamic acid at its C-terminus. One skilled in the art will understand that the C-terminus of a barcode fragment, when cleavably fused within a polypeptide (such as an ELNN), can refer to the "last" (or most C-terminal) amino acid residue within the barcode fragment, even if other non-barcode amino acid residues are located C-terminal to the barcode fragment within the polypeptide (e.g., an ELNN). In some embodiments, the barcode fragment has an N-terminal amino acid immediately preceded by a glutamic acid residue. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not include a (second) glutamic acid residue at a position other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by a proline. In some embodiments, the barcode fragment is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, this distance being 10 to 150, or 10 to 125 amino acids. In some embodiments, the barcode fragment is located within, or at, 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids of the N-terminus of the polypeptide, or at a position within a range between any of the foregoing. In some embodiments, the barcode fragment is located within 200 amino acids, within 150 amino acids, within 100 amino acids, or within 50 amino acids of the N-terminus of the polypeptide.In some embodiments, the barcode fragment is located at a position that is 10-200, 30-200, 40-150, or 50-100 amino acids from the N-terminus of the polypeptide. In some embodiments, the barcode fragment is located within 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the C-terminus of the polypeptide, or at a position within a range between any of the foregoing. In some embodiments, the barcode fragment is located within 200 amino acids, within 150 amino acids, within 100 amino acids, or within 50 amino acids of the C-terminus of the polypeptide. In some embodiments, the barcode fragment is located at a position that is 10 to 200, 30 to 200, 40 to 150, or 50 to 100 amino acids from the C-terminus of the polypeptide. In some embodiments, the barcode fragment (barcode, BAR) is characterized in that it (i) does not contain a glutamic acid, if present, immediately adjacent to another glutamic acid in the ELNN, (ii) has a glutamic acid at its C-terminus, (iii) has an N-terminal amino acid immediately preceded by a glutamic acid residue, and (iv) is located at a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids or 10 to 125 amino acids in length. In some embodiments, the barcode fragment is in an ELNN and (i) does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide, (ii) does not include a glutamic acid in the ELNN that is immediately adjacent to another glutamic acid, (iii) has a glutamic acid at its C-terminus, (iv) has an N-terminal amino acid immediately preceded by a glutamic acid residue, and (v) is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150, or 10 to 125 amino acids in length. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not include a glutamic acid residue at any position other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by a proline.Depending on the context herein, the term "distance," when referring to a position within a polypeptide sequence, can refer to the number of amino acid residues from the N-terminus of the polypeptide to the N-most amino acid residue of the barcode fragment, or from the C-terminus of the polypeptide to the C-most amino acid residue of the barcode fragment. In some embodiments, for a barcoded ELNN fused to a biologically active polypeptide, at least one barcode fragment (or at least two barcode fragments, or three barcode fragments) contained in the barcoded ELNN is located at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the biologically active polypeptide. In some embodiments, the barcode fragment is at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids in length. In some embodiments, the barcode fragment is at least 4 amino acids in length. In some embodiments, the barcode fragment is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids in length, or within a range between any of the foregoing values. In some embodiments, the barcode fragment is 4-20, 5-15, 6-12, or 7-10 amino acids in length. In some embodiments, the barcode fragment herein comprises an amino acid sequence identified by SEQ ID NOs: 68-79 and 1010-1027 in Table 2.

[0371] [Table 2]

[0372] In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments releasable from a chimeric polypeptide described herein upon complete digestion of the chimeric polypeptide by a non-mammalian protease, which in some embodiments is Glu-C.

[0373] In some embodiments, the chimeric polypeptides disclosed herein comprise a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0374] In some embodiments, the chimeric polypeptides disclosed herein have the following amino acid sequence: PE.GSX n PE.SG, PE.GSX n SE.GG, PE.GSX n SE.TG, PE.GSX n SE.SA, PE.SGX n PE.SG, PE.SGX n SE.GG, PE.SGX n SE.TG, PE.SGX n SE.SA and PE.TPX n PE.SG, PE.TPX n SE.GG, PE.TPX n SE.TG, PE.TPX n wherein each "." is a Glu-C cleavage site and n is any integer between 0 and 50. In some embodiments, the chimeric polypeptides disclosed herein comprise at least one of the following amino acid sequences: PE.SGX n PE.SG, PE.GSX n SE.GG, PE.TPX n SE.TG, PE.SGX n In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is any integer from 5 to 15. In some embodiments, X nis SGPGTGTSATPE, SGPGSGPGTSE, SGPGTTPGTTPE, SGPGTPPTSTPE, SGPGTSPSATPE, SGPGTGSAGTPE, SGPGTGGAGTPE, SGPGTSPGATPE, SGPGTSGSGTPE, SGPGTSSASTPE, SGPGTGAGTTPE, SGPGTGSTSTPE, TPGSEPATSGSE, GSAPGTSTEPSE, SGPGTAGSGTPE, SGPGTSSGGTPE, SGPGTAGPATPE, SGPGTPGTGTPE, SGPGTGGPTTPE, or SGPGTGSGSTPE.

[0375] In some embodiments, the chimeric polypeptide has the following amino acid sequence: SGPE.SGPGX n SGPE.SGPG, SGPE.SGPGX n ATPE.SGPG, SGPE.SGPGX n GTSE.SATP, SGPE.SGPGX n TTPE.SGPG, SGPE.SGPGX n STPE.SGPG, SGPE.SGPGX n GTPE.SGPG, SGPE.SGPGX n GTPE.TPGS, SGPE.SGPGX n GTPE.TPGS, SGPE.SGPGX n SGSE.TGTP, SGPE.SGPGX n GTPE.GSAP, SGPE.SGPGX n EPSE.SATP, ATPE.SGPGX n SGPE.SGPG, ATPE.SGPGX n ATPE.SGPG, ATPE.SGPGX n GTSE.SATP, ATPE.SGPGX n TTPE.SGPG, ATPE.SGPGX n STPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n GTPE.TPGS, ATPE.SGPGX n SGSE.TGTP, ATPE.SGPGX nGTPE.GSAP、ATPE.SGPGX n EPSE.SATP、GTSE.SATPX n SGPE.SGPG、GTSE.SATPX n ATPE.SGPG、GTSE.SATPX n GTSE.SATP、GTSE.SATPX n TTPE.SGPG、GTSE.SATPX n STPE.SGPG、GTSE.SATPX n GTPE.SGPG、GTSE.SATPX n GTPE.TPGS、GTSE.SATPX n SGSE.TGTP、GTSE.SATPX n GTPE.GSAP、GTSE.SATPX n EPSE.SATP、TTPE.SGPGX n SGPE.SGPG、TTPE.SGPGX n ATPE.SGPG、TTPE.SGPGX n GTSE.SATP、TTPE.SGPGX n TTPE.SGPG、TTPE.SGPGX n STPE.SGPG、TTPE.SGPGX n GTPE.SGPG、TTPE.SGPGX n GTPE.TPGS、TTPE.SGPGX n SGSE.TGTP、TTPE.SGPGX n GTPE.GSAP、TTPE.SGPGX n EPSE.SATP、STPE.SGPGX n SGPE.SGPG、STPE.SGPGX n ATPE.SGPG、STPE.SGPGX n GTSE.SATP、STPE.SGPGX n TTPE.SGPG、STPE.SGPGX n STPE.SGPG、STPE.SGPGX n GTPE.SGPG、STPE.SGPGX n GTPE.TPGS、STPE.SGPGX n SGSE.TGTP、STPE.SGPGX nGTPE.GSAP、STPE.SGPGX n EPSE.SATP、GTPE.SGPGX n SGPE.SGPG、GTPE.SGPGX n ATPE.SGPG、GTPE.SGPGX n GTSE.SATP、GTPE.SGPGX n TTPE.SGPG、GTPE.SGPGX n STPE.SGPG、GTPE.SGPGX n GTPE.SGPG、GTPE.SGPGX n GTPE.TPGS、GTPE.SGPGX n SGSE.TGTP、GTPE.SGPGX n GTPE.GSAP、GTPE.SGPGX n EPSE.SATP、GTPE.TPGSX n SGPE.SGPG、GTPE.TPGSX n ATPE.SGPG、GTPE.TPGSX n GTSE.SATP、GTPE.TPGSX n TTPE.SGPG、GTPE.TPGSX n STPE.SGPG、GTPE.TPGSX n GTPE.SGPG、GTPE.TPGSX n GTPE.TPGS、GTPE.TPGSX n SGSE.TGTP、GTPE.TPGSX n GTPE.GSAP、GTPE.TPGSX n EPSE.SATP、SGSE.TGTPX n SGPE.SGPG、SGSE.TGTPX n ATPE.SGPG、SGSE.TGTPX n GTSE.SATP、SGSE.TGTPX n TTPE.SGPG、SGSE.TGTPX n STPE.SGPG、SGSE.TGTPX n GTPE.SGPG、SGSE.TGTPX n GTPE.TPGS、SGSE.TGTPX n SGSE.TGTP、SGSE.TGTPX nGTPE.GSAP, SGSE.TGTPX n EPSE.SATP, GTPE.GSAPX n SGPE.SGPG, GTPE.GSAPX n ATPE.SGPG, GTPE.GSAPX n GTSE.SATP, GTPE.GSAPX n TTPE.SGPG, GTPE.GSAPX n STPE.SGPG, GTPE.GSAPX n GTPE.SGPG, GTPE.GSAPX n GTPE.TPGS, GTPE.GSAPX n SGSE.TGTP, GTPE.GSAPX n GTPE.GSAP, GTPE.GSAPX n EPSE.SATP, EPSE.SATPX n SGPE.SGPG, EPSE.SATPX n ATPE.SGPG, EPSE.SATPX n GTSE.SATP, EPSE.SATPX n TTPE.SGPG, EPSE.SATPX n STPE.SGPG, EPSE.SATPX n GTPE.SGPG, EPSE.SATPX n GTPE.TPGS, EPSE.SATPX n SGSE.TGTP, EPSE.SATPX n GTPE.GSAP or EPSE.SATPX n EPSE.SATP, where each "." is a Glu-c cleavage site and n is any integer between 0 and 50. In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences: SGPE.SGPGX n ATPE.SGPG, ATPE.SGPGX n GTSE.SATP, ATPE.SGPGX n TTPE.SGPG, ATPE.SGPGX n STPE.SGPG, ATPE.SGPGX n ATPE.SGPG, ATPE.SGPGX nGTPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n ATPE.SGPG, GTPE.SGPGX n GTPE.SGPG, GTPE.SGPGX n STPE.SGPG, GTPE.SGPGX n TTPE.SGPG, GTPE.SGPGX n STPE.SGPG, GTPE.TPGSX n SGSE.TGTP, GTPE.GSAPX n EPSE.SATP, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n GTPE.SGPG, ATPE.SGPGX n ATPE.SGPG, ATPE.SGPGX n GTPE.SGPG, TTPE.SGPGX n TTPE.SGPG or STPE.SGPGX n STPE.SGPG, where each "." is a Glu-C cleavage site and n is any integer between 0 and 30. In some embodiments, n is any integer between 1 and 20. In some embodiments, n is any integer between 5 and 15. In some embodiments, n is any integer between 3 and 7. In some embodiments, n is any integer between 5 and 10. In some embodiments, n is 9. In some embodiments, n is 4. In some embodiments, n is any integer between 5 and 15. In some embodiments, X n is PGTGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTAGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST. nis TGTS, SGP, TTPG, TPPT, TSPS, TGSA, TGGA, TSPG, TSGS, TSSA, TGAG, TGST, EPAT, GTST, TAGS, TSSG, TAGP, TPGT, TGGP, or TGSG.

