Compositions targeting prostate-specific membrane antigen (PSMA), and methods for preparing and using the 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 of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 444,839, filed on 10 February 2023, and U.S. Provisional Patent Application No. 63 / 499,031, filed on 28 April 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Prostate cancer is the second most common cancer in men, and it is estimated that one in nine men in the United States will develop it at some point in their lives. Treatment with radiation, radical prostatectomy, or active surveillance for localized disease (Gleason score less than 6, PSA less than 10 ng / ml) is successful in controlling the disease in its early stages, however, recurrence occurs in 20-50% of men. Patients whose cancer continues to progress despite first-line androgen deprivation therapy (ADT) develop castration-resistant prostate cancer (CRPC), which often metastasizes to the bones, brain, liver, and lungs (metastatic castration-resistant prostate cancer, mCRPC). Chemotherapy such as docetaxel and cabazitaxel has shown improved survival in this population, but there is no treatment for mCRPC.

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

[0004] Immunotherapy has shown mixed success, including in relation to prostate cancer. While the first cell-based immunotherapy, cyplucel-T (PROVENGE®), was approved for mCRPC in 2010, checkpoint blockade targeting the immunosuppressive receptors PD-1 and CTLA-4 has shown little reduction in response rates in prostate cancer compared to other solid tumor malignancies. 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, which has been shown to be necessary for a response to checkpoint blockade inhibitors.

[0005] There has been a long-standing, unmet need for therapeutic interventions for PSMA-expressing tumors, including immunologically cold tumors. [Overview of the Initiative]

[0006] This disclosure provides, in particular, antigen-binding molecules having binding specificity to PSMA, antigen-binding molecules having binding specificity to CD3, and bispecific antigen-binding molecules that bind to both PSMA and CD3, for use in therapeutic settings where specific targeting of PSMA-expressing cells and T cell-mediated killing are desired. The embodiments disclosed herein address a long-standing unmet need for PSMA-targeted cancer therapies, including T cell engagers (TCEs) with increased therapeutic index. The embodiments of this disclosure also address a long-standing, yet unmet need for therapeutic intervention for immunologically cold tumors expressing PSMA, such as solid tumors. Also included are, for example, protease-cleavable linkers, barcode fragments, antibody domain linkers, and activatable TCEs (including those that do not bind to 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 embodiments of this disclosure include compounds, compositions, and methods for increasing the therapeutic response of a target to checkpoint inhibitors (e.g., PD-1 or CTLA-4 inhibitors such as anti-PD1 antibodies or anti-CTLA4 antibodies). In some embodiments, the compounds provided herein recruit and activate effector T cells in a major histocompatibility complex-independent manner via CD3 engagement on T cells. In some embodiments, the compounds are bispecific TCEs that are administered in an inactive form and activated at and / or within the tumor site. In some embodiments, the bispecific TCEs are administered before initiating checkpoint inhibitor therapy, concurrently with checkpoint inhibitor therapy, or after discontinuing checkpoint inhibitor therapy.

[0008] 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 that specifically binds to prostate-specific membrane antigen (PSMA) and a second antigen-binding domain that binds to cluster of differentiation 3 (CD3), wherein the first antigen-binding domain is VHH or the second antigen-binding domain is Fab or scFV, and the chimeric polypeptide further comprises a mask polypeptide conjugated to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the mask polypeptide and the bispecific antibody domain, wherein the mask polypeptide is capable of reducing the 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 the tumor.

[0009] 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 that specifically binds to a protease-specific membrane antigen (PSMA) and a second antigen-binding domain that binds to the differentiated antigen group 3 T cell receptor (CD3), and the chimeric polypeptide further comprises a mask polypeptide, which is conjugated to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the mask polypeptide and the bispecific antibody domain, thereby enabling the mask polypeptide to reduce the binding of the bispecific antibody domain to CD3 or PSMA, and the protease-cleavable release segment is not cleavable by legmine in human plasma, or legmine cleaves the protease-cleavable release segment in human plasma at a rate less than 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmine.

[0010] In some embodiments, the chimeric polypeptide includes a structural arrangement defined from the N-terminus to the C-terminus 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 hyphen is a covalent linkage or polypeptide linker.

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

[0012] In some embodiments, the linker further includes 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 by (i) at least 90% of its amino acids being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) containing 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 long.

[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 can be cleaved by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

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

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

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

[0021] Certain aspects of the present disclosure are chimeric polypeptides comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain having binding specificity to a cancer cell antigen and a second antigen-binding domain having binding specificity to an effector cell antigen expressed on an effector cell, and the chimeric polypeptide further comprises a first ELNN, which is conjugated to the first antigen-binding domain via a first linker comprising a first protease-cleavable release segment (RS1) located between the first ELNN and the first antigen-binding domain, thereby enabling the first ELNN to reduce the binding of the first antigen-binding domain to the cancer cell antigen, and RS1 The present invention relates to a chimeric polypeptide comprising: 1 being cleavable by at least one protease present in the tumor, the chimeric polypeptide further comprising a second ELNN which is a second ELNN conjugated to the second antigen-binding domain via a second linker containing a second protease-cleavable release segment (RS2) located between the second ELNN and the second antigen-binding domain, thereby enabling the second ELNN to reduce the binding of the first antigen-binding domain to the effector cell antigen, RS2 being cleavable by at least one protease present in the tumor, the first ELNN having a shorter amino acid sequence than the second ELNN, and the cancer cell antigen being not HER2.

[0022] In some embodiments, the chimeric polypeptide includes a structural arrangement defined as (ELNN1)-(linker 1)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 2)-(ELNN2), (ELNN1)-(linker 1)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 2)-(ELNN2), (ELNN2)-(linker 2)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 1)-(ELNN1), or (ELNN2)-(linker 2)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 1)-(ELNN1), where each is individually a covalent bond or polypeptide linker.

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

[0024] In some embodiments, linker 1 further includes a first spacer (spacer 1). In some embodiments, linker 2 further includes 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 includes a structural arrangement defined as (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 polypeptide linker.

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

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

[0029] Certain aspects of this disclosure are chimeric polypeptides comprising a bispecific antibody domain, comprising a formula from N-terminus to C-terminus including: 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 to cancer cell antigens, and the second antigen-binding domain is expressed on effector cells. The chimeric polypeptide has binding specificity to an antigen cell antigen, each comprising a covalent linkage or polypeptide linker, wherein Mask 1 is a polypeptide capable of reducing the binding of a first antigen-binding domain to its target, and Mask 2 is a polypeptide capable of reducing the binding of a second antigen-binding domain to its target, wherein if the chimeric polypeptide contains formula 1, Spacer 1 consists of A, E, G, S, P, and / or T residues, if the chimeric polypeptide contains formula 2, Spacer 2 consists of A, E, G, S, P, and / or T residues, and if the chimeric polypeptide contains formula 3, Spacer 1 and / or Spacer 2 consist of A, E, G, S, P, and / or T residues, and the chimeric polypeptide is targeted in which the cancer cell antigen is not HER2.

[0030] In some embodiments, each - is individually a covalent linkage. 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, cancer cell antigens include 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, 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), Muellerian 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 (Cora These include antigens such as 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 the differentiation antigen group 3 T cell receptor (CD3).

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

[0034] In some embodiments, the second antigen-binding domain has binding specificity to 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 acid long.

[0040] In some embodiments, spacer 1 and / or spacer 2 contain 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 can be cleaved by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0043] A chimeric polypeptide according to any one of claims 42 to 47, wherein spacer 1 and / or spacer 2 include amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the sequences listed in Table C. In some embodiments, spacer 1 and / or spacer 2 include amino acid sequences having at least 85% identity with respect to the sequences listed in Table C. In some embodiments, spacer 1 and / or spacer 2 include amino acid sequences having at least 90% identity with respect to the sequences listed in Table C. In some embodiments, spacer 1 and / or spacer 2 include amino acid sequences having at least 91% identity with respect to the sequences listed in Table C. In some embodiments, spacer 1 and / or spacer 2 include amino acid sequences having at least 92% identity with respect to the sequences listed in Table C. In some embodiments, Spacer 1 and / or Spacer 2 include an amino acid sequence having at least 93% identity with the sequences listed in Table C. In some embodiments, Spacer 1 and / or Spacer 2 include an amino acid sequence having at least 94% identity with the sequences listed in Table C. In some embodiments, Spacer 1 and / or Spacer 2 include an amino acid sequence having at least 95% identity with the sequences listed in Table C. In some embodiments, Spacer 1 and / or Spacer 2 include an amino acid sequence having at least 96% identity with the sequences listed in Table C. In some embodiments, Spacer 1 and / or Spacer 2 include an amino acid sequence having at least 97% identity with the sequences listed in Table C. In some embodiments, Spacer 1 and / or Spacer 2 include an amino acid sequence having at least 98% identity with the sequences listed in Table C. In some embodiments, Spacer 1 and / or Spacer 2 include an amino acid sequence having at least 99% identity with the sequences listed in Table C. In some embodiments, spacer 1 and / or spacer 2 contain amino acid sequences that have 100% identity with the sequences listed in Table C.

[0044] In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 85% identity with respect to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 90% identity with respect to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 91% identity with respect to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 92% identity with respect to GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 93% identity with GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 94% identity with GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 94% identity with GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 95% identity with GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 96% identity with GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 97% identity with GTSESATPES or GTATPESGPG.In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 98% identity with GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 99% identity with GTSESATPES or GTATPESGPG. In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having 100% identity with 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 long, and the amino acid sequence of the second ELNN is about 582 amino acids long.

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

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

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

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

[0050] In some embodiments, the antibody domain linker consists of G and S amino residues. In some embodiments, the antibody domain linker is approximately 9 residues long. In some embodiments, the antibody domain linker contains the amino acid sequence GGGGSGGGS.

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

[0052] In some embodiments, the linker is characterized by (i) at least 90% of its amino acids being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) containing 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 domain and the VH domain is 25-35 amino acids long.

[0054] In some embodiments, the linker between the VL domain and the VH domain contains 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 domain and the VH domain consist of A, E, G, S, P, and / or T.

[0056] In some embodiments, the linker between the VL domain and the VH domain can be cleaved 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 contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0058] In some embodiments, the linker between the VL domain and the VH domain includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 including three VHH complementarity determining regions (CDRs), the three VHH CDRs comprising CDR1, CDR2, and CDR3 of a VHH domain containing the following amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0060] In some embodiments, the second antigen-binding domain comprises a VL domain containing three VL CDRs, the three VL CDRs comprising CDR1, CDR2, and CDR3 of a VL domain containing the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSX1GAVTX2SNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX3YYCALWYX4NLWVFGGGTKLTVL, where 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, and the three VL CDRs comprise CDR1, CDR2, and CDR3 of a 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, and the three VH CDRs comprise the following amino acid sequence: EVQLX5ESGGGX6VQPGGSLX7LSCAASGFTFX8TYAMNWVRQAPGKGLEWVX9RIRX ,

[0063] , , 11 , 16 , 10 , 14 , 15 , 12 , 16 , 13 , 14 , 11 ، 15 , 12 , 13 KX 11 NNYATYYADSVKX 12 RFTISRDDSKNTX 13 YLQMNX 14 LKTEDTAVYYCVRHX 15 NFGNSYVSWFAX 16 WGQGTLVTVSS, and comprise CDR1, CDR2, and CDR3 of a VH domain, wherein 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, X 10 corresponds to T or S, X 11 corresponds to R or Y, X 12 corresponds to G or D, X 13 corresponds to V or A, X 14 corresponds to S or N, X 15 corresponds to E or G, X 16 corresponds to H or Y.

[0063] In some embodiments, the second antigen-binding domain comprises a VH domain containing three VH CDRs, the three VH CDRs comprising CDR1, CDR2, and CDR3 of a VH domain containing 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 / 7 Includes a pair of VL domain amino acid sequence number / VH domain amino acid sequence number selected from the group consisting of 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 is a VHH containing 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 CDR:GRTFGIYVWG; and a VHH containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to CDR1;AMSWSGSNRKVSDSVKG. VHH containing amino acid sequences that have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to CDR2; and AASNKEYGRTWYDFNESDY. (ii) a VHH containing CDR3, and the second antigen-binding domain is 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 CDR:RSSX1GAVTX2SNYAN (wherein X1 corresponds to T or N, and X2 corresponds to T or S), or 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 VL domain CDR1;GTNKRAP. VL domain CDR2;ALWYX4NLWV (wherein X4 corresponds to S or P) containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VL domain CDR3;GFTFX8TYAMN (wherein X8 corresponds to S or N) containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VL domain CDR3;GFTFX8TYAMN (wherein X8 corresponds to S or N) containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VL domain CDR1;RIRX 10 KX 11 NNYATYYADSVKX 12 (In the formula, X 10 However, it corresponds to T or S, X 11 However, it corresponds to R or Y, and X12 VH domain CDR2;HX containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to (corresponding to G or D) 15 NFGNSYVSWFAX 16 (In the formula, X 15 However, it corresponds to E or G, and X 16 The VH domain CDR3 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to H or Y.

[0066] In some embodiments, (i) the first antigen-binding domain is a VHH containing 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 CDR:GRTFGIYVWG; and a VHH containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to CDR1;AMSWSGSNRKVSDSVKG. VHH containing amino acid sequences that have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to CDR2; and AASNKEYGRTWYDFNESDY. (ii) a VHH containing CDR3, and the second antigen-binding domain contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs:RSSNGAVTSSNYAN; VL domain CDR2 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV VL domain CDR3 containing an amino acid sequence having 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity; VH domain CDR1 containing 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; VH domain CDR2 containing 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 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to HENFGNSYVSWFAH.

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

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

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

[0070] In some embodiments, (i) the first antigen-binding domain is a VHH CDR1 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with respect to the following CDRs:GRTFGIYVWG; VHH CDR2 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with respect to (ii) a VHH containing CDR3, and the second antigen-binding domain contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs:RSSNGAVTSSNYAN; VL domain CDR2 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV VL domain CDR3 containing an amino acid sequence having 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity; VH domain CDR1 containing 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; VH domain CDR2 containing 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 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to HENFGNSYVSWFAH.

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

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

[0073] In some embodiments, the second antigen-binding domain comprises a VL domain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to ELVVTQEPSLTVSPGGTVTLTCRSSX1GAVTX2SNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX3YYCALWYX4NLWVFGGGTKLTVL, where 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 includes 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, with respect 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 16The formula comprises a VH domain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with X5 corresponding to V or L, X6 corresponding to I or L, X7 corresponding to R or K, X8 corresponding to S or N, X9 corresponding to G or A, and X 10 However, it corresponds to T or S, X 11 However, it corresponds to R or Y, and X 12 However, it corresponds to G or D, and X 13 However, it corresponds to V or A, X 14 However, it corresponds to S or N, and X 15 However, it corresponds to E or G, and X 16 However, it corresponds to H or Y.

[0076] In some embodiments, the second antigen-binding domain includes 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, with respect to EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

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

[0078] In some embodiments, the second antigen-binding domain includes 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, with respect to ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS.

[0079] In some embodiments, the first antigen-binding domain includes 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, with respect to the amino acid sequences 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 SNK list 22 VHH containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to YGRTWYDFNESDYWGQGTQVTVSS, wherein X 17 , X 18 , X 19 , X20 , X 21 , and X6 each correspond individually to any naturally occurring amino acid. In some embodiments, X 17 It corresponds to M or W, and X 18 This corresponds to M or I, and X 19 This corresponds to F or Y, and X 20 This corresponds to A or G, and X 21 It corresponds to A or G, and / or X 22 These correspond to L, W, R, D, E, or G.

[0081] In some embodiments, the first antigen-binding domain includes 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, with respect to QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0082] In some embodiments, the RS includes a protease cleavage site that can be cleaved by at least one protease listed in Table 7.

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

[0084] In some embodiments, RS can be disconnected by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.

[0085] In some embodiments, RS is not cleavable by legmine. In some embodiments, RS is not cleavable by legmine in human blood, plasma, or serum. In some embodiments, RS is not cleavable during incubation with legmine at a concentration of about 1 nM or less for about 20 hours. In some embodiments, RS is not cleavable during incubation with legmine at a concentration of about 1 nM or less in human blood, plasma, or serum for about 20 hours.

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

[0087] In some embodiments, RS1 and / or RS2 include protease cleavage that can be cleaved by at least one protease listed in Table 7.

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

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

[0090] In some embodiments, RS1 and / or RS2 are not cleavable by legmine. In some embodiments, RS1 and / or RS2 are not cleavable by legmine in human blood, plasma, or serum. In some embodiments, RS1 and / or RS2 are not cleavable during incubation with legmine at a concentration of about 1 nM or less for about 20 hours. In some embodiments, RS1 and / or RS2 are not cleavable during incubation with legmine at a concentration of about 1 nM or less in human blood, plasma, or serum for about 20 hours.

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

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

[0094] In some embodiments, RS1 and / or RS2 include 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 releasing segment is a first protease-cleavable releasing segment (RS1), and the chimeric polypeptide further comprises a second mask polypeptide and a second protease-cleavable releasing segment (RS2), wherein the second mask polypeptide is conjugated to the second antigen-binding domain via a second protease-cleavable releasing segment (RS2) located between the second mask polypeptide and the second antigen-binding domain, thereby reducing the 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, the first mask polypeptide is bound to a first antigen-binding domain, and the second mask polypeptide is bound to a second antigen-binding domain.

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

[0099] In some embodiments, the first ELNN and the second ELNN are each 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 contains 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 further characterized in that (i) each contains at least 100 amino acid residues, and (ii) each contains a plurality of non-overlapping sequence motifs having a length of 9 to 14 amino acids, the plurality of non-overlapping sequence motifs comprising a set of non-overlapping sequence motifs, and each non-overlapping sequence motif in the set of non-overlapping sequence motifs is repeated at least twice in the ELNN.

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

[0102] In some embodiments, each of the first ELNN and the second ELNN contains at least four amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, each of the amino acids in the first ELNN and the second ELNN consists 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 long, and the amino acid sequence of the second ELNN is about 582 amino acids long.

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

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

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

[0108] In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that can be released 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 includes 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 It includes at least one of EPSE.SATP, In the formula, each "." is a Glu-C cleavage site, and n is any integer from 0 to 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 Includes at least one of STPE.SGPG, In the formula, each "." represents a Glu-C cleavage site, and n is any integer between 0 and 30.

[0111] 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.

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

[0113] In some embodiments, X n These are 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 acids of the chimeric polypeptide are included in the barcode fragment.

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

[0116] In some embodiments, the chimeric polypeptide comprises a first ELNN containing a first barcode fragment and a second ELNN containing a second barcode fragment, wherein neither the first nor the second barcode fragment contains glutamic acid directly 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 contains 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 preceding glutamic acid.

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

[0120] In some embodiments, at least one of the barcode segments does not contain the second glutamate at any position other than the C-terminus of the barcode segment, unless proline follows immediately after the second glutamate.

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

[0122] In some embodiments, at least one of the barcode segments is at least 4 amino acids long.

[0123] In some embodiments, at least one of the barcode segments is 4-20, 5-15, 6-12, or 7-10 amino acid long.

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

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

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

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

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

[0131]

[0132]

[0133] Certain embodiments of this disclosure relate 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 frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted before administration.

[0134] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0135] Certain aspects of this disclosure relate to polynucleotide sequences encoding chimeric polypeptides described herein.

[0136] Certain aspects of this disclosure relate to expression vectors comprising polynucleotides as described herein.

[0137] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0138] Certain aspects of this disclosure relate to methods for producing chimeric polypeptides as described herein. In some embodiments, the methods further include isolating the chimeric polypeptide from host cells.

[0139] Certain aspects of this disclosure relate to methods for treating cancer in subjects that require treatment, the method comprising the step of administering an effective amount of a chimeric polypeptide described herein to the subject.

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

[0141] In some embodiments, the method further includes administering docetaxel to the target.

[0142] In some embodiments, the method further includes administering a checkpoint inhibitor to the target. 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 cemiprimab.

[0143] Certain aspects of this disclosure relate to linker polypeptides containing amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides containing amino acid sequences having at least 85% identity with respect to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides containing amino acid sequences having at least 90% identity with respect to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides containing amino acid sequences having at least 91% identity with respect to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 92% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 93% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 94% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 95% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 96% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 97% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 98% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having at least 99% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of this disclosure relate to linker polypeptides comprising an amino acid sequence having 100% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

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

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

[0146] Certain aspects of this disclosure relate 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, with respect to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

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

[0148] In some embodiments, the antigen-binding polypeptide is 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 the N-terminus of the VH domain.

[0151] Certain aspects of this disclosure relate to pharmaceutical compositions comprising a linker polypeptide or antigen-binding polypeptide as described herein and at least one pharmaceutically acceptable excipient.

[0152] 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.

[0153] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0154] Certain aspects of this disclosure relate to a linker or a polynucleotide sequence encoding an antigen-binding polypeptide as described herein.

[0155] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0156] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0157] Certain aspects of this disclosure relate to methods for producing the linker or antigen-binding polypeptide described herein. In some embodiments, the method further includes isolating the linker or antigen-binding polypeptide from host cells.

[0158] Certain embodiments of this disclosure relate to isolated polypeptides comprising a protease-cleavable amino acid sequence including sequence:EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S.

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

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

[0161] Certain embodiments of this disclosure relate to pharmaceutical compositions comprising isolated polypeptides described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions are in liquid form or frozen. In some embodiments, the pharmaceutical compositions are formulated as lyophilized powders or cakes that are reconstituted before administration.

[0162] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0163] Certain aspects of this disclosure relate to polynucleotide sequences encoding isolated polypeptides described herein.

[0164] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0165] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0166] Certain embodiments of this disclosure relate to methods for producing isolated polypeptides as described herein. In some embodiments, the methods further include isolating the isolated polypeptide from host cells.

[0167] Certain embodiments of this disclosure relate to a fusion protein comprising a protease-cleavable amino acid sequence comprising the sequence:EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N, and the protease-cleavable amino acid sequence ligates a first polypeptide portion to a second polypeptide portion. In some embodiments, X is S.

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

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

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

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

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

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

[0174] In some embodiments, the fusion protein comprises a single polypeptide chain comprising a first polypeptide, followed by a protease-cleavable amino acid sequence, and then a second polypeptide moiety, in the direction from the N-terminus to the C-terminus. In some embodiments, the fusion protein comprises a single polypeptide chain comprising a second polypeptide, followed by a protease-cleavable amino acid sequence, and then a first polypeptide moiety, in the direction from the N-terminus to the C-terminus.

[0175] Certain embodiments of this disclosure relate 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 frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted before administration.

[0176] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0177] Certain aspects of this disclosure relate to polynucleotide sequences encoding fusion proteins described herein.

[0178] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0179] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0180] Certain aspects of this disclosure relate to methods for producing fusion proteins as described herein. In some embodiments, the methods further include isolating the fusion protein from a host cell.

[0181] Certain aspects of this disclosure relate to ELNN polypeptides comprising the following amino acid sequence: ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP.

[0182] Certain aspects of this disclosure relate to ELNN polypeptides comprising the following amino acid sequence:

[0183] Certain aspects of this disclosure relate to fusion proteins comprising the ELNN polypeptide described herein.