[0376] In some embodiments, barcodes are designed to have improved analytical properties. In some embodiments, such barcodes can be released by relatively mild concentrations of non-mammalian proteases, such as Glu-C. This facilitates better detection, e.g., via LC / MS, and also allows for measurement of peptides generated from the cleavable linker, thereby allowing for measurement of cleavage products, e.g., using LC / MS.

[0377] In some embodiments of fusion proteins comprising an ELNN, the fusion protein has a single polypeptide chain, wherein the polypeptide chain comprises a barcode fragment located within the polypeptide chain that is 10-200 amino acids or 10-125 amino acids from the N-terminus or C-terminus of the polypeptide chain. In some embodiments, the fusion protein (e.g., paTCE) comprises a first ELNN and a second ELNN, wherein the first ELNN is N-terminal to the bispecific domain and the first barcode fragment is located within 200, 150, 100, or 50 amino acids of the N-terminus of the fusion protein. In some embodiments, the second ELNN is C-terminal to the bispecific antibody domain and the second barcode fragment is located within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.

[0378] In some embodiments, the ELNN further comprises one or more additional barcode fragments, each of which differs in sequence and molecular weight from all other peptide fragments releasable from the polypeptide upon complete digestion of the polypeptide by a protease. In some embodiments, the barcoded ELNN comprises only one barcode fragment. In some embodiments, the barcoded ELNN comprises a set of barcode fragments including a first barcode fragment, such as those described herein. In some embodiments, the set of barcode fragments includes a second barcode fragment (or additional barcode fragments), such as those described herein. In some embodiments, the set of barcode fragments includes a third barcode fragment, such as those described herein.

[0379] A set of fused barcode fragments within an N-terminal ELNN may be referred to as an N-terminal set of barcodes (the "N-terminal set"). A set of fused barcode fragments within a C-terminal ELNN may be referred to as a C-terminal set of barcodes (the "C-terminal set"). In some embodiments, the N-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal set further comprises a third barcode fragment. In some embodiments, the C-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal set further comprises a third barcode fragment. In some embodiments, a polypeptide comprises a set of barcode fragments comprising a first barcode fragment, a further (second) barcode fragment, and at least one additional barcode fragment, wherein each barcode fragment of the set of barcode fragments (1) is part of a second ELNN and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by a protease.

[0380] Included herein is a mixture comprising a plurality of polypeptides of varying lengths, the mixture comprising a first set of polypeptides and a second set of polypeptides. In some embodiments, each polypeptide in the first set of polypeptides comprises a barcode fragment that (a) is releasable from the polypeptide by digestion with a protease and (b) has a sequence and molecular weight that differs from all other fragments releasable from the first set of polypeptides. In some embodiments, the second set of polypeptides lacks the barcode fragment of the first set of polypeptides (e.g., due to cleavage). In some embodiments, both the first set of polypeptides and the second set of polypeptides each comprise a reference fragment that is (a) common to the first set of polypeptides and the second set of polypeptides and (b) releasable by digestion with a protease. In some embodiments, the ratio of the first set of polypeptides to the polypeptides comprising the reference fragment is greater than 0.70. In some embodiments, the ratio of the first set of polypeptides to the polypeptides comprising the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the protease is a protease that cleaves C-terminal to glutamic acid residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the variable length polypeptides include polypeptides comprising at least one ELNN, such as any described herein. In some embodiments, the first set of polypeptides includes full-length polypeptides, and the barcode fragment is a portion of the full-length polypeptide. In some embodiments, the full-length polypeptide is any (fusion) polypeptide described herein above or elsewhere herein. In some embodiments, the variable length polypeptides in the mixture differ from each other due to N-terminal truncation, C-terminal truncation, or both N- and C-terminal truncation of the full-length polypeptide.In some embodiments, the first set of polypeptides and the second set of polypeptides may differ in one or more pharmacological properties.

[0381] The present disclosure also provides methods for assessing the relative abundance of a first set of polypeptides in a mixture containing polypeptides of varying lengths versus a second set of polypeptides in the mixture, wherein (1) each polypeptide in the first set of polypeptides shares a barcode fragment that occurs once and only once among the polypeptides; and (2) each polypeptide in the second set of polypeptides lacks the barcode fragment shared by the first set of polypeptides, and each individual polypeptide in both the first and second set of polypeptides each comprises a reference fragment. In some embodiments, the method includes contacting the mixture with a protease to produce a plurality of proteolytic fragments resulting from cleavage of the first set of polypeptides and the second set of polypeptides, wherein the plurality of proteolytic fragments comprises a plurality of reference fragments and a plurality of barcode fragments. In some embodiments, the method may further include determining the ratio of the amount of the barcode fragment to the amount of the reference fragment, thereby assessing the relative abundance of the first set of polypeptides versus the second set of polypeptides. In some embodiments, the barcode fragment occurs no more than once among each polypeptide in the first set of polypeptides. In some embodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the plurality of proteolytic fragments comprises a plurality of reference fragments and a plurality of barcode fragments. In some embodiments, the protease cleaves the first and second sets of polypeptides (or polypeptides of various lengths) C-terminal to glutamic acid residues that are not followed by proline residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, determining the ratio of the amount of barcode fragment to the amount of reference fragment comprises identifying the barcode fragment and the reference fragment from the mixture after contacting with the protease. In some embodiments, the barcode fragment and the reference fragment are identified based on their respective masses. In some embodiments, the barcode fragment and the reference fragment are identified via mass spectrometry.

[0382] In some embodiments, the barcode fragments and the reference fragments are identified via liquid chromatography-mass spectrometry (LC-MS). In some embodiments, determining the ratio of the barcode fragments to the reference fragments comprises isobaric labeling. In some embodiments, determining the ratio of the barcode fragments to the reference fragments comprises spiking the mixture with one or both of an isotopically labeled reference fragment and an isotopically labeled barcode fragment. In some embodiments, the polypeptides of various lengths comprise polypeptides comprising at least one ELNN described herein above or elsewhere herein. In some embodiments, the ELNN is characterized in that it (i) comprises at least 100 or at least 150 amino acids, (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P), and (iii) comprises at least four different types of amino acids that are G, A, S, T, E, or P. In some embodiments, the barcode fragment, if present, is part of an ELNN. In some embodiments, the mixture of polypeptides of varying lengths comprises any of the polypeptides described herein above or elsewhere herein. In some embodiments, the polypeptides of varying lengths include full-length polypeptides and truncated fragments thereof. In some embodiments, the polypeptides of varying lengths consist primarily of full-length polypeptides and truncated fragments thereof. In some embodiments, the polypeptides of varying lengths in the mixture differ from each other due to N-terminal truncation, C-terminal truncation, or both N-terminal and C-terminal truncation of the full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide described herein above or elsewhere. In some embodiments, the ratio of the amount of barcode fragment to reference fragment is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.

[0383] Quantification based on isobaric labeling of peptides In some embodiments, isobaric labeling can be used to determine the ratio of barcode fragments to reference fragments. Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics, in which peptides or proteins (or portions thereof) are labeled with various chemical groups that are isobaric (identical in mass) but vary in terms of the distribution of heavy isotopes around their structure. In some embodiments, these tags, commonly referred to as tandem mass tags, are designed so that upon high-energy collision-induced dissociation (CID) during tandem mass spectrometry, the mass tag is cleaved at a specific linker region, thereby generating reporter ions of different masses. Some of the most common isobaric tags are amine-reactive tags.

[0384] Exemplary barcoded ELNN polypeptides Included herein are ELNNs that include barcode fragments that are part of the ELNN.

[0385] The amino acid sequences of exemplary barcoded ELNNs containing one barcode (e.g., SEQ ID NOs: 8002-8003, 8005-8009, and 8013-8022), two barcodes (e.g., SEQ ID NOs: 8001, 8004, and 8012), or three barcodes (e.g., SEQ ID NO: 8011) are shown in Table 3a, with barcodes identified in bold. In some embodiments, of these exemplary barcoded ELNNs, 12 (SEQ ID NOs: 8001-8003, 8008-8009, 8011, 8015-8019, and 8022) are fused to a biologically active protein (such as a TCE) at the C-terminus of the biologically active protein, and 10 (SEQ ID NOs: 8004-8007, 8010, 8012-8014, 8020, and 8021) are fused at the N-terminus of the biologically active protein. In some embodiments, the ELNNs have at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 8001-8022 in Table 3a.

[0386] [Table 3-1]

[0387] [Table 3-2]

[0388] [Table 3-3]

[0389] [Table 3-4]

[0390] [Table 3-5]

[0391] Table 3-6

[0392] Table 3-7

[0393] Table 3-8

[0394] Table 3-9

[0395] In some embodiments, barcoded ELNNs can be obtained by making one or more mutations in an existing ELNN, such as any listed in Table 3b, according to one or more of the following criteria: minimizing sequence changes in the ELNN, minimizing amino acid composition changes in the ELNN, substantially maintaining the net charge of the ELNN, substantially maintaining (or improving) the low immunogenicity of the ELNN, and substantially maintaining (or improving) the pharmacokinetic properties of the ELNN. In some embodiments, the ELNN sequence has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:601-659 listed in Table 3b. In some embodiments, an ELNN sequence having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) but less than 100% sequence identity to any of SEQ ID NOs:601-659 listed in Table 3b is obtained by one or more mutations (e.g., fewer than 10, fewer than 8, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 mutations) of the corresponding sequence from Table 3b. In some embodiments, the one or more mutations comprise a deletion of a glutamic acid residue, an insertion of a glutamic acid residue, a substitution of a glutamic acid residue, or a substitution with a glutamic acid residue, or any combination thereof. In some embodiments, if an ELNN sequence differs from any one of SEQ ID NOs:601-659 listed in Table 3b but has at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence in Table 3b comprises a deletion of a glutamic acid residue, an insertion of a glutamic acid residue, a substitution of a glutamic acid residue, or a substitution with a glutamic acid residue, or any combination thereof.In some such embodiments, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the differences between an ELNN sequence and the corresponding sequence in Table 3b include substitutions of glutamic acid residues, or substitutions with glutamic acid residues, or both.

[0396] As used herein, a "first amino acid substitution" refers to the replacement of a first amino acid residue with a second amino acid residue, with the second amino acid residue occupying its place at the substitution position in the resulting sequence. For example, a "glutamic acid substitution" refers to the replacement of a glutamic acid (E) residue with a non-glutamic acid residue (e.g., serine (S)).

[0397] [Table 4-1]

[0398] [Table 4-2]

[0399] [Table 4-3]

[0400] [Table 4-4]

[0401] [Table 4-5]

[0402] [Table 4-6]

[0403] [Table 4-7]

[0404] [Table 4-8]

[0405] [Table 4-9]

[0406] [Table 4-10]

[0407] [Table 4-11]

[0408] [Table 4-12]

[0409] In some embodiments, to construct the sequences of barcoded ELNNs, amino acid mutations are performed on ELNNs of intermediate lengths relative to those in Table 3b, as well as on ELNNs of lengths longer than those in Table 3b, such as those in which one or more 12-mer motifs from Table 1 are added to the N- or C-terminus of the generic ELNNs of Table 3b.

[0410] Additional examples of existing ELNNs that can be used in accordance with the present disclosure are U.S. Patent Publication Nos. 2010 / 0239554(A1), 2010 / 0323956(A1), 2011 / 0046060(A1), 2011 / 0046061(A1), 2011 / 0077199(A1), or 2011 / 0172146(A1), or International Patent Publication No. 201 0091122(A1), 2010144502(A2), 2010144508(A1), 2011028228(A1), 2011028229(A1), 2011028344(A2), 2014 / 011819(A2), or 2015 / 023891, each of which is incorporated herein by reference.

[0411] In some embodiments, a barcoded ELNN fused within a polypeptide chain adjacent to the N-terminus of the polypeptide chain ("N-terminal ELNN") may be attached to a His-tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate purification of the fusion polypeptide. In some embodiments, a barcoded ELNN fused within a polypeptide chain at the C-terminus of the polypeptide chain ("C-terminal ELNN") may contain or be attached to the sequence EPEA at the C-terminus to facilitate purification of the fusion polypeptide. In some embodiments, the fusion polypeptide comprises both an N-terminally barcoded ELNN and a C-terminally barcoded ELNN, wherein the N-terminally barcoded ELNN is linked at its N-terminus to a His tag of the sequence HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49), and the C-terminally barcoded ELNN is linked at its C-terminus to the sequence EPEA, thereby facilitating purification of the fusion polypeptide to, for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatographic methods known in the art, including but not limited to, IMAC chromatography, C-tagXL affinity matrix, and other such methods.

[0412] The barcode fragments described herein may be cleavably fused within an ELNN and may be releasable (i.e., configured to be released) from the ELNN upon digestion of the polypeptide by a protease. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves C-terminal to glutamic acid residues that are not followed by proline. In some embodiments, the barcoded ELNN (the ELNN containing the barcode fragment therein) is designed to achieve high efficiency, precision, and accuracy of protease digestion. For example, in some embodiments, adjacent Glu-Glu (EE) residues in the ELNN sequence may result in diverse cleavage patterns upon Glu-C digestion. Thus, when a Glu-C protease is used for barcode release, the barcoded ELNN or barcode fragment may not contain a Glu-Glu (EE) sequence. Additionally, the dipeptide Glu-Pro (EP) sequence, if present in the fusion polypeptide, may not be cleaved by the Glu-C protease during the barcode release process.

[0413] Structural configuration of activatable TCE In some embodiments, the fusion protein comprises a single BsAb in the form of a TCE and a single ELNN. In some embodiments, such fusion proteins may have at least the following configurations: (TCE)-(ELNN), (TCE)-(ELNN), (ELNN)-(TCE), (TCE)-(linker)-(ELNN), (ELNN)-(linker)-(TCE), each listed in the N-terminal to C-terminal direction.