[0184] Certain embodiments of this disclosure relate to pharmaceutical compositions comprising an ELNN polypeptide or fusion protein as 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 this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0186] Certain aspects of this disclosure relate to polynucleotide sequences encoding ELNN polypeptides or fusion proteins as described herein.

[0187] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0188] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0189] Certain aspects of this disclosure relate to methods for producing the ELNN polypeptide or the fusion protein described herein.

[0190] The method of claim 288, further comprising, in some embodiments, isolating an ELNN polypeptide or fusion protein from a host cell.

[0191] Certain aspects of this disclosure relate to barcode fragments containing amino acid sequences 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, SGPGTGSTSTPE, TPGSEPATSGSE, GSAPGTSTEPSE, SGPGTAGSGTPE, SGPGTSSGGTPE, SGPGTAGPATPE, SGPGTPGTGTPE, SGPGTGGPTTPE, or SGPGTGSGSTPE.

[0192] Certain aspects of this disclosure relate to barcode fragments containing amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to SGPGSGPGTSE or SGPGTSPSATPE.

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

[0194] Certain aspects of this disclosure relate to fusion proteins comprising barcode fragments as described herein.

[0195] Certain aspects of this disclosure relate to fusion proteins containing a Glu-C cleavage site which includes one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0196] Certain aspects of this disclosure include 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 The subject is a fusion protein containing at least one of EPSE.SATP, wherein each "." in the formula is a Glu-c cleavage site and n is any integer from 0 to 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 The formula includes at least one of STPE.SGPG, where each "." is a Glu-C cleavage site and n is any integer between 0 and 30.

[0198] 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.

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

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

[0201] Certain embodiments of this disclosure relate to pharmaceutical compositions comprising a barcode fragment or fusion protein as 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.

[0202] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0203] Certain aspects of this disclosure relate to polynucleotide sequences encoding barcode fragments or fusion proteins described herein.

[0204] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0205] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0206] Certain aspects of this disclosure relate to methods for producing barcode fragments or fusion proteins as described herein. In some embodiments, the method further includes isolating the barcode fragment or fusion protein from a host cell.

[0207] Certain aspects of this disclosure relate 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 aspects of this disclosure relate to antibodies or antigen-binding fragments thereof that specifically bind to PSMA and include an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the following CDR:GRTFGIYVWG; and VHH1;AMSWSGSNRKVSDSVKG. The target is an antibody or its antigen-binding fragment containing a VHH CDR3 that has an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to CDR2 and AASNKEYGRTWYDFNESDY.

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

[0210] Certain aspects of this disclosure include antibodies or antigen-binding fragments thereof that specifically bind to PSMA, and which include VHH CDR1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the following CDRs:GRTFGIYVWG; VHH CDR2 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with AMSWSGSNRK; and VHH CDR2 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with AASNKEYGRTWYDFNESDY. The target is antibodies or their antigen-binding fragments, including CDR3.

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

[0212] Certain aspects of this disclosure relate to antibodies or antigen-binding fragments thereof that specifically bind to PSMA and include VHH containing 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 this disclosure relate to antibodies or antigen-binding fragments thereof that specifically bind to PSMA and include 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, with respect to PSMA.2, PSMA.3, PSMA.5, PSMA.6, PSMA.262, or PSMA.263.

[0215] Certain aspects of this disclosure are antibodies or antigen-binding fragments thereof that specifically bind to PSMA, and QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVX 17 GWFRQAPGKEREFVGAX 18 SWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYX 19 CX 20 X 21 SNK list 22 VHH containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to YGRTWYDFNESDYWGQGTQVTVSS, wherein X 17 , X 18 , X 19 , X 20 , X 21 X6, and X6, each individually, target an antibody or its antigen-binding fragment corresponding to any naturally occurring amino acid. In some embodiments, X 17 It corresponds to M or W, and X 18 This corresponds to M or I, and X 19 This corresponds to F or Y, and X 20 This corresponds to A or G, and X 21 It corresponds to A or G, and / or X 22 These correspond to L, W, R, D, E, or G.

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

[0217] 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 (i) the VL domain comprises a VL CDR of the amino acid sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), or (ii) the VH domain comprises a VH CDR of the amino acid sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

[0218] Certain aspects of this disclosure relate to an anti-CD3 antibody or its antigen-binding fragment having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the following CDR:RSSNGAVTSSNYAN, or at least 85%, 90%, 91%, 92%, 93%, or 94% identity to the VL domain CDR1;GTNKRAP. VL domain CDR2 containing an amino acid sequence having 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, ALWYPNLWV; VL domain CDR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, GFTFSTYAMN VH domain CDR1;RIRTKRNNYATYYADSVKG containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, or 1 The target is an anti-CD3 antibody or its antigen-binding fragment containing one or more of the following: a VH domain CDR2 containing an amino acid sequence having 00% identity; and / or a VH domain CDR3 containing 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 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following FR:ELVVTQEPSLTVSPGGTVTLTC, and an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the VL domain FR1;WVQQKPGQAPRGLIG. VL domain FR3 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to VL domain FR2;GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC; and VL domain F contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to VL domain F VH domain FR2;RFTISRDDS contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VH domain FR1;WVRQAPGKGLEWVG, which. It comprises one or more of the following: VH domain FR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to KNTVYLQMNSLKTEDTAVYYCVR; and / or VH domain FR4 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to WGQGTLVTVSS.

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

[0221] In some embodiments, the VL domain contains 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 includes a VH domain.

[0223] In some embodiments, the VH domain contains 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 this disclosure include an antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising a VL domain and a VH domain, wherein the VL domain amino acid sequence number / VH domain amino acid sequence number pairs are 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 The target is an antibody or its antigen-binding fragment selected from the group consisting of 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 a 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 scFv.

[0229] In some embodiments, the VL domain is the N-terminus of the VH domain in scFv.

[0230] In some embodiments, the VL domain is the C-terminus of the VH domain in the scFv.

[0231] In some embodiments, scFv includes a linker between the VL domain and the VH domain, the linker consisting of A, E, G, S, P, and / or T residues.

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

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

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

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

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

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

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

[0239] 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.

[0240] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0241] Certain aspects of this disclosure relate to polynucleotide sequences encoding antibodies or antigen-binding fragments thereof as described herein.

[0242] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0243] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0244] Certain aspects of this disclosure relate to methods for producing antibodies or antigen-binding fragments thereof as described herein. In some embodiments, the methods further include isolating the antibody or antigen-binding fragment from host cells.

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

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

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

[0248] Certain embodiments of this disclosure relate to a pharmaceutical composition comprising a multispecific antibody 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.

[0249] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0250] Certain aspects of this disclosure relate to polynucleotide sequences encoding multispecific antibodies described herein.

[0251] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0252] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0253] Certain aspects of this disclosure relate to methods for producing multispecific antibodies as described herein. In some embodiments, the methods further include isolating the multispecific antibodies from host cells.

[0254] Certain aspects of the present disclosure are T cell engagers comprising a first antigen-binding domain that binds to prostate-specific membrane antigen (PSMA) and a second antigen-binding domain that binds to the differentiated antigen group 3 T cell receptor (CD3), wherein the first antigen-binding domain comprises a VHH 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 ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGT A VL domain containing 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 EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG The target is a T cell engager comprising a VH domain containing 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 embodiments of this disclosure relate to a pharmaceutical composition 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 frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted before administration.

[0256] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0257] Certain aspects of this disclosure relate to polynucleotide sequences encoding T cell engagers described herein.

[0258] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0259] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0260] Certain aspects of this disclosure relate to methods for producing T cell engagers as described herein. In some embodiments, the methods further include isolating the T cell engagers from host cells.

[0261] Certain aspects of the present disclosure relate 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, TCE contains anti-PSMA VHH comprising the following amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).

[0264] In some embodiments, TCE contains 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 embodiments of this disclosure relate to pharmaceutical compositions comprising a 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 before administration.

[0266] Certain aspects of this disclosure relate to injection devices comprising the pharmaceutical compositions described herein. In some embodiments, the injection device includes a syringe.

[0267] Certain aspects of this disclosure relate to polynucleotide sequences encoding paTCE as described herein.

[0268] Certain aspects of this disclosure relate to expression vectors comprising the polynucleotide sequences described herein.

[0269] Certain aspects of this disclosure involve host cells containing the expression vector described herein.

[0270] Certain aspects of this disclosure relate to methods for producing paTCE as described herein. In some embodiments, the method further includes isolating paTCE from host cells.

[0271] Certain aspects of this disclosure relate to chimeric polypeptides, isolated polypeptides, fusion proteins, antigen-binding polypeptides, antibodies or antigen-binding fragments thereof that specifically bind to PSMA, antibodies or antigen-binding fragments thereof that specifically bind to CD3, multispecific antibodies, T cell engagers, or paTCEs produced by the methods described herein.

[0272] Certain aspects of this disclosure relate to a polynucleotide sequence encoding the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is an amino acid sequence other than N.

[0273] In some embodiments, polynucleotides are vectors.

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

[0275] Certain aspects of this disclosure relate to cell lines expressing an exogenous polynucleotide comprising the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is an amino acid sequence other than N.

[0276] In some embodiments, the exogenous polypeptide is the 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 located in a bioreactor.

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

[0280] In some embodiments, the cells are mammalian cells.

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

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

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

[0284] Certain embodiments of this disclosure relate to non-human animals comprising an exogenous polypeptide having the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than 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 this disclosure relate to fusion proteins comprising an anti-PSMA antibody or fragment described herein and a biologically active protein.

[0286] Certain aspects of this disclosure relate 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 includes cytokines, enzymes, hormones, growth factors, chemotherapy polypeptides, antiviral polypeptides, or toxins.

[0288] Certain aspects of this disclosure relate to immunoconjugates comprising an anti-PSMA antibody or fragment described herein and a compound.

[0289] Certain aspects of this disclosure relate to immunoconjugates comprising an anti-CD3 antibody or fragment described herein and a compound.

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

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

[0292] In some embodiments, the compound includes a toxin, a radioactive molecule, a contrast agent, or a drug.

[0293] This disclosure relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising a heavy chain variable region (VH) containing three heavy chain CDRs and a light chain variable region (VL) containing three light chain CDRs, wherein the three heavy chain CDRs are derived from CD3 from EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311). The present invention provides an antibody or its antigen-binding fragment comprising R1, CDR2, and CDR3, wherein the three light chain CDRs comprise CDR1, CDR2, and CDR3 from ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), and the CDRs are identified by Kabat definition, Chothia definition, AbM definition, IMGT definition, or contact definition. In some embodiments, the antibody is scFv.

[0294] This specification includes antigen-binding proteins comprising (i) a light chain variable domain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, identical to a light chain variable domain sequence comprising a CDR sequence 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%, or 100%, identical to a heavy chain variable domain sequence comprising a CDR sequence 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 scFv.

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

[0296] This specification includes isolated antibodies or antigen-binding fragments thereof that specifically bind to CD3, comprising the amino acid sequence DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 1043), comprising a heavy chain variable region containing three heavy chain CDRs, and a light chain variable region containing three light chain CDRs, wherein the three heavy chain CDRs are EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIR The isolated antibody or its antigen-binding fragment contains CDR1, CDR2, and CDR3 from TKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311), and the three light chain CDRs contain CDR1, CDR2, and CDR3 from ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361). In some embodiments, the heavy chain CDR1 includes GFTFSTYAMN, the heavy chain CDR2 includes RIRTKRNNYATYYADSVKG, the heavy chain CDR3 includes HENFGNSYVSWFAH, the light chain CDR1 includes RSSNGAVTSSNYAN, the light chain CDR2 includes GTNKRAP, and the light chain CDR3 includes ALWYPNLWV. In some embodiments, the heavy chain variable region includes EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).In some embodiments, the light chain variable region includes ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361). In some embodiments, the heavy chain variable region includes EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311), and the light chain variable region includes ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0297] In some embodiments, as included herein, is a K of about 300 nM or less, measured by surface plasmon resonance, for example. D The antibody or antigen-binding fragment thereof specifically binds to CD3 (for example, 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). In some embodiments, the antibody or antigen-binding portion thereof has a K content of about 200 nM or less, about 150 nM or less, about 100 nM or less, or about 75 nM or less. D It exhibits this characteristic.

[0298] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD3, comprising a heavy chain variable region and a light chain variable region (for example, 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), wherein the antibody or antigen-binding fragment thereof has (a) an affinity (K) for CD3 of about 100 nM or less. D(b) an affinity for CD3 of approximately 300 nM or less (K D A heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (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 shown in ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), or (c) affinity for CD3 of about 75 nM or less (K DAn antibody or its antigen-binding fragment comprising a heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (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 shown in ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

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

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

[0301] This specification includes isolated antibodies or antigen-binding fragments thereof that specifically bind to PSMA, and which include the amino acid sequence of (SEQ ID NO: 1044), which includes a VHH region containing three VHH CDRs from QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549).In some embodiments, VHH CDR1 includes GRTFGIYVWG, VHH CDR2 includes AMSWSGSNRK, and VHH CDR3 includes AASNKEYGRTWYDFNESDY. In some embodiments, the VHH region includes QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (Sequence ID 549).

[0302] In some embodiments, as included herein, is a K of about 300 nM or less, measured by surface plasmon resonance, for example. D The antibody or antigen-binding portion specifically binds to PSMA (for example, a protein having the amino acid sequence of SEQ ID NO: 549). In some embodiments, the antibody or antigen-binding portion has a K content of about 200 nM or less, about 150 nM or less, about 100 nM or less, or about 50 nM or less. D It exhibits this characteristic.

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

[0304] This disclosure includes a bispecific T cell engager comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human CD3 as provided herein (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), and (ii) an antibody or antigen-binding fragment thereof that specifically binds to human PSMA as provided herein (SEQ ID NO: 549).

[0305] Various features of this disclosure are specifically described in the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0306] [Figure 1A] An example of a non-restrictive schematic diagram of paTCE is shown. [Figure 1B]Figure 1A shows schematic diagrams of fully unmasked paTCE (uTCE) from the exemplary paTCE shown, as well as the individually masked metabolites paTCE(1x-N) and paTCE(1x-C). [Figure 2A] The biophysical characterization data of the PMSA.2 variant antibody is shown. Figure 2A shows the concentration of the PMSA.2 variant antibody. Figure 2B shows the relative binding of the PMSA.2 variant antibody to PSMA. Figure 2C shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 65°C. The AC clone numbers of the tested uTCE are shown in the figures. These experiments used uTCE, not paTCE. [Figure 2B] The biophysical characterization data of the PMSA.2 variant antibody is shown. Figure 2A shows the concentration of the PMSA.2 variant antibody. Figure 2B shows the relative binding of the PMSA.2 variant antibody to PSMA. Figure 2C shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 65°C. The AC clone numbers of the tested uTCE are shown in the figures. These experiments used uTCE, not paTCE. [Figure 2C] The biophysical characterization data of the PMSA.2 variant antibody is shown. Figure 2A shows the concentration of the PMSA.2 variant antibody. Figure 2B shows the relative binding of the PMSA.2 variant antibody to PSMA. Figure 2C shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 65°C. The AC clone numbers of the tested uTCE are shown in the figures. These experiments used uTCE, not paTCE. [Figure 2D]The biophysical characterization data of the PMSA.2 variant antibody is shown. Figure 2A shows the concentration of the PMSA.2 variant antibody. Figure 2B shows the relative binding of the PMSA.2 variant antibody to PSMA. Figure 2C shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 62°C. Figure 2D shows the thermal stability of the PMSA.2 variant antibody, measured by monomer concentration (pM) at 65°C. The AC clone numbers of the tested uTCE are shown in the figures. These experiments used uTCE, not paTCE. [Figure 3A] The biophysical characterization data for the PMSA.3 variant antibody are shown. Figure 3A shows the relative binding of the PMSA.3 variant antibody to PSMA. Figures 3B and 3C show the thermal stability of the PMSA.3 variant antibody, measured by monomer concentration (Figure 3B) or aggregate concentration (Figure 3C) (pM) at 63.5°C. The AC clone numbers of the tested uTCE are shown in the figures. These experiments used uTCE, not paTCE. [Figure 3B] The biophysical characterization data for the PMSA.3 variant antibody are shown. Figure 3A shows the relative binding of the PMSA.3 variant antibody to PSMA. Figures 3B and 3C show the thermal stability of the PMSA.3 variant antibody, measured by monomer concentration (Figure 3B) or aggregate concentration (Figure 3C) (pM) at 63.5°C. The AC clone numbers of the tested uTCE are shown in the figures. These experiments used uTCE, not paTCE. [Figure 3C] The biophysical characterization data for the PMSA.3 variant antibody are shown. Figure 3A shows the relative binding of the PMSA.3 variant antibody to PSMA. Figures 3B and 3C show the thermal stability of the PMSA.3 variant antibody, measured by monomer concentration (Figure 3B) or aggregate concentration (Figure 3C) (pM) at 63.5°C. The AC clone numbers of the tested uTCE are shown in the figures. These experiments used uTCE, not paTCE. [Figure 4]The PTE scores for representative PSMA and CD3 variants are shown. The graphs show molecules containing known antidrug antibodies (ADAs) and their corresponding PTE scores. A higher score indicates a higher likelihood of possessing a putative T-cell epitope. [Figure 5-1] EpiScreen® DC: Shows T cell proliferation in a T cell immunogenicity assay. PSMA.350 and positive control KLH were tested. For each donor's data point, each bar represents day 9, day 10, day 11, and day 12, from left to right. [Figure 5-2] EpiScreen® DC: Shows T cell proliferation in a T cell immunogenicity assay. PSMA.350 and positive control KLH were tested. For each donor's data point, each bar represents day 9, day 10, day 11, and day 12, from left to right. [Figure 6A] This shows the PTE score evaluation using the internal PTE algorithm v22 for anti-CD3 pool 2 antibodies. [Figure 6B] This shows the percentage of residual antibody after the thermal stability assay. [Figure 7A] The amino acid sequences of RSR-2295 and RSR-3213, and the alignment of proteases capable of cleaving them are shown. [Figure 7B] This shows in vitro protease digestion of paTCE using RSR-2295 or RSR-3213. The RSR-3213 sequence is modified to substantially reduce cleavage by legmine. [Figure 8A] Figure 8A shows the relative plasma stability of paTCE using RSR-2295 or RSR-3213, measured on day 0 and day 7. In Figure 8A, RSR-2295 was measured using SCy5.5 fluorophores, and RSR-3213 was measured using SCy7.5 fluorophores. In Figure 8B, RSR-2295 was measured using SCy7.5 fluorophores, and RSR-3213 was measured using SCy5.5 fluorophores. [Figure 8B]Figure 8A shows the relative plasma stability of paTCE using RSR-2295 or RSR-3213, measured on day 0 and day 7. In Figure 8A, RSR-2295 was measured using SCy5.5 fluorophores, and RSR-3213 was measured using SCy7.5 fluorophores. In Figure 8B, RSR-2295 was measured using SCy7.5 fluorophores, and RSR-3213 was measured using SCy5.5 fluorophores. [Figure 8C] The bar graph shows the truncated in vivo results observed from tumor homogenates from three different mouse tumor models. For each set of bars in the bar graph (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] This shows the percentage of the total of the three metabolites + paTCE (paTCE, 1x-N, 1x-C, and uTCE) when using RSR-2295 or RSR-3213. [Figure 9] This shows the relative tumor uptake of paTCE using RSR-2295 or RSR-3213. The plasma:tumor ratio was calculated in three different mouse tumor models (four mice per tumor model). Each of the three tumor models contains "Mouse 1", each of the three tumor models contains "Mouse 2", each of the three tumor models contains "Mouse 3", and each of the three tumor models contains "Mouse 4". [Figure 10] The graph shows the PSMA-transfected CHO cell binding activity to human PSMA or cyno-PSMA between AC3092 (AMX-500-P1) and AC3896 (AMX-500-P4, also simply referred to as AMX-500 herein). Surface binding was detected with a labeled secondary antibody specific to anti-CD3 scFv. [Figure 11A]The relative in vitro cytotoxicity dose-response curves for LNCaP PSMAhigh cells (Figure 11A) and 22Rv1 PSMAlow cells (Figure 11B) are shown. [Figure 11B] The relative in vitro cytotoxicity dose-response curves for LNCaP PSMAhigh cells (Figure 11A) and 22Rv1 PSMAlow cells (Figure 11B) are shown. [Figure 12A] The relative in vitro cytotoxicity dose-response curves for LNCaP PSMAhigh cells (Figures 12A and 12B) and 22Rv1 PSMAlow cells (Figure 12C) using three different donor human PBMC samples are shown. [Figure 12B] The relative in vitro cytotoxicity dose-response curves for LNCaP PSMAhigh cells (Figures 12A and 12B) and 22Rv1 PSMAlow cells (Figure 12C) using three different donor human PBMC samples are shown. [Figure 12C] The relative in vitro cytotoxicity dose-response curves for LNCaP PSMAhigh cells (Figures 12A and 12B) and 22Rv1 PSMAlow cells (Figure 12C) using three different donor human PBMC samples are shown. [Figure 13A] Graphs of 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) are shown. Figure 13A shows the assay results from donor 1. Figure 13B shows similar results from donors 2-5. [Figure 13B] Graphs of 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) are shown. Figure 13A shows the assay results from donor 1. Figure 13B shows similar results from donors 2-5. [Figure 14A]Graphs of 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) are shown. Figure 14A shows the assay results from donor 1. Figure 14B shows similar results from donors 2 and 3. [Figure 14B] Graphs of 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) are shown. Figure 14A shows the assay results from donor 1. Figure 14B shows similar results from donors 2 and 3. [Figure 15-1] Graphs showing 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) are shown. Cells were co-incubated with PBMCs in a 10:1 ratio. 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] Graphs showing 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) are shown. Cells were co-incubated with PBMCs in a 10:1 ratio. 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]Graphs showing 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) are shown. Cells were co-incubated with PBMCs in a 10:1 ratio. 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] Graphs showing 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) are shown. Cells were co-incubated with PBMCs in a 10:1 ratio. 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] Graphs showing 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) are shown. Cells were co-incubated with PBMCs in a 10:1 ratio. 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] Graphs of CD69, CD25, and PD-1 expression on CD4+ T cells from LNCaP PSMAhigh / PBMC co-cultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite) are shown. LNCaP cells were co-incubated with PBMCs in a 10:1 ratio. PBMCs were collected from donor 1. [Figure 17] The graphs show the expression of CD69, CD25, and PD-1 on CD8+ T cells from LNCaP PSMAhigh / PBMC co-cultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). LNCaP cells were co-incubated with PBMCs in a 10:1 ratio. PBMCs were collected from donor 1. [Figure 18-1] Graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC co-cultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite) are shown. LNCaP cells were co-incubated with PBMCs in a 10:1 ratio. PBMCs were collected from donor 2. [Figure 18-2] Graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC co-cultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite) are shown. LNCaP cells were co-incubated with PBMCs in a 10:1 ratio. PBMCs were collected from donor 2. [Figure 19-1] Graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC co-cultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite) are shown. LNCaP cells were co-incubated with PBMCs in a 10:1 ratio. PBMCs were collected from donor 3. [Figure 19-2]Graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from LNCaP PSMAhigh / PBMC co-cultures incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite) are shown. LNCaP cells were co-incubated with PBMCs in a 10:1 ratio. PBMCs were collected from donor 3. [Figure 20A] This shows the relative target binding of AMX-500 (Figure 20A) and AMX-500-P7 (AC3934, Figure 20B) to approximately 6,000 different HEK293T membrane proteins. [Figure 20B] This shows the relative target binding of AMX-500 (Figure 20A) and AMX-500-P7 (AC3934, Figure 20B) to approximately 6,000 different HEK293T membrane proteins. [Figure 21] The graph shows the tumor volume from human prostate tumor mouse models. Tumor mouse models were generated using 22Rv1 PSMAlow cells, LNCaP PSMAhigh cells, or C4-2 PSMAhigh cells. For the 22Rv1 model, AMX-500paTCE was administered at 2 mg / kg, 16 nmol / kg, and AMX-500 non-mask 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] This graph shows the tumor volume from a human prostate tumor mouse model using LNCaP PSMAhigh cells. [Figure 23] This graph shows the tumor volume from a human prostate tumor mouse model using 22Rv1 PSMAlow cells. [Figure 24]The graphs show tumor volume from human prostate tumor mouse models administered with AMX-500, the anti-PD-1 antibody pembrolizumab, or a combination of AMX-500 and pembrolizumab. [Figure 25] This shows the tissue distribution of AMX-500 in a mouse tumor model. [Modes for carrying out the invention]

[0307] There is a significant unmet need in cancer therapy for PSMA-targeted bispecific therapeutic modalities effective against solid tumors, particularly those residing in an immunologically cold microenvironment. 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 tissue have hindered the development of broad-spectrum therapies.