[0414] In some embodiments, the fusion protein comprises a C-terminal ELNN and, optionally, a linker (such as those described herein, e.g., in Table C) between the ELNN and the TCE. In some embodiments, such a fusion protein has Formula I (shown from N-terminus to C-terminus): (TCE)-(linker)-(ELNN)(I) where TCE is as described herein, the linker is a linker sequence (such as those described herein, e.g., in Table C) comprising 1 to about 50 amino acid residues that may optionally include a TCE-releasing segment (e.g., as described herein), and the ELNN can be any ELNN described herein.

[0415] In some embodiments, the fusion protein comprises an N-terminal ELNN and, optionally, a linker (such as those described herein, e.g., in Table C) between the ELNN and the TCE. In some embodiments, such a fusion protein has Formula II (shown N-terminal to C-terminal): (ELNN)-(linker)-(TCE)(II) where TCE is as described herein, the linker is a linker sequence (such as those described herein, e.g., in Table C) comprising 1 to about 50 amino acid residues that may optionally include a TCE-releasing segment (e.g., as described herein), and the ELNN can be any ELNN described herein.

[0416] In some embodiments, the fusion protein comprises both an N-terminal ELNN and a C-terminal ELNN. In some embodiments, such a fusion protein has Formula III: (ELNN)-(linker)-(TCE)-(linker)-(ELNN)(III) where TCE is as described herein, each linker is a linker sequence (such as those described herein, e.g., in Table C) having 1 to about 50 amino acid residues that may individually optionally include a TCE-releasing segment (e.g., as described herein), and each ELNN may individually be any ELNN described herein.

[0417] The present disclosure provides BsAbs (eg, TCEs) comprising one or more sequences disclosed in any one of Tables 6a-6g herein.

[0418] Of particular interest are BsAbs (e.g., TCEs) for which increased pharmacokinetic parameters, increased solubility, increased stability, masked activity, or some other improved pharmaceutical property is desired, or for which increased terminal half-life improves efficacy and / or safety. Thus, paTCE fusion protein compositions are prepared with a variety of objectives in mind, including improving the therapeutic efficacy of TCEs by, for example, increasing in vivo exposure or increasing the length of time that TCE remains within the therapeutic window when administered to a subject, compared to TCEs that are not linked to any ELNNs.

[0419] For example, it will be understood that various amino acid substitutions (particularly conservative amino acid substitutions) can be made in the bispecific sequences to create variants without departing from the spirit of the present disclosure with respect to the biological activity or pharmacological properties of TCE. Examples of conservative amino acid substitutions in polypeptide sequences are shown in Table 4. In addition, variants can also include polypeptides in which one or more amino acid residues have been added or deleted, for example, at the N- or C-terminus of the full-length native amino acid sequence of TCE that retain at least some of the biological activity of the native peptide.

[0420] In some embodiments, sequences that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more activity compared to the corresponding original TCE sequence are considered suitable for inclusion in a subject paTCE. In some embodiments, TCEs found to retain a suitable level of activity can be linked to one or more ELNN polypeptides having at least about 80% sequence identity (e.g., at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity) to a sequence from Tables 3a-3b.

[0421] [Table 5]

[0422] The present disclosure provides an ELLNNylated TCE (such as a paTCE) that targets PSMA, wherein the TCE is a bispecific antibody (e.g., a bispecific TCE) that specifically binds to PSMA in one portion of the bispecific TCE and specifically binds to CD3 in another portion of the bispecific TCE.

[0423] In some embodiments, the ELLNized TCE comprises (1) a first portion comprising a first binding domain and a second binding domain, (2) a second portion comprising a release segment, and (3) a third portion comprising an unstructured polypeptide mask (sometimes referred to herein as a masking portion).

[0424] In some embodiments, the ELLNylated TCE has the configuration of Formula Ia (shown N-terminus to C-terminus): (First part)-(Second part)-(Third part)(Ia) wherein the first portion is a bispecific antibody domain comprising the two antigen-binding domains described above, wherein the first binding domain has specific binding affinity for PSMA (e.g., expressed on cancer cells) and the second binding domain has specific binding affinity for CD3 (e.g., expressed on effector cells), the second portion comprises a release segment (RS) cleavable by a mammalian protease, and the third portion is a masking moiety that functions to mask a biological property of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that is cleavable by proteases present in the tumor microenvironment.

[0425] In some embodiments where the first portion comprises a binding domain comprising a VHH and a binding domain comprising a VL and a VH, the binding domains of the first portion may be ordered as follows: (VL-VH)1-(VHH)2, or (VH-VL)1-(VHH)2, or (VHH)1-(VL-VH)2, or (VHH)1-(VH-VL)2 (where "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (e.g., as described herein). In some embodiments in which the first portion comprises two binding domains, each comprising a VL and a VH, the binding domains of the first portion may be in the order (VL-VH)1-(VL-VH)2, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (where "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are connected by a polypeptide linker (e.g., as described herein).

[0426] In some embodiments, the domain that binds to PSMA is a VHH.

[0427] In some embodiments, the binding domain of the first portion comprises a sequence provided in Tables 6a-6g, where Tables 6a-e set forth sequences that bind to CD3, and Tables 6f-h set forth sequences that bind to PSMA, the RS sequence comprises a sequence provided in Tables 8a-8b (e.g., as described herein), and the masking moiety is ELNN. In some embodiments, the masking moiety is ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the moieties are linked by chemical conjugation.

[0428] In some embodiments, the fusion protein has the configuration of Formula IIa (shown N-terminus to C-terminus): (Third Part)-(Second Part)-(First Part)(IIa) wherein the first portion is bispecific comprising two antigen-binding domains, the first binding domain having specific binding affinity for PSMA (e.g., expressed on cancer cells) and the second binding domain having specific binding affinity for CD3 (e.g., expressed on effector cells), the second portion comprises a release segment (RS) cleavable by a mammalian protease, and the third portion is a masking moiety that functions to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that is universally cleavable in the tumor microenvironment.

[0429] In some embodiments where the first portion comprises a binding domain comprising a VHH and a binding domain comprising a VL and a VH, the binding domains of the first portion may be ordered as follows: (VL-VH)1-(VHH)2, or (VH-VL)1-(VHH)2, or (VHH)1-(VL-VH)2, or (VHH)1-(VH-VL)2 (where "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (e.g., as described herein). In some embodiments in which the first portion comprises two binding domains, each comprising a VL and a VH, the binding domains of the first portion may be in the order (VL-VH)1-(VL-VH)2, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (where "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are connected by a polypeptide linker (e.g., as described herein).

[0430] In some embodiments, the domain that binds to PSMA is a VHH.

[0431] In some embodiments, the binding domain of the first portion comprises a sequence provided in Tables 6a-6g, where Tables 6a-e set forth sequences that bind to CD3, and Tables 6f-h set forth sequences that bind to PSMA, the RS sequence comprises a sequence provided in Tables 8a-8b (e.g., as described herein), and the masking moiety is ELNN. In some embodiments, the masking moiety is ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the moieties are linked by chemical conjugation.

[0432] In some embodiments, the paTCE composition has the configuration of Formula IIIa (shown N-terminus to C-terminus): (5th part)-(4th part)-(1st part)-(2nd part)-(3rd part)(IIIa) wherein the first portion is bispecific comprising two antigen-binding domains, the first binding domain has specific binding affinity for PSMA (e.g., expressed on cancer cells), the second binding domain has specific binding affinity for CD3 (e.g., expressed on effector cells), the second portion comprises a release segment (RS) that is cleavable by a mammalian protease, the third portion is a masking moiety that functions to mask a biological property of the bispecific antibody domain, the fourth portion comprises a release segment (RS) that is cleavable by a mammalian protease that may be the same as or different from the second portion, and the fifth portion is a masking moiety that may be the same as or different from the third portion.

[0433] In some embodiments where the first portion comprises a binding domain comprising a VHH and a binding domain comprising a VL and a VH, the binding domains of the first portion may be ordered as follows: (VL-VH)1-(VHH)2, or (VH-VL)1-(VHH)2, or (VHH)1-(VL-VH)2, or (VHH)1-(VH-VL)2 (where "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (e.g., as described herein). In some embodiments in which the first portion comprises two binding domains, each comprising a VL and a VH, the binding domains of the first portion may be in the order (VL-VH)1-(VL-VH)2, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (where "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are connected by a polypeptide linker (e.g., as described herein).

[0434] In some embodiments, the domain that binds to PSMA is a VHH.

[0435] In some embodiments, the binding domain of the first portion comprises a sequence provided in Tables 6a-6g, where Tables 6a-e set forth sequences that bind to CD3, and Tables 6f-h set forth sequences that bind to PSMA, each RS sequence individually comprises a sequence provided in Tables 8a-8b (e.g., as described herein), and each masking moiety individually is an ELNN. In some embodiments, each masking moiety is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising a group of sequences set forth in Tables 3a-3b. In some embodiments, the paTCE is a recombinant fusion protein. In some embodiments, one or more portions of the paTCE are linked by chemical conjugation.

[0436] Provided herein are compositions that advantageously provide PSMA-targeted bispecific therapeutics that have greater selectivity, longer half-lives, and produce lower toxicity and fewer side effects upon cleavage by proteases found in target tissues or diseased tissues, resulting in an improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions are useful for treating cancer. In some embodiments, when paTCE is in proximity to a target tissue or cell that harbors or secretes a protease capable of cleaving RS, the bispecific binding domain is liberated from the ELNN by the action of the protease, removing the steric hindrance barrier and freeing the TCE to exert its pharmacological effects. This property is particularly advantageous in treating immunologically cold tumors that express PSMA. In some embodiments, the paTCE provided herein is activated in the target tissue, which is a solid tumor of an organ or system.

[0437] Binding domain In some embodiments, the binding domains provided herein comprise one or more full-length antibodies or one or more antigen-binding fragments thereof. Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide that contains a portion or portions of an antibody that specifically binds to an antigen. Antigen-binding fragments of antibodies can be derived from a complete antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. The terms binding domain and antibody domain are used interchangeably herein.

[0438] In some embodiments, single-chain binding domains are used, including but not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single domain antibodies, VHH, single-chain antibody molecules (scFv), and diabodies, which are capable of binding to effector cells and ligands or receptors associated with antigens of diseased tissues or cells, such as cancer, tumor, or other malignant tissue.

[0439] In some embodiments, the binding domain is a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to a first target and a second antigen-binding domain that specifically binds to a second target. In some embodiments, the first antigen-binding domain is a first antigen-binding fragment (e.g., an ISVD such as an scFv or VHH) and the second antigen-binding domain is a second antigen-binding fragment (e.g., an ISVD such as an scFv or VHH).

[0440] In some embodiments, the antigen binding fragments (AF) (e.g., the first antigen binding fragment (AF1) and / or the second antigen binding fragment (AF2)) may (each independently) be chimeric, humanized, or human. The antigen binding fragments (AF) (e.g., the first antigen binding fragment (AF1) and / or the second antigen binding fragment (AF2)) may (each independently) be Fv, Fab, Fab', Fab'-SH, linear antibody, VHH, or scFv.

[0441] In some embodiments, one or both antigen-binding fragments (e.g., the first and / or second antigen-binding fragments) may be configured as (Fab')2 or single-chain diabodies. In some embodiments, a bispecific antibody comprises a first binding domain that has binding specificity for a cancer cell marker and a second binding domain that has binding specificity for an effector cell antigen. In some embodiments, the binding domain for a tumor cell target is a variable domain of a T-cell receptor that has been engineered to bind to MHC loaded with a peptide fragment of a protein overexpressed by tumor cells.

[0442] In some embodiments, a paTCE is designed to provide a wide therapeutic window by taking into account the location of target tissue proteases, the presence of the same proteases in healthy tissues not intended to be targeted, and the presence of target ligands in healthy tissues but greater presence in unhealthy target tissues. The "therapeutic window" refers to the difference between the minimum effective dose and the maximum tolerated dose for a given therapeutic composition. In some embodiments, to help achieve a wide therapeutic window of a TCE, the binding domain of the TCE is masked by the proximity of a masking (e.g., ELNN) moiety such that the binding affinity of the intact composition for one or both of the ligands is reduced compared to a composition that has been cleaved by a mammalian protease, thereby releasing the first moiety from the masking effect of the masking moiety.

[0443] In some embodiments, a complete antigen recognition and binding site comprises a dimer of one heavy chain variable domain (VH) and one light chain variable domain (VL). Within each VH and VL chain are three complementarity-determining regions (CDRs) that interact to define an antigen-binding site on the surface of the VH-VL dimer; the six CDRs of the binding domain confer antigen-binding specificity to the antibody or single-chain binding domain. The framework sequences flanking the CDRs have a tertiary structure that is largely conserved in native immunoglobulins across species, and the framework residues (FRs) function to hold the CDRs in their proper orientation. In some embodiments, constant domains are not required for binding function but can help stabilize VH-VL interactions. In some embodiments, binding sites can be pairs of VH-VL, VH-VH, or VL-VL domains from either the same or different immunoglobulins; however, it is generally preferred to create single-chain binding domains using the respective VH and VL chains from the parent antibody. In some embodiments, the order of the VH and VL domains within a polypeptide chain is not limited, so long as the VH and VL domains are arranged in a way that allows the antigen-binding site to fold properly. Thus, in some embodiments, a single-chain binding domain comprising a VH and a VL (e.g., in an scFv) can have the VH and VL arranged as VL-VH or VL-VH.

[0444] In some embodiments, the V chain configuration is VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen) )-VH(effector cell antigen)-VL(effector cell antigen), VHH(cancer cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VHH(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen)-VHH(effector cell antigen), or VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VHH(effector cell antigen).

[0445] In some embodiments, the sequences are as follows: VH(effector cell antigen)-VL(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VH(effector cell antigen)-VL(effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen) The following are possible: effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VHH(effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VHH(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VHH(cancer cell surface antigen), or VH(effector cell antigen)-VL(effector cell antigen)-VHH(cancer cell surface antigen).

[0446] As used herein, "N-terminally" or "C-terminally," and grammatical variations thereof, refer to relative positions within the primary amino acid sequence, rather than absolute N- or C-terminal placement of the bispecific single chain antibody. Thus, as a non-limiting example, a first binding domain "located C-terminally to" a second binding domain indicates that the first binding domain is located on the carboxyl side of the second binding domain within the bispecific single chain antibody, and does not exclude the possibility that additional sequences, e.g., a linker and / or another compound such as an ELNN, a His tag, or a radioisotope, may be located at the C-terminus of the bispecific single chain antibody.

[0447] In some embodiments, a paTCE comprises a first portion comprising a first binding domain and a second binding domain, wherein each of the binding domains is an scFv, and each scFv comprises one VL and one VH. In some embodiments, a paTCE composition comprises a first portion comprising a first binding domain and a second binding domain, wherein one of the binding domains is an scFv and the other binding domain is a VHH. In some embodiments, the CD3 binding domain can be an scFV (e.g., comprising a sequence shown in any of Tables 6a-e), and the second binding domain is a VHH that binds to PSMA. In some embodiments, a paTCE comprises a first portion comprising a first binding domain and a second binding domain, wherein the binding domains are in a diabody configuration, one domain comprises one VHH region, and the other domain comprises one VL region and one VH region. Exemplary PSMA-binding VHH binding domains are shown in Table 6f. In some embodiments, the paTCE comprises a first portion comprising a first binding domain and a second binding domain, wherein the binding domains are in a diabody configuration and each domain comprises one VL region and one VH region. Exemplary PSMA-binding VH and VL regions can be derived from the sequences shown in Table 6g.

[0448] In a non-limiting example, a TCE may comprise a sequence exhibiting at least about 80% sequence identity, or alternatively, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an antibody sequence identified herein. In some embodiments, the TCE comprises a bispecific sequence (e.g., a BsAb) comprising a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity for a tumor-specific marker or cancer cell antigen and has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 17 and the second binding domain has specific binding affinity for an effector cell and exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the paired VL and VH sequences of an anti-CD3 antibody disclosed in any of Tables 6a-e herein.

[0449] In some embodiments, the TCE can comprise a binding domain (e.g., a VH and / or VL amino acid sequence) of or derived from an anti-CD3 antibody. Non-limiting examples of anti-CD3 antibodies include OKT3 (also known as muromonab) and humanized anti-CD3 monoclonal antibody (hOKT31(Ala-Ala)) (KC Herold et al., New England Journal of Medicine 346:1692-1698, 2002), as well as fragments and derivatives thereof that selectively bind to CD3. Additional examples are described in U.S. Patent Nos. 5,885,573, 6,491,916, and U.S. Patent Application Publication No. 2021 / 0054077(A1), the entire contents of each of which are incorporated herein by reference. Additional non-limiting examples of anti-CD3 antigen sequences include the sequences of pasotuxizumab (also known as AMG-212) and acapatamab (also known as AMG-160).