[0308] While not bound by any scientific theory, TCEs form crosslinks between T cells and tumor cells, activating T cell-mediated tumor cell killing and initiating a cytokine amplification cascade. This cytokine amplification cascade promotes further tumor cell killing and can potentially provide long-term immunity. T cells activated by TCEs release cell-soluble perforin / granzymes in a manner independent of antigen-MHC recognition. This creates a twofold 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. Cytokine responses include, among others, an increase in interferon-g, which stimulates CD8 T cell activity and antigen presentation by APCs; an increase in IL-2, which leads to increased proliferation of activated T cells; and an increase in CXCL9 and 10 responses, which increases T cell recruitment. The release of tumor antigens and the initiation of the cytokine response together result in the activation of the endogenous T cell response, which potentially triggers epitope diffusion and induces long-term immunity.

[0309] One toxic challenge with TCEs stems from the fact that many tumor targets are expressed to some extent in healthy tissues, and normal cells can also produce cytokine responses that lead to cytokine release syndrome (CRS). These two potent responses in healthy tissues to TCE-induced T cell activation often result in an overall lack of acceptable therapeutic indices for these drugs.

[0310] This disclosure addresses an unmet need and provides a protease-activatable TCE (paTCE) that is superior in one or more embodiments to conventional antibody or bispecific therapeutics that are active at injection, including improved terminal phase half-life, targeted delivery, and / or reduced damage to healthy tissue, and improved therapeutic ratio.

[0311] This specification includes compounds, compositions, and methods that overcome the shortcomings of existing TCEs by providing PSMA-targeted paTCEs (referred to herein as PSMA-paTCEs and exemplified as AMX-500).

[0312] AMX-500 contains the amino acid sequence described as Sequence ID No. 1000. While not bound by any scientific theory, it is understood that the paTCE described herein leverages the dysregulated protease activity present in tumors against healthy tissue, enabling an expansion of the therapeutic index. The paTCE core comprises antigen-binding domains, one of which targets CD3 and the other targets PSMA. The two antigen-binding domains may, in exemplary embodiments, 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 may be used.

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

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

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

[0316] In some embodiments, in addition to localized activation, the short half-life of the unmasked TCE form further broadens the therapeutic index while providing T-cell immune efficacy that improves the eradication of solid tumors. In some embodiments, the release site 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 utilizing enhanced protease activity that is upregulated at 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 their meanings unless otherwise specified.

[0318] As used herein and in the claims, the singular forms "a," "an," and "the" include multiple referents unless otherwise clearly indicated. For example, the term "cell" includes multiple cells, including mixtures thereof, unless otherwise clearly indicated by the context.

[0319] Furthermore, as used herein, “and / or” is to be interpreted as a specific disclosure of each of two particular features or components, whether or not they are accompanied by the other. Accordingly, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include 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] Wherever an aspect is described in this specification with the word “including,” it is understood that other similar aspects are also provided, described in terms of “consisting of” and / or “primarily from.”

[0321] Numerical ranges include both ends of the number defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino-carboxyl direction. The headings provided herein are not limitations of the various aspects of this disclosure. Therefore, the terms defined immediately thereunder are more fully defined by referring to the entirety of this specification.

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

[0323] With respect to naturally occurring compounds, the term “isolated” refers to a compound (i.e., a polypeptide or polynucleotide) that does not exist in its natural state (e.g., does not contain naturally associated components in nature, but contains them to varying degrees). No specific level of purification is required. For example, isolated polypeptides can be readily extracted from their natural or natural environment. Recombinant-produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this disclosure, as are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any preferred technique. “Isolate” and “isolated” may also, depending on the context, indicate the degree of separation from the original source or environment.

[0324] The term “polypeptide” refers to any polymer of two or more amino acids. Therefore, 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 all included within the definition of “polypeptide.” The term “polypeptide” also encompasses amino acid polymers modified by any other operation (e.g., post-translational modification), such as disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or conjugation with labeling components. Depending on the context, the term “polypeptide” may also be used to refer to a protein containing two or more polymers of two or more amino acids.

[0325] "Host cells" include individual cells (e.g., in culture) containing exogenous polynucleotides. Host cells may include offspring of a single host cell. Offspring may not necessarily be completely identical to the original parent cell (in morphology or in the genome of the whole DNA complement) due to naturally occurring or genetically engineered mutations.

[0326] A “fusion” or “chimeric” polypeptide or protein comprises a first polypeptide moiety linked to a second polypeptide moiety that is not naturally linked in nature. In some embodiments, these moieties may typically exist in separate proteins and be combined in the fusion polypeptide, or they may typically exist in the same protein but be arranged in a new configuration in the fusion polypeptide, or these moieties may be combined from different sources. In some embodiments, a fusion or chimeric protein comprises two or more moieties that do not exist in nature (e.g., human-made, designed, or generated moieties such as binding domains, masks, linkers, barcodes, and other polypeptides provided herein). Chimeric proteins may be produced, for example, by chemosynthesis or by recombinant expression (e.g., including producing and translating polynucleotides in which peptide regions are encoded in a desired relationship).

[0327] The terms “conjugated,” “linked,” “fused,” and “fused” 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 the second polypeptide. Similarly, “sequence identity” between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide with the sequence of the second polynucleotide. The terms “identical%”, “identity%”, or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids (where applicable) that are identical in the optimal alignment between the sequences being compared. This percentage may be purely statistical, and the differences between the two sequences may, but may not, be randomly distributed over the entire length of the sequences being compared. The comparison of two sequences is usually performed by comparing the sequences with respect to a segment or “window of comparison” after the optimal alignment in order to identify local regions of the corresponding sequences. For example, optimal alignment for comparison can be performed manually, or with the help of local homology algorithms by Smith and Waterman, 1981, Ads App.Math.2, 482, by Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443, by identity search algorithms by Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA 88, 2444, or with the help of computer programs using algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).In some embodiments, the percentage of identity between two sequences is determined using the BLASTN or BLASTP algorithm available on the United States National Center for Biotechnology Information (NCBI) website (e.g., 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 expected threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) a match / mismatch score set to 1, -2; (v) a gap cost set linearly; and (vi) a filter for low complexity regions used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word size set to 3; (iii) maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to exist: 11, extended: 1; and (vi) a conditional composition score matrix adjustment. Where 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 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 described in 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 in this disclosure, the percentage of identity is naturally calculated over the entire length of the reference polypeptide sequence, and parameters are set so that a homology gap of up to 5% of the total number of amino acids in the reference sequence is acceptable.

[0329] As used herein, the terms “mask polypeptide,” “mask,” and “masking portion” refer to polypeptides capable of reducing the binding of an antigen-binding domain (e.g., an antibody) to a target antigen, in the context of fusion proteins (such as chimeric polypeptides) provided herein. Exemplary mask polypeptides include, but are not limited to, the ELNN polypeptides described herein. Additional mask polypeptides include polypeptides comprising albumin, proline, serine, and alanine; coiled-coil domains; albumin-binding domains; Fc domains; and binding domains specific to the conserved region of the antibody-variable domain. 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 elongated polypeptide comprising a substantially non-repeating sequence (e.g., polypeptide motif) that is not naturally occurring, primarily composed of small hydrophilic amino acids, which has little to no secondary or tertiary structure under physiological conditions. ELNN polypeptides include unstructured hydrophilic polypeptides comprising a repeating motif of six natural amino acids (G, A, P, E, S, and / or T). In some embodiments, ELNN polypeptides comprise multiple motifs of the six natural amino acids (G, A, P, E, S, T), where the motifs are identical or combinations of different motifs. In some embodiments, when ELNN polypeptides are ligated to proteins, including T cell engagers disclosed herein, they can confer certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties. Such desirable properties include, but are not limited to, improved pharmacokinetic parameters and solubility characteristics, as well as improved therapeutic index. ELNN polypeptides are known in the art, and a non-exclusive description 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 these are incorporated herein by reference.

[0331] In some embodiments, the repeatability of an ELNN sequence refers to trimer repeatability and can be measured by a computer program or algorithm, or by other means known in the art. In some embodiments, the trimer repeatability of an ELNN can be evaluated by determining the number of occurrences of overlapping trimer sequences within the polypeptide. For example, a polypeptide of 200 amino acid residues has 198 overlapping 3-amino acid sequences (trimers), but the number of unique trimer sequences depends on the amount of repeatability in the sequence. In some embodiments, a score (hereinafter, "subsequence score") can be generated that reflects the degree of trimer repeatability in the entire polypeptide sequence. In this context, the "subsequence score" means the sum of occurrences of each unique trimer frame across 200 consecutive amino acid sequences of the polypeptide divided by the absolute number of unique trimer subsequences within the 200 amino acid sequences. Examples of such subsequence scores derived from the first 200 amino acids of repeating and non-repeat polypeptides are presented in Example 73 of International Patent Application Publication No. 2010 / 091122(A1), which is incorporated by reference in whole.

[0332] In some embodiments, in the context of ELNNs, “substantially non-repeating sequences” means ELNN sequences in which (1) there are few or no examples of four identical amino acids in the sequence of the ELNN sequence, and (2) the ELNN has a subsequence score of 12 or 10 or less (as defined in the preceding paragraphs herein), or there is no pattern in the order of the sequence motifs constituting the polypeptide sequence from N-terminus to C-terminus.

[0333] A “vector” is a nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. In some embodiments, the vector self-replicates in a suitable host. The term includes vectors that primarily function for the insertion of DNA or RNA into cells, vector replicaters that primarily function 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 two or more of the above functions. An “expression vector” is a polynucleotide that, when introduced into a suitable host cell, can be used for the transcription of mRNA which is translated into polypeptides. 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 “therapeutic” or “to treat” and “to improve” may be used interchangeably herein. These terms refer to approaches to obtain beneficial or desirable outcomes, including but not limited to therapeutic benefits. “Therapeutic benefits” means the eradication or improvement of the underlying disorder being treated. In some embodiments, the therapeutic benefit is achieved by the eradication or improvement of one or more physiological symptoms associated with the underlying disease state, such that improvement is observed in the subject, even though the subject may still suffer from the underlying disorder. In some embodiments, the therapeutic benefit includes slowing or halting the growth of one or more tumors. In some embodiments, the therapeutic benefit includes reducing the size of one or more tumors. In some embodiments, the therapeutic benefit includes eradicating one or more tumors from the subject. In some embodiments, the therapeutic benefit includes causing the death of cancer cells.