[0450] In some embodiments, the TCE can comprise a binding domain (e.g., a VH and / or VL amino acid sequence) of or derived from an anti-PSMA antibody. Non-limiting examples of anti-PSMA sequences include those of pasotuxizumab and acapatamab.

[0451] In some embodiments, the TCE is pasotuxizumab. In some embodiments, the TCE is acapatamab.

[0452] The present disclosure provides immunoglobulin single variable domains (ISVDs) that bind to PSMA. The present disclosure further provides nucleic acids encoding the ISVDs or polypeptides, as well as vectors, hosts, and methods for producing these ISVDs or polypeptides. Also provided are multispecific polypeptides comprising an ISVD according to the present disclosure and at least one CD3-binding domain comprising paTCE. Included are methods of treatment utilizing an ISVD or polypeptide according to the present disclosure. In some embodiments, the ISVD is a heavy chain ISVD. In some embodiments, the ISVD is a VHH, a humanized VHH, or a camelized VH.

[0453] In some embodiments, the ISVD is a VHH.

[0454] Also provided are nucleic acid molecules encoding the ISVDs or polypeptides of the disclosure, or vectors comprising the nucleic acids.

[0455] The present disclosure also relates to non-human hosts or host cells transformed or transfected with nucleic acids or vectors encoding the ISVDs or polypeptides disclosed herein.

[0456] The present disclosure further relates to compositions, such as pharmaceutical compositions, comprising the ISVDs or polypeptides disclosed herein.

[0457] Included herein are methods for producing an ISVD or polypeptide disclosed herein, the methods comprising: a. expressing a nucleic acid sequence encoding an ISVD or polypeptide in a host cell or host organism, or in another expression system, and then, optionally, b. isolating and / or purifying the ISVD or polypeptide.

[0458] Provided herein are compositions and polypeptides comprising an ISVD for use as pharmaceuticals. In some embodiments, the polypeptide or composition is for use in treating a proliferative disease. In some embodiments, the proliferative disease is cancer.

[0459] The present disclosure also provides methods of treatment, comprising administering to a subject in need thereof a composition or polypeptide comprising an ISVD. In some embodiments, the method of treatment is for treating a proliferative disease. In some embodiments, the proliferative disease is cancer.

[0460] Included herein are compositions and polypeptides comprising an ISVD for use in the preparation of a medicament. In some embodiments, the medicament is used in the treatment of a proliferative disease. In some embodiments, the proliferative disease is cancer.

[0461] The term "immunoglobulin single variable domain" (ISVD) defines an immunoglobulin molecule in which an antigen-binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (Fab, Fab', F(ab')2, scFv, di-scFv, etc.), in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in antigen-binding site formation, whereas in ISVDs, only three CDRs from a single domain contribute to antigen-binding site formation.

[0462] In view of the above definition, the antigen-binding domain of a conventional four-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule as known in the art), or a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulfide-linked Fv, or an scFV fragment, or diabodies derived from such conventional four-chain antibodies (all as known in the art) are not normally considered to be immunoglobulin single variable domains, since in these cases binding to the respective epitope of an antigen does not normally occur by one (single) immunoglobulin domain, but by a pair of (associated) immunoglobulin domains such as a light and heavy chain variable domain, i.e. by a VH-VL pair of immunoglobulin domains which together bind to the respective epitope of the antigen.

[0463] In contrast, an immunoglobulin single variable domain is capable of specifically binding to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH, or a single VL domain.

[0464] Thus, the single variable domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it is possible to form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, where the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0465] The immunoglobulin single variable domain (ISVD) can be a heavy chain ISVD such as a VH, VHH, including a camelized VH or a humanized VHH. In some embodiments, it is a VHH, including a camelized VH or a humanized VHH. The heavy chain ISVD can be derived from a traditional four-chain antibody or a heavy chain antibody.

[0466] For example, the immunoglobulin single variable domain may be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb", or a dAb (or an amino acid sequence suitable for use as a dAb); other single variable domain, or any suitable fragment of any of these.

[0467] In some embodiments, the immunoglobulin single variable domain may be a Nanobody® molecule or a suitable antigen-binding fragment thereof. Nanobody® is a registered trademark of Ablynx N.V.

[0468] "VHH domains," also known as VHHs, VHH regions, VHH antibody fragments, and VHH antibodies, were originally described as antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al. Nature 363:446-448, 1993). The term "VHH domain" was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (referred to herein as "VH domains," "VH regions," and "VH") and the light chain variable domains present in conventional four-chain antibodies (referred to herein as "VL domains," "VL regions," and "VL"). For a further description of VHHs, see the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0469] Typically, the production of immunoglobulins involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening naive or synthetic libraries, for example, by phage display.

[0470] The generation of immunoglobulin sequences has been extensively described in various publications, including WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001. In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The repertoire of VHHs obtained from immunization is further screened for VHHs that bind to the target antigen.

[0471] In these cases, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from natural sources or during recombinant production.

[0472] Immunization and / or screening for immunoglobulin sequences can be carried out using peptide fragments of such antigens.

[0473] The technology can use immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The technology also includes fully human, humanized, or chimeric sequences. For example, the technology includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs, as described by Ward et al. (See, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). In some embodiments, the technology also makes use of fused immunoglobulin sequences (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see Conrath et al., J. Biol. Chem., Vol. 276, 10, 7346-7350, 2001, and also, for example, WO 96 / 34103 and WO 99 / 23221), e.g., forming multivalent and / or multispecific constructs, and immunoglobulin sequences comprising tags or other functional moieties, e.g., toxins, labels, radiochemicals, etc., that are derivable from the immunoglobulin sequences of the technology.

[0474] "Humanized VHH" includes amino acid sequences that correspond to the amino acid sequence of a naturally occurring VHH domain, but in which one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (especially in the framework sequences) have been "humanized," i.e., humanized by replacing them with one or more of the amino acid residues present at the corresponding positions in a VH domain from a conventional four-chain antibody from a human (e.g., as shown above). This can be carried out in a manner known per se, as will be clear to those skilled in the art, for example, based on the further explanations herein and the prior art (e.g., WO 2008 / 020079). Again, it should be noted that such humanized VHH can be obtained in any suitable manner known per se, and is therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as starting material.

[0475] "Camelized VH" comprises an amino acid sequence which corresponds to the amino acid sequence of a naturally occurring VH domain, but which has been "camelized", i.e., camelized by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody with one or more of the amino acid residues present at the corresponding positions in a VHH domain of a heavy-chain antibody. This can be performed in a manner known per se, as will be clear to those skilled in the art, for example based on the further explanations herein and the prior art (e.g., WO 2008 / 020079). Such "camelizing" substitutions are preferably inserted at amino acid positions forming and / or present at the VH-VL interface and / or at the so-called Camelidae hallmark residues as defined herein (see, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). In some embodiments, the VH sequence used as starting material or starting point for generating or designing a camelized VH is preferably a VH sequence from a mammal, e.g. a human VH sequence, such as a VH3 sequence. However, it should be noted that such a camelized VH can be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides obtained using as starting material a polypeptide comprising a naturally occurring VHH domain.

[0476] In some embodiments, the structure of an immunoglobulin single variable domain sequence can be considered to be composed of four framework regions ("FRs"), referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively, interrupted by three complementarity-determining regions ("CDRs"), referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0477] As further described in paragraph q) of pages 58 and 59 of WO 08 / 020079, the amino acid residues of immunoglobulin single variable domains may be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from the family Camelidae in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195, see e.g., Figure 2 of this publication). It should be noted that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than permitted by the Kabat numbering). This generally means that the Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. In some embodiments, the total number of amino acid residues in the VH and VHH domains ranges from 110 to 135. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0478] The determination of the CDR regions can also be carried out according to different methods.

[0479] In some embodiments, VHH CDR sequences were determined according to the AbM definition described in Martin 2010 (In: Kontermann and Dubel (Eds.) 2010, Antibody Engineering, vol. 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 comprises amino acid residues 1-25, CDR1 comprises amino acid residues 26-35, FR2 comprises amino acid residues 36-49, CDR2 comprises amino acid residues 50-58, FR3 comprises amino acid residues 59-94, CDR3 comprises amino acid residues 95-102, and FR4 comprises amino acid residues 103-113.

[0480] In some embodiments, the CDR sequences are determined according to Kabat (Martin 2010, In: Kontermann and Dubel (eds.), Antibody Engineering Vol. 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 of the immunoglobulin single variable domain comprises amino acid residues at positions 1 to 30, CDR1 of the immunoglobulin single variable domain comprises amino acid residues at positions 31 to 35, FR2 of the immunoglobulin single variable domain comprises amino acid residues at positions 36 to 49, CDR2 of the immunoglobulin single variable domain comprises amino acid residues at positions 50 to 65, FR3 of the immunoglobulin single variable domain comprises amino acid residues at positions 66 to 94, CDR3 of the immunoglobulin single variable domain comprises amino acid residues at positions 95 to 102, and FR4 of the immunoglobulin single variable domain comprises amino acid residues at positions 103 to 113.

[0481] In some embodiments, FR1 comprises amino acid residues 1 to 25, CDR1 comprises amino acid residues 26 to 35, FR2 comprises amino acids 36 to 49, CDR2 comprises amino acid residues 50 to 58, FR3 comprises amino acid residues 59 to 94, CDR3 comprises amino acid residues 93 to 102, and FR4 comprises amino acid residues 103 to 113.

[0482] In some embodiments, FR1 comprises amino acid residues 1 to 25, CDR1 comprises amino acid residues 26 to 35, FR2 comprises amino acids 36 to 49, CDR2 comprises amino acid residues 50 to 58, FR3 comprises amino acid residues 59 to 94, CDR3 comprises amino acid residues 93 to 102, and FR4 comprises amino acid residues 103 to 126.

[0483] In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to those skilled in the art, e.g., on the basis of standard handbooks and the further disclosures and references mentioned herein.

[0484] In some embodiments, the framework sequences are immunoglobulin framework sequences or appropriate combinations of framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences can be framework sequences derived from a light chain variable domain (e.g., a VL sequence) and / or a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In some embodiments, the framework sequences are either framework sequences derived from a VHH sequence (which may optionally be partially or fully humanized), or are conventional camelized VH sequences (as defined herein).

[0485] In some embodiments, the framework sequences present in the ISVD sequences used in the technology may contain one or more of the hallmark residues (as defined herein) such that the ISVD sequence is a VHH, including a humanized or camelized VH. Some non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0486] Again, it is also possible to use any suitable fragment (or combination of fragments) of the foregoing, such as a fragment containing one or more CDR sequences suitably adjacent to and / or linked through one or more framework sequences (e.g., in the same order as those CDR and framework sequences may be present in the full-sized immunoglobulin sequence from which the fragment is derived), as generally described herein for immunoglobulin sequences.

[0487] However, it should be noted that the technology is not limited with respect to the origin of the ISVD sequence (or the nucleotide sequence used to express it), nor with respect to the manner in which the ISVD sequence or nucleotide sequence is produced or obtained (or produced or obtained). Thus, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In particular, but non-limiting aspects, the ISVD sequences are naturally occurring sequences (from any appropriate species), or synthetic or semi-synthetic sequences, including, but not limited to, "humanized" immunoglobulin sequences (as disclosed herein) (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, in particular partially or fully humanized VHH sequences), "camelized" immunoglobulin sequences (as disclosed herein), and immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR-grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art; or any suitable combination of any of the foregoing.

[0488] Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, for example a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using techniques for DNA synthesis known per se.

[0489] As mentioned above, the ISVD may be an ISVD or a suitable fragment thereof. For a general description of ISVDs, see the further description below and the references cited herein. In this regard, however, it should be noted that this specification and the prior art primarily describe ISVDs of the so-called "VH3 class" (i.e., ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class, such as DP-47, DP-51, or DP-29). However, it should be noted that the technology in its broadest sense can generally use any kind of ISVD, and also, for example, ISVDs belonging to the so-called "VH4 class" (i.e., ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class, such as DP-78), as described, for example, in WO 2007 / 118670.

[0490] Generally, an ISVD (especially a VHH sequence, including (partially) humanized and camelized VH sequences) may be characterized by the presence of one or more "hallmark residues" (as described herein) in one or more of the framework sequences (again, as further described herein). Thus, in general, an ISVD may have the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and one or more of the hallmark residues are as further defined herein.

[0491] In some embodiments, the ISVD has the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and the framework sequences are as further defined herein.

[0492] In some embodiments, the ISVD has the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to the Kabat numbering are selected from the hallmark residues referred to in Table 5 below.

[0493] [Table 6]

[0494] In some embodiments, the technology provided herein uses an ISVD capable of binding to PSMA. In the context of the present technology, "binding to" a particular target molecule has its ordinary meaning in the art, as understood in the context of antibodies and their respective antigens.

[0495] In some embodiments, the ISVD (such as a VHH) or multispecific multivalent polypeptide exhibits reduced binding by pre-existing antibodies in human serum. To this end, in some embodiments, the polypeptide exhibits a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in the ISVD. For example, the following sequence:

[0496] [Table 7] will return the following array:

[0497] [Table 8] It can be modified to be:

[0498] In some embodiments, the polypeptide exhibits an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the ISVD (e.g., the C-terminal ISVD of a fusion protein or an ISVD that is not fused to any other polypeptide). The C-terminus of the ISVD is typically VTVSS (SEQ ID NO: 574). For example, the following sequence: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549) will return the following array:

[0499] [Table 9] It can be modified to be any one of:

[0500] In some embodiments, the polypeptide exhibits a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD.

[0501] For example, the following array:

[0502] [Table 10] will return the following array:

[0503] [Table 11] It can be modified to be any one of:

[0504] In some embodiments, the ISVD presents a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) on ​​at least the ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 575), VQVSS (SEQ ID NO: 576), VTVKS (SEQ ID NO: 577), VTVQS (SEQ ID NO: 578), VKVKS (SEQ ID NO: 579), VKVQS (SEQ ID NO: 580), VQVKS (SEQ ID NO: 581), or VQVQS (SEQ ID NO: 582), such that after the addition of a single alanine, the C-terminus of the polypeptide exhibits, for example, the sequence VTVSSA (SEQ ID NO: 583), VKVSSA (SEQ ID NO: 584), VQVSSA (SEQ ID NO: 585), VTVKSA (SEQ ID NO: 586), VTVQSA (SEQ ID NO: 587), VKVKSA (SEQ ID NO: 588), VKVQSA (SEQ ID NO: 589), VQVKSA (SEQ ID NO: 590), or VQVQSA (SEQ ID NO: 591), preferably VTVSSA (SEQ ID NO: 583).

[0505] In some embodiments, the polypeptide exhibits a valine (V) at amino acid position 11 (according to Kabat numbering) and a leucine (L) at amino acid position 89 in at least the C-terminal ISVD, and optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and exhibits a (preferably naturally occurring) amino acid extension at the C-terminus of the C-terminal ISVD, such as one to five single alanine (A) extensions (such that the C-terminus of the polypeptide consists, for example, of the sequence VTVSSA (SEQ ID NO: 583), VKVSSA (SEQ ID NO: 584), or VQVSSA (SEQ ID NO: 585), preferably VTVSSA (SEQ ID NO: 583)). For further information in this regard, see, for example, WO 2012 / 175741 and WO 2015 / 173325.

[0506] As will be apparent from the above and further description herein, the ISVDs of the present technology can be used as "building blocks" to form polypeptides of the present technology that combine one or more desirable properties or biological functions in one molecule, e.g., by suitably combining them with other groups, residues, moieties, or binding units to form compounds or fusion proteins described herein (such as, but not limited to, the bivalent / trivalent / tetravalent / multivalent and bi / tri / quadruplex / multispecific polypeptides of the present technology described herein). Polypeptides having multiple ISVDs are also referred to herein as "ISVD constructs" or "ISVD formats."

[0507] The terms "specificity," "specifically binding," or "specific binding" refer to the number of different target molecules, such as antigens from the same organism, to which a particular binding unit, such as an ISVD (e.g., VHH) or scFv, can bind with sufficiently high affinity (see below). "Specificity," "specifically binding," or "specific binding" are used herein interchangeably with "selectivity," "selectively binding," or "selective binding." Binding units, such as VHHs and scFvs, preferably bind specifically to their designated targets.

[0508] The specificity / selectivity of a binding unit can be determined based on affinity, which indicates the strength or stability of a molecular interaction. Affinity is generally expressed in units of moles / liter (or M), K D is given by

[0509] Affinity is a measure of the binding strength between a moiety and a binding site on a target molecule, K D The lower the value of , the stronger the binding strength between the target molecule and the targeting moiety.