[0334] As used herein, the term “therapeutic dose” means the amount of a biologically active agent (e.g., a fusion protein provided herein as part of a pharmaceutical composition) that, when administered to a subject in a single or repeated dose, is capable of having any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease condition or state. Such effect does not need to be absolute to be beneficial. A disease condition may refer to a disorder or disease, such as cancer or the symptoms of cancer.

[0335] Antigen-binding domain, cleavage sequence, barcode fragment, and fusion polypeptide This disclosure provides, in particular, novel and useful anti-PSMA antibodies, novel and useful anti-CD3 antibodies, cleavage sequences, barcode fragments, and fusion proteins comprising them. Included herein are fusion polypeptides comprising (i) one or more mask polypeptides (e.g., ELNN), (ii) a bispecific antibody (BsAb, e.g., TCE) linked to the mask polypeptide, and (iii) one or more protease-cleavable release segments (RS), wherein the RS is located between the mask polypeptide and the BsAb.

[0336] In some embodiments, the anti-PSMA antibody provided herein has the following sequence: Includes a VHH domain containing a CDR of a VHH domain including QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (Sequence ID 549).

[0337] In some embodiments, the anti-CD3 antibody provided herein has the sequence: VH domain containing CDR of VH domain containing EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (Sequence ID 311) and / or array: Includes a VL domain containing a CDR of a VL domain including ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (Sequence ID 361).

[0338] Also provided is a BsAb comprising, for example, the anti-PSMA antibody and / or anti-CD3 antibody disclosed herein. In some embodiments, the bispecific antibody comprises the anti-PSMA VHH region disclosed herein. In some embodiments, the BsAb comprises the VH and VL regions of the anti-CD3 antibody disclosed herein. In some embodiments, the BsAb comprises the anti-PSMA VHH region disclosed herein and an anti-CD3 scFV comprising the 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 the ELNN described herein). In some embodiments, the polypeptide further comprises a second ELNN (such as the ELNN described herein). In some embodiments, the polypeptide comprises an ELNN ("N-terminal ELNN") at or near its N-terminus. In some embodiments, the polypeptide comprises an ELNN ("C-terminal ELNN") at or near its C-terminus. 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, where a first ELNN is bound to the N-terminus of the BsAb by a first RS, and a second ELNN is bound 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] This specification includes polypeptide sequences that can be used, for example, to link one polypeptide moiety to another in a fusion protein. For example, useful linkers are provided that are cleaved by multiple proteases but not by legmine. In some embodiments, such linkers may be used outside the context of antibodies, such as those described herein.

[0342] In some embodiments, the fusion polypeptide (e.g., one or more ELNNs of paTCE, and / or other parts of the fusion polypeptide such as a linker or spacer sequence) may include one or more barcode fragments (e.g., as described herein) that are capable of releasing (e.g., configured to release) 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 has a different sequence and molecular weight from all other peptide fragments (including all other barcode fragments, if present) that are capable of releasing from the polypeptide upon complete digestion of the polypeptide by the protease, thereby making it unique and allowing its presence to be detected by techniques such as mass spectrometry.

[0343] Elongated recombinant polypeptide (ELNN) Chain length and amino acid composition In some embodiments, ELNN contains at least 100 or at least 150 amino acids. In some embodiments, ELNN has an amino acid length of 100 to 3,000 or 150 to 3,000. In some embodiments, ELNN has an amino acid length of 100 to 1,000 or 150 to 1,000. In some embodiments, ELNN has 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, and a few The amino acid length is at least (approximately) 850, at least (approximately) 900, at least (approximately) 950, at least (approximately) 1,000, at least (approximately) 1,100, at least (approximately) 1,200, at least (approximately) 1,300, at least (approximately) 1,400, at least (approximately) 1,500, at least (approximately) 1,600, at least (approximately) 1,700, at least (approximately) 1,800, at least (approximately) 1,900, or at least (approximately) 2,000. In some embodiments, ELNN is at most (approximately) 100, at most (approximately) 150, at most (approximately) 200, at most (approximately) 250, at most (approximately) 300, at most (approximately) 350, at most (approximately) 400, at most (approximately) 450, at most (approximately) 500, at most (approximately) 550, at most (approximately) 600, at most (approximately) 650, at most (approximately) 700, at most (approximately) 750, at most (approximately) 800, many The amino acid length is at most (approximately) 850, at most (approximately) 900, at most (approximately) 950, at most (approximately) 1,000, at most (approximately) 1,100, at most (approximately) 1,200, at most (approximately) 1,300, at most (approximately) 1,400, at most (approximately) 1,500, at most (approximately) 1,600, at most (approximately) 1,700, at most (approximately) 1,800, at most (approximately) 1,900, or at most (approximately) 2,000.In some embodiments, ELNN is approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 800. The amino acid lengths are approximately 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000, or within a range between any two of the above. In some embodiments, at least 90% of the amino acid residues of 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 of ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, 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, 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, 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, ELNN consists of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, ELNN comprises the amino acids G, A, S, T, E, and P. In some embodiments, ELNNs (e.g., ELNN1, ELNN2, etc.) are characterized by (i) containing at least 100 or at least 150 amino acids, (ii) at least 90% of the amino acid residues of the ELNN being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P), and (iii) containing 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, the ELNN-containing fusion polypeptide comprises a first ELNN and a second ELNN. In some embodiments, the sum of the total number of amino acids in the first ELNN and the total number of amino acids in 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-repeating array motifs In some embodiments, the ELNN comprises or is formed from a plurality of non-repeating sequence motifs. In some embodiments, at least one of the non-repeating sequence motifs is repeating (or repeats at least twice in the ELNN). In some embodiments, the ELNN comprises at least one other non-repeating sequence motif that is non-repeating (or found only once in the ELNN). In some embodiments, the plurality of non-repeating sequence motifs comprises (a) a set of (repeating) non-repeating sequence motifs in which each non-repeating sequence motif in the set is repeated at least twice in the ELNN, and (b) a non-repeating (non-repeating) sequence motif that occurs (or is found) only once in the ELNN. In some embodiments, each non-repeating sequence motif is 9–14 (or 10–14, or 11–13) amino acid length. In some embodiments, each non-repeating sequence motif is 12 amino acid length. In some embodiments, the multiple non-repeating sequence motifs include a set of non-repeating (repeating) sequence motifs, where each non-repeating sequence motif in the set of non-repeating sequence motifs is (1) repeated at least twice in the ELNN and (2) 9 to 14 amino acid lengths. In some embodiments, the set of (repeating) non-repeating sequence motifs includes dodecamer sequence motifs identified herein by sequence numbers 179-200 and 1715-1722 in Table 1. In some embodiments, the set of (repeating) non-repeating sequence motifs includes dodecamer sequence motifs identified herein by sequence numbers 186-189 in Table 1. In some embodiments, the set of (repeating) non-repeating sequence motifs includes at least two, at least three, or all four of the dodecamer sequence motifs of sequence numbers 186-189 in Table 1. In some embodiments, the ELNN further includes sequences other than the dodecamer sequence motifs shown in Table 1. In some embodiments, the ELNN includes sequences not in Table 1, such as ASSATPESGP, GSGPGTSESATP, or GTSESATP. In some embodiments, ELNN includes sequences not listed in Table 1, such as ATPESGP, GTSPSATPESGP, or GTSESAGEPEA. In some embodiments, ELNN includes barcode sequences.

[0345] [Table 1] * This indicates individual motif sequences that, when used together in various permutations, create a "family sequence".

[0346] Confirmation of unstructured polypeptides In various embodiments, the ELNN components (or multiple ELNN components) of a fusion protein have an unstructured three-dimensional structure under physiological conditions, regardless of the polymer length (e.g., elongated length). For example, the ELNN is characterized by a large degree of structural freedom of the peptide backbone. In some embodiments, the ELNN is characterized by the absence of long-range interactions as determined by NMR. In some embodiments, the disclosure provides an ELNN that, under physiological conditions, resembles the structure of a denatured sequence with little secondary structure. In some embodiments, the ELNN may substantially lack secondary structure under physiological conditions. As used in this context, “little secondary structure” means that, as measured or determined by the means described herein, less than 50% of the ELNN amino acid residues of the ELNN contribute to the secondary structure. As used in this context, “substantially secondary” means that, as measured or determined by the means described herein, 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 the secondary structure.

[0347] Various methods have been established in the art to identify the presence or absence of secondary and tertiary structures in a given polypeptide. In some embodiments, the secondary structure of an ELNN 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 structures can also be predicted for polypeptide sequences through 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), U.S. Patent Application Publication No. 20030228309(A1) (the entire contents of which are incorporated herein by reference), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553). For a given sequence, the algorithm can predict whether some or no secondary structure is present, expressed, for example, as the total and / or percentage of residues in sequences that form an alpha-helix or beta-sheet, or as the percentage of residues in sequences that are predicted to result in random coil formation (lack of secondary structure).

[0348] In some embodiments, the ELNN used in the fusion protein composition may have an alpha-helix percentage in the range of 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 in the range of 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 in the range of 0% to less than about 5% and a beta-sheet percentage in the range of 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition has an alpha-helix percentage of less than about 2% and a beta-sheet percentage of less than about 2%. In some embodiments, the ELNN of the fusion protein composition may have a high random coil percentage, as determined by the GOR algorithm. In some embodiments, the ELNN 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%, as determined by the GOR algorithm.

[0349] Net charge In some embodiments, the ELNN polypeptide may have unstructured characteristics conferred by the incorporation of net-charged amino acid residues and / or a reduction in 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 ELNN in the composition may be greater than +0.1 or less than -0.1 charge / residue. In some embodiments, the net charge of the ELNN may 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] Since most tissues and surfaces in humans or animals have a net negative charge, ELNNs can be optionally 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. While not bound by any particular theory, ELNNs can adopt an open conformation due to the electrostatic repulsive forces between individual amino acids of the ELNN polypeptide, which carry a high net negative charge individually and are distributed across the sequence of the ELNN polypeptide. Such a distribution of net negative charge in the extended sequence length of ELNN can result in an unstructured conformation, which in turn can lead to an effective increase in the hydrodynamic radius. Therefore, in some embodiments, ELNNs contain glutamine such that glutamine accounts for about 8, 10, 15, 20, 25, or even about 30% of the amino acids in the sequence. The ELNNs in the compositions of this disclosure generally have no positively charged amino acids or have a low content of positively charged amino acids. In some embodiments, the ELNN may have less than 10% positively charged amino acid residues, or less than 7%, 5%, or 2% positively charged amino acid residues. However, the disclosure envisions a polypeptide in which a limited number of positively charged amino acids, such as lysine, can be incorporated into the ELNN to enable conjugation between the epsilonamine of lysine and a reactive group on a peptide, a linker crosslink, or a reactive group on a drug or small molecule that is conjugated to the ELNN skeleton.

[0351] In some embodiments, ELNN may contain charged residues separated by other residues such as serine or glycine, which may result in better expression or purification behavior. Based on net charge, ELNN of the composition in question may have isoelectric points (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, ELNN has isoelectric points between 1.5 and 4.5. In some embodiments, ELNN incorporated into a paTCE fusion protein carries a net negative charge under physiological conditions, contributing to an unstructured three-dimensional structure and reduced binding of ELNN components to mammalian proteins and tissues.

[0352] Since hydrophobic amino acids can confer structure to polypeptides, in some embodiments, the hydrophobic amino acid content in ELNN is less than 5%, less than 2%, or less than 1%. In some embodiments, ELNN does not contain hydrophobic amino acids. In some embodiments, the amino acid content of methionine and tryptophan in the ELNN components of the paTCE fusion protein is less than 5%, less than 2%, most preferably less than 1%. In some embodiments, ELNN has a sequence having less than 10% positively charged amino acid residues, or about less than 7%, about less than 5%, or about less than 2% positively charged amino acid residues, with the sum of methionine and tryptophan residues being less than 2% of the total ELNN sequence, and the sum of asparagine and glutamine residues being less than 10% of the total ELNN sequence. In some embodiments, ELNN does not contain methionine or tryptophan residues.

[0353] Increased hydrodynamic radius In some embodiments, ELNNs may have a high hydrodynamic radius, conferring a correspondingly increased apparent molecular weight to the paTCE fusion protein incorporating the ELNN. Conjugation of ELNNs to a BsAb (e.g., TCE) sequence may result in a paTCE composition having an increased hydrodynamic radius, increased apparent molecular weight, and increased apparent molecular weight factor compared to a BsAb (e.g., TCE) not conjugated to an ELNN. For example, in some therapeutic applications where an extended half-life is desirable, one or more ELNNs with a high hydrodynamic radius may be incorporated into a fusion protein containing a BsAb (e.g., TCE) to effectively expand the hydrodynamic radius of the fusion protein beyond a 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 the circulating protein. In some embodiments, the hydrodynamic radius of a protein is determined by its molecular weight and its structure, including its shape and compactness. While not bound by any particular theory, ELNNs can adopt an open conformation due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility conferred by certain amino acids in a sequence lacking the potential to confer secondary structure. In some embodiments, the open, elongated, and unstructured conformation of an ELNN polypeptide has a larger proportional hydrodynamic radius compared to polypeptides of comparable sequence length and / or molecular weight that have a secondary structure and / or tertiary structure, such as a typical globular protein. Methods for determining the hydrodynamic radius, such as the use of size exclusion chromatography (SEC) as described in U.S. Patents 6,406,632 and 7,294,513, are well known in the art.In some embodiments, the addition of increasing the ELNN length results in a proportional increase in the parameters of hydrodynamic radius, apparent molecular weight, and apparent molecular weight factor, allowing for the adjustment of paTCE to a desired characteristic cutoff apparent molecular weight or hydrodynamic radius. Thus, in some embodiments, the paTCE fusion protein can be configured with an ELNN such that the fusion protein may 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 ELNN in the paTCE fusion protein may result in a reduction in the renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal phase half-life, an increase in mean residence time, and / or a decrease in renal clearance rate.

[0354] In some embodiments, ELNNs of selected length and sequence (or multiple ELNNs, such as two ELNNs) can be selectively incorporated into paTCE to produce a fusion protein having an apparent molecular weight 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 under physiological conditions. In some embodiments, selective ligation of ELNNs (or multiple ELNNs, such as two ELNNs) of selected length and sequence to BsAb (e.g., TCE) can yield a paTCE fusion protein having, under physiological conditions, 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 apparent molecular weight factors, or at least 20 or more apparent molecular weight factors. In some embodiments, the paTCE fusion protein has, under physiological conditions, an apparent molecular weight factor of 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 of greater than about 6 under physiological conditions.

[0355] Increased terminal phase half-life In some embodiments, the fusion polypeptide containing an ELNN (such as paTCE) has a terminal phase half-life that is at least twice as long, at least three times longer, at least four times longer, or at least five times longer compared to the corresponding biologically active polypeptide that is not linked to an ELNN.

[0356] In some embodiments, administering a therapeutically effective dose of paTCE fusion protein to a subject in need results in at least a twofold, at least threefold, at least fourfold, or at least fivefold increase in the time the fusion protein remains within the therapeutic window compared to the corresponding BsAb (e.g., TCE) not linked to ELNN when administered to the subject at an equivalent dose.

[0357] In some embodiments, the TCE released from paTCE upon protease cleavage contains one or more short polypeptides (e.g., amino acids with an amino acid length of about 30, 25, 20, 15, 14, 13, 12, 11, 10, or less) that do not contain amino acids other than G, A, P, E, S, and / or T. For example, the short polypeptides that do not contain amino acids other than G, A, P, E, S, and / or T may be incorporated into one or more spacer or linker sequences of the TCE and / or into one or more spacer or linker sequences that remain as part of the TCE after cleavage. In some embodiments, the TCE released from paTCE contains GTSESATPES on the N-terminal side of the TCE (e.g., the nearest amino acid in the sequence is within the 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 released from paTCE is GTATPESGPG on the C-terminal side of the TCE (for example, the nearest amino acid in the sequence is at the 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 includes an internal linker (for example, between the VL and VH regions of scFV) containing a polypeptide sequence that does not contain 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 low immunogenicity or are substantially non-immunogenic. Several factors, such as substantially non-repeating sequences, unstructured three-dimensional structures, high solubility, low degree of autoaggregation or absence of autoaggregation, low degree of proteolytic sites or absence of proteolytic sites in the sequence, and low degree of epitopes or absence of epitopes in ELNNs, may contribute to the low immunogenicity of ELNNs.

[0359] Those skilled in the art will generally understand that polypeptides having highly repeating short amino acid sequences (e.g., sequences of 200 amino acids in length containing a limited set of trimers or tetramers with an average of 20 or more repeats) and / or polypeptides having consecutive repeating amino acid residues (e.g., sequences of pentamers or hexamers having identical amino acid residues) tend to aggregate, form higher-order structures, or form contacts to produce crystalline or pseudocrystalline structures.

[0360] In some embodiments, the ELNN sequence is substantially non-repeating, (1) the ELNN sequence does not have three consecutive amino acids of the same type unless the amino acid is serine, in which case three or fewer consecutive amino acids may be serine residues, and (2) the ELNN does not contain three amino acid sequences (trimers) that occur more than 16, 14, 12, or 10 times within the sequence of at least 200 amino acids in length of the ELNN (e.g., the entire span of the ELNN which is at least the length of the amino acids). While not bound by any scientific theory, such substantially non-repeating sequences allow for the design of long sequences of ELNNs with a relatively low frequency of charged amino acids, which are less prone to aggregation and therefore more likely to aggregate if the sequence or amino acid residues are more repeating.

[0361] Structural epitopes can be formed by regions on the protein surface composed of multiple discontinuous amino acid sequences of a protein antigen. While not bound by any scientific theory, the precise folding of a protein can transform these sequences into a clearly defined, stable spatial configuration or epitope that can be recognized as "foreign" by the host humoral immune system, leading to antibody production against the protein and / or triggering a cell-mediated immune response. In the latter case, the immune response to the protein in an individual is largely influenced by T cell epitope recognition, which correlates with the peptide bond specificity of the individual's HLA-DR allotype. Engagement of MHC class II peptide complexes by congeneral T cell receptors on the T cell surface, along with cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T cell. This activation can lead to cytokine release, further activating other lymphocytes such as B cells to produce antibodies, or activating T killer cells as a complete cellular immune response.

[0362] While not 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) can depend on several factors, most notably its primary sequence. In some embodiments, lower immunogenicity can be achieved by designing an ELNN that resists antibody processing in antigen-presenting cells and / or by selecting a sequence that does not bind sufficiently to the MHC receptor. In some embodiments, an ELNN-containing fusion protein has a substantially non-repeating ELNN polypeptide designed to reduce binding to the MHC II receptor and to avoid the formation of epitopes for T cell receptor or antibody binding, resulting in lower immunogenicity. While not bound by any scientific theory, avoidance of immunogenicity is, in part, a direct result of the flexibility of the ELNN's conformation, i.e., the lack of secondary structure due to the selection and order of amino acid residues. Of particular interest, for example, are sequences that tend to adopt a compactly folded conformation in aqueous solution or under physiological conditions that may produce conformational epitopes. Administration of ELNN-containing fusion proteins using conventional therapeutic practices and dosages generally does not result in the formation of neutralizing antibodies against ELNN and can reduce the immunogenicity of BsAb (e.g., TCE) fusion partners in paTCE compositions.

[0363] In some embodiments, the ELNN used in the target fusion protein may substantially omit epitopes recognized by human T cells. Excluding such epitopes for the purpose of generating less immunogenic proteins has been previously disclosed, see, for example, International Publications 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 be oligomerized 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 for the generation of non-human 6-15 mers, particularly 9-mer sequences, and then modifying the design of the ELNN sequence to exclude or disrupt the epitope sequences. In some embodiments, ELNNs become substantially non-immunogenic by limiting the number of ELNN epitopes predicted to bind to MHC receptors. Along with the reduction in the number of epitopes capable of binding to MHC receptors, there is a simultaneous reduction in the potential for T cell activation and T cell helper function, a reduction in B cell activation or upregulation, and a reduction in antibody production. The low degree of predicted T cell epitopes can be determined 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), which is incorporated entirely by reference. A form of TEPITOPE score for a given peptide frame in a protein is disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555. The score should be at least above 20log, approximately 10 to approximately -10 (10e 10 K D ~10e -10 K DThis range corresponds to the binding constraints of and can be reduced by avoiding hydrophobic amino acids such as M, I, L, V, or F that can function as anchor residues during peptide display on MHC. In some embodiments, the ELNN components incorporated into paTCE do not have T cell epitopes predicted by a TEPITOPE score of about -5 or greater, or -6 or greater, or -7 or greater, or -8 or greater, or a TEPITOPE score of -9 or greater. As used herein, the score of "-9 or greater" includes TEPITOPE scores from 10 to -9 (inclusive), but does not include a score of -10, as -10 is less than -9.

[0364] In some embodiments, ELNNs, including those incorporated into a target paTCE fusion protein, can be made substantially non-immunogenic by limiting known proteolytic sites from the ELNN sequence, reducing the processing of the ELNN into small peptides that can bind to MHC II receptors. In some embodiments, the ELNN sequence can be made substantially non-immunogenic by using a sequence that substantially lacks secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Thus, the reduction of the TEPITOPE score and the elimination of known proteolytic sites from the ELNN can make ELNN compositions containing the ELNN of a paTCE fusion protein composition substantially unbound by mammalian receptors, including receptors of the immune system. In some embodiments, the ELNN of a paTCE fusion protein has a Kb greater than 100 nM for mammalian receptors. D Binding, or a K+500 nM K to mammalian cell surface or circulating polypeptide receptors. D Alternatively, K greater than 1 μM D It may have.

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

[0366] Compared to these less repeating sequences (such as those with three consecutive identical amino acids), the additional features of certain ELNNs having substantially non-repeating sequences are that non-repeating ELNNs may form weaker contact (e.g., monovalent interactions) with antibodies, thereby resulting in the possibility of lower immunoclearance, allowing paTCE compositions to persist for an increased period in circulation.

[0367] In some embodiments, biologically active polypeptides containing ELNNs (BsAbs, e.g., TCE) exhibit lower immunogenicity compared to fusion polypeptides not linked to any ELNN, and this immunogenicity is confirmed by measuring the production of IgG antibodies that selectively bind to the biologically active polypeptide after administration of equivalent doses to the subject.

[0368] Barcode fragment In some embodiments, the polypeptide (e.g., a fusion polypeptide, or a portion thereof such as ELNN) comprises one or more barcode fragments (e.g., a first, second, or third barcode fragment) that can be released from the polypeptide upon digestion by 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 terms “barcode fragment” (or “barcode” or “barcode sequence”) may refer to either a portion of a polypeptide cleavably fused within the polypeptide, or a peptide fragment resulting from its release from the polypeptide.

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

[0370] In some embodiments, the barcode fragment does not contain the N-terminal or C-terminal amino acids of the fusion polypeptide. As described herein, in some embodiments, the barcode fragment is releaseable (e.g., configured to be releaseable) during Glu-C digestion of the fusion polypeptide. In some embodiments, the barcode fragment is located in ELNN and does not contain a glutamate directly adjacent to another glutamate (if present) in ELNN. In some embodiments, the barcode fragment has glutamate at its C-terminus. Those skilled in the art will understand that if the C-terminus of the barcode fragment is cleavably fused within a polypeptide (such as ELNN), it can refer to the "last" (or most C-terminal) amino acid residue in the barcode fragment, even if other non-barcode amino acid residues are located C-terminal to the barcode fragment within the polypeptide (e.g., ELNN). In some embodiments, the barcode fragment has an N-terminal amino acid immediately preceded by a glutamate residue. In some embodiments, the glutamate residue preceding the N-terminal amino acid is not directly adjacent to another glutamate residue. In some embodiments, the barcode fragment does not contain a (second) glutamate residue at any position other than the C-terminus of the barcode fragment, unless proline immediately follows glutamate. In some embodiments, the barcode fragment is located at a certain distance from either the N-terminus or the C-terminus of the polypeptide, where this distance is 10 to 150 or 10 to 125 amino acids. 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 N-terminus of the polypeptide, or within a range of any of the aforementioned. In some embodiments, the barcode fragment is located within 200 amino acids, 150 amino acids, 100 amino acids, or 50 amino acids from the N-terminus of the polypeptide.In some embodiments, the barcode fragment is located at a position 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 within a range between any of the above. In some embodiments, the barcode fragment is located within 200, 150, 100, or 50 amino acids from the C-terminus of the polypeptide. In some embodiments, the barcode fragment is located at a position 10-200, 30-200, 40-150, or 50-100 amino acids from the C-terminus of the polypeptide. In some embodiments, the barcode fragment (BAR) is characterized by (i) not containing a glutamic acid directly adjacent to another glutamic acid, if present in ELNN, (ii) having a glutamic acid at its C-terminus, (iii) having an N-terminal amino acid immediately preceding a glutamic acid residue, and (iv) being located at a certain distance from either the N-terminus or the C-terminus of the polypeptide, with a distance of 10-150 amino acids or 10-125 amino acids in length. In some embodiments, the barcode fragment is located in the ELNN and (i) does not contain the N-terminal or C-terminal amino acid of the polypeptide, (ii) does not contain a glutamic acid directly adjacent to another glutamic acid in the ELNN, (iii) has glutamic acid at its C-terminus, (iv) has an N-terminal amino acid immediately preceding a glutamic acid residue, and (v) is located at a certain distance from either the N-terminus or C-terminus of the polypeptide, the distance being 10 to 150 or 10 to 125 amino acid lengths. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not directly adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not contain glutamic acid residues at any position other than the C-terminus of the barcode fragment unless a proline immediately follows the glutamic acid.Depending on the context herein, when referring to a location within a polypeptide sequence, the term “distance” may refer to the number of amino acid residues from the N-terminus of the polypeptide to the most N-terminal amino acid residue of the barcode fragment, or from the C-terminus of the polypeptide to the most C-terminal 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, or three) contained in the barcoded ELNN is located at 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 acid lengths. In some embodiments, the barcode fragment is at least 4 amino acid lengths. 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, or 25 amino acid lengths, or within a range of any of the aforementioned values. In some embodiments, the barcode fragment is 4–20, 5–15, 6–12, or 7–10 amino acid lengths. In some embodiments, as used herein, the barcode fragment comprises amino acid sequences identified by SEQ ID NOs. 68–79 and SEQ ID NOs. 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 that can be released from the chimeric polypeptide described herein upon complete digestion of the chimeric polypeptide by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

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

[0374] In some embodiments, the chimeric polypeptide disclosed herein has 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 The formula comprises at least one of SE.SA, where each "." is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptide disclosed herein has the following amino acid sequence:PE.SGX n PE.SG, PE.GSX n SE.GG, PE.TPX n SE.TG, PE.SGX n Includes at least one of SE.SA. 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 nThese are 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 The formula comprises at least one of EPSE.SATP, where each "." is a Glu-c cleavage site and n is any integer from 0 to 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 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 The formula includes at least one of STPE.SGPG, where each "." is a Glu-C cleavage site and n is any integer from 0 to 30. 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. In some embodiments, n is any integer from 5 to 15. In some embodiments, X n This is PGTGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST. In some embodiments, X nThese are 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, the barcode is designed to have improved analytical properties. In some embodiments, such barcodes may be released by a non-mammalian protease such as Glu-C at a relatively low concentration. This facilitates better detection, for example, through LC / MS, and also enables the measurement of peptides produced from the cleavable linker, thereby enabling the measurement of the cleavage product, for example, using LC / MS.

[0377] In some embodiments of fusion proteins containing ELNNs, the fusion protein has a single polypeptide chain, and the polypeptide chain contains a barcode fragment located within the polypeptide chain at a position 10 to 200 amino acids or 10 to 125 amino acids from the N-terminus or C-terminus of the polypeptide chain. In some embodiments, the fusion protein (e.g., paTCE) contains a first ELNN and a second ELNN, the first ELNN being on the N-terminal side of the bispecificity domain, and the first barcode fragment being located within 200, 150, 100, or 50 amino acids from the N-terminus of the fusion protein. In some embodiments, the second ELNN is on the C-terminal side of the bispecificity antibody domain, and the second barcode fragment is located within 200, 150, 100, or 50 amino acids from the C-terminus of the chimeric polypeptide.

[0378] In some embodiments, the ELNN further comprises one or more additional barcode fragments, each of which has a different sequence and molecular weight from all other peptide fragments that can be released 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 comprises a second barcode fragment (or further barcode fragments), such as those described herein. In some embodiments, the set of barcode fragments comprises 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 the N-terminal set of the barcode ("N-terminal set"). A set of fused barcode fragments within a C-terminal ELNN may be referred to as the C-terminal set of the barcode ("C-terminal set"). In some embodiments, the N-terminal set includes a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal set further includes a third barcode fragment. In some embodiments, the C-terminal set includes a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal set further includes a third barcode fragment. In some embodiments, the polypeptide includes 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 in 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 can be released from the polypeptide upon complete digestion of the polypeptide by a protease.

[0380] This specification includes mixtures comprising multiple polypeptides of varying lengths, wherein the mixture comprises a set of first polypeptides and a set of second polypeptides. In some embodiments, each polypeptide in the set of first polypeptides includes a barcode fragment having a sequence and molecular weight different from all other fragments that can be released from the set of first polypeptides (a) by protease digestion and (b) having a sequence and molecular weight different from all other fragments that can be released from the set of first polypeptides. In some embodiments, the set of second polypeptides lacks the barcode fragment of the set of first polypeptides (e.g., due to cleavage). In some embodiments, both the set of first polypeptides and the set of second polypeptides each include a reference fragment that is common to both the set of first polypeptides and the set of second polypeptides and (b) can be released by protease digestion. In some embodiments, the ratio of the set of first polypeptides to the polypeptide containing the reference fragment is greater than 0.70. In some embodiments, the ratio of the set of first polypeptides to the polypeptide containing the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, a 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 at the C-terminal side of the glutamic acid residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, polypeptides of varying lengths include polypeptides containing at least one ELNN, such as any of those described herein. In some embodiments, the first set of polypeptides includes a full-length polypeptide, and the barcode fragment is part of the full-length polypeptide. In some embodiments, the full-length polypeptide is any of those described above herein or any other part of this specification (fusion) polypeptide. In some embodiments, polypeptides of varying lengths in the mixture differ from one another due to N-terminal cleavage, C-terminal cleavage, or both N-terminal and C-terminal cleavage 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 disclosure also provides a method for evaluating the relative amount of a set of first polypeptides to a set of second polypeptides in a mixture containing polypeptides of varying lengths, wherein (1) each polypeptide in the set of first polypeptides shares a barcode fragment that occurs only once and only once in the polypeptide, and (2) each polypeptide in the set of second polypeptides lacks a barcode fragment shared by the set of first polypeptides, and each individual polypeptide in both the set of first polypeptides and the set of second polypeptides contains a reference fragment. In some embodiments, the method comprises contacting the mixture with a protease to produce a plurality of proteolytic fragments resulting from the cleavage of the set of first polypeptides and the set of second polypeptides, wherein the plurality of proteolytic fragments contain a plurality of reference fragments and a plurality of barcode fragments. In some embodiments, the method may further comprise determining the ratio of the amount of barcode fragments to the amount of reference fragments, thereby evaluating the relative amount of the set of first polypeptides to the set of second polypeptides. In some embodiments, the barcode fragment occurs once or less in each polypeptide of the set of first polypeptides. In some embodiments, a reference fragment is generated once or less for each polypeptide in the first set of polypeptides and the second set of polypeptides. In some embodiments, multiple proteolytic fragments include multiple reference fragments and multiple barcode fragments. In some embodiments, the protease cleaves the first and second sets of polypeptides (or polypeptides of varying lengths) at the C-terminal side of a glutamic acid residue that is not followed by a proline residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the step of determining the ratio of the amount of barcode fragments to the amount of reference fragments includes identifying the barcode fragments and reference fragments from the mixture after contact with the protease. In some embodiments, the barcode fragments and reference fragments are identified based on their respective masses. In some embodiments, the barcode fragments and reference fragments are identified by mass spectrometry.

[0382] In some embodiments, the barcode fragment and the reference fragment are identified via liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the step of determining the ratio of the barcode fragment to the reference fragment includes isobaric labeling. In some embodiments, the step of determining the ratio of the barcode fragment to the reference fragment includes spiking the mixture with one or both of the isotopically labeled reference fragment and the isotopically labeled barcode fragment. In some embodiments, polypeptides of varying lengths include polypeptides comprising at least one ELNN described above or elsewhere herein. In some embodiments, the ELNN is characterized by (i) comprising at least 100 or at least 150 amino acids, (ii) at least 90% of the amino acid residues of the ELNN being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P), and (iii) comprising at least four different types of amino acids, which are G, A, S, T, E, or P. In some embodiments, the barcode fragment, if present, is part of the ELNN. In some embodiments, the mixture of polypeptides of varying lengths includes polypeptides described above or elsewhere in this specification. In some embodiments, the polypeptides of varying lengths include full-length polypeptides and their cleavage fragments. In some embodiments, the polypeptides of varying lengths consist mainly of full-length polypeptides and their cleavage fragments. In some embodiments, the polypeptides of varying lengths in the mixture differ from one another due to N-terminal cleavage, C-terminal cleavage, or both N-terminal and C-terminal cleavage of the full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide described above or elsewhere in this specification. In some embodiments, the ratio of barcode fragments to reference fragments 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 a barcode fragment to a reference fragment. Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics in which a peptide or protein (or part thereof) is 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 during high-energy collision-induced dissociation (CID) in tandem mass spectrometry, the mass tag is cleaved at a specific linker region, thereby producing reporter ions of different masses. Some of the most common isobaric tags are amine-reactive tags.

[0384] Exemplary barcoded ELNN polypeptide This specification includes an ELNN that contains a barcode fragment, which is part of the ELNN.

[0385] Table 3a shows 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 NOs. 8011), with the 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 TCE) at the C-terminus of a biologically active protein, and 10 (SEQ ID NOs. 8004-8007, 8010, 8012-8014, 8020, and 8021) are fused to the N-terminus of a biologically active protein. In some embodiments, ELNN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequences identified herein by sequence numbers 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 introducing one or more mutations into an existing ELNN, such as any of those listed in Table 3b, according to one or more of the following criteria: minimizing sequence changes in the ELNN, minimizing changes in amino acid composition 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 with any one of sequence numbers 601-659 listed in Table 3b. In some embodiments, ELNN sequences 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 sequence numbers 601-659 listed in Table 3b are obtained by one or more mutations from the corresponding sequences in Table 3b (e.g., mutations less than 10, less than 8, less than 6, less than 5, less than 4, less