[0510] Typically, the binding units used in this technology (such as ISVD or scFv) are 10 -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12moles / liter or less, more preferably 10 -8 ~10 -12 moles / liter K D bind to their targets.

[0511] In some embodiments, 10 -4 moles / liter K D A value of 10 is considered non-specific. -4 moles / liter less than K D The values ​​are considered to be specific.

[0512] The K of a biological interaction, such as the binding of an antibody sequence to an antigen, that is considered specific D is typically in the range of 10000 nM or 10 μM to 0.001 nM or 1 pM or less.

[0513] Therefore, specific / selective binding can be measured using the same measurement method, e.g., SPR, to determine whether a binding unit (or a polypeptide comprising it) is more than 10 -5 ~10 -12 moles / liter K D Binds to PSMA at a value of 10 -4 moles / liter K D This may mean that different values ​​bind to different targets.

[0514] Thus, the ISVD preferably exhibits at least half the binding affinity, e.g., at least the same binding affinity, for human PSMA compared to an ISVD consisting of the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549), where the binding affinity is measured using the same method, such as SPR.

[0515] Specific binding to a particular target from a particular species does not exclude that the binding unit can also specifically bind to a similar target from a different species, e.g., specific binding to human PSMA does not exclude that the binding unit (or a polypeptide comprising it) can also specifically bind to PSMA from cynomolgus monkeys.

[0516] Specific binding of a binding unit to a designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as their different variations known per se in the art; and other techniques mentioned herein.

[0517] The dissociation constant can be, for example, an actual or apparent dissociation constant, as will be apparent to one skilled in the art. Methods for determining dissociation constants will be apparent to one skilled in the art and include, for example, the techniques mentioned below.

[0518] The affinity of a molecular interaction between two molecules can be measured through essentially different known techniques, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al. 2001, Intern. Immunology 13:1551-1559). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule is detected by k on , k off measurements, hence K DThe immobilized molecules are passed over the immobilized molecules under flow conditions that result in a value. This can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Jonsson et al. (1995, J. Mol. Recognit. 8:125-131), and Jonsson et al. (1991, Anal. Biochem. 198:268-277).

[0519] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the term "biolayer interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). Changes in the number of molecules bound to the biosensor chip cause a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor chip surface. Interactions can be measured in real time, allowing association and dissociation rates, as well as affinity, to be determined. BLI can be performed, for example, using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0520] Alternatively, affinity can be measured in a Kinetic Exclusion Assay (KinExA) (e.g., Drake et al. 2004, Anal. Biochem., 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrated solution of antibody / antigen complexes is passed through a column containing beads pre-coated with the antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecules. Detection of the antibody (or antigen) captured in this way is achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0521] The GYROLAB® Immunoassay System provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).

[0522] In some embodiments, the ISVD provided herein has a variability of at least about 10 as measured, e.g., by SPR, e.g., performed on a ProteOn XPR36 instrument at 25°. 3 M -1 s -1 , at least about 10 4 M -1 s -1 , and at least about 10 5 M -1 s -1 an on-rate constant (k) for binding to human PSMA selected from the group consisting of on )

[0523] In some embodiments, the ISVDs provided herein have a variability of at least about 10 as measured, e.g., by SPR, e.g., as performed on a ProteOn XPR36 instrument at 25°C. 3 M -1s -1 , at least about 10 4 M -1 s -1 , and at least about 10 5 M -1 s -1 k for binding to non-human primate PSMA selected from the group consisting of on It has.

[0524] In some embodiments, the ISVDs provided herein are at most about 10, as measured, for example, by SPR, such as performed on a ProteOn XPR36 instrument, preferably at 25° C. -2 s -1 , at most about 10 -3 s -1 , and at most about 10 -4 s -1 k for binding to human PSMA selected from the group consisting of off It has.

[0525] In some embodiments, the ISVDs provided herein are at most about 10 as measured, e.g., by SPR, e.g., as performed on a ProteOn XPR36 instrument, e.g., at 25°C. -1 s -1 , at most about 10 -2 s -1 , at most about 10 -3 s -1 , and at most about 10 -4 s -1 k for binding to non-human primate PSMA selected from the group consisting of off It has.

[0526] In some embodiments, the ISVDs provided herein are at most about 10 as measured, e.g., by SPR, e.g., as performed on a ProteOn XPR36 instrument, e.g., at 25°C. -6 M, at most about 10 -7 M, at most about 10 -8 M, at most about 10 -8 M, and at most about 10 -9Affinity (K) for binding to human PSMA selected from the group consisting of D )

[0527] In some embodiments, the ISVDs provided herein are at most about 10 as measured, e.g., by SPR, e.g., as performed on a ProteOn XPR36 instrument, e.g., at 25°C. -6 M, at most about 10 -7 M, at most about 10 -8 K for binding to non-human primate PSMA selected from the group consisting of M D It has.

[0528] In some embodiments, the PSMA-binding ISVD of the present technology has the same or a lower off rate constant (k off In some embodiments, the ISVD of the present technology binds to human PSMA with the same or lower k compared to the ISVD of QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549). off binds to non-human primate PSMA.

[0529] In some embodiments, the paTCE comprises a binding domain that is an scFv and a binding domain that is a VHH. In some embodiments, the scFv comprises VL and VH domains and specifically binds to an effector cell antigen (such as CD3), and the VHH domain specifically binds to a cancer cell antigen (such as PSMA). In some embodiments, the scFv comprises six CDRs. In some embodiments, the scFv comprises VH and VL regions that comprise amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the VL and VH sequences of a paired anti-CD3 antibody identified in Table 6a. In some embodiments, the scFv comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions of the VL and VH sequences of the paired anti-CD3 antibodies identified in Table 6a. In some embodiments, the VHH is derived from an anti-PSMA antibody identified as an antibody listed in Table 6f. In some embodiments, the VHH comprises an amino acid sequence that is at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to or identical to a VHH sequence disclosed in Table 6f. In some embodiments, the VHH comprises the CDR-1, CDR-2, and CDR-3 regions of a VHH sequence in Table 6f. In some embodiments, the scFv comprises VH and VL regions comprising amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to or identical to the VL and VH sequences of the paired anti-PSMA antibodies identified in Table 6g. In some embodiments, the scFv comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions of the VL and VH sequences of the paired anti-PSMA antibodies identified in Table 6g.

[0530] In some embodiments, the paTCE comprises a first binding domain that is an scFv and a second binding domain that is also an scFv. In some embodiments, the scFv comprises VL and VH domains derived from a monoclonal antibody with binding specificity for a tumor-specific marker or a cancer cell antigen and an effector cell antigen, respectively. In some embodiments, the first and second binding domains each comprise six CDRs derived from a monoclonal antibody with binding specificity for a cancer cell marker, such as a tumor-specific marker, and an effector cell antigen, respectively. In some embodiments, the first binding domain and the second binding domain of the first portion of the subject composition can have three, four, five, or six CDRs within each binding domain. In some embodiments, the paTCE comprises a first binding domain and a second binding domain, each comprising a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, each of which is derived from a monoclonal antibody capable of binding to a tumor-specific marker or an antigen of a cancer cell, and an effector cell antigen, respectively.

[0531] In some embodiments, the second binding domain comprises VH and VL regions derived from a monoclonal antibody capable of binding to human CD3. In some embodiments, the second binding domain comprises an scFv comprising VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or identity to the VL and VH sequences of a paired anti-CD3 antibody identified in Table 6a. In some embodiments, the second domain comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, each of which is derived from a monoclonal antibody identified herein as an antibody set forth in Table 6a. In some embodiments, the VH and / or VL domains may be configured as an scFv or diabody.

[0532] In some embodiments, the paTCE comprises a first binding domain that is a diabody and a second binding domain that is also a diabody, hi some embodiments, the diabody comprises VL and VH domains derived from monoclonal antibodies that have binding specificity for a tumor-specific marker or an antigen of cancer cells, and an effector cell antigen, respectively.

[0533] In some embodiments, the present disclosure provides paTCE compositions in which the second binding domain of the diabody comprises a VH region and a VL region, each of which exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to the VL and VH sequences of huUCHT1 in Table 6a. In some embodiments, the second domain of the diabody of the composition is derived from an anti-CD3 antibody described herein. In some embodiments, the anti-CD3 diabody is linked to an anti-PSMA-binding VHH sequence disclose...

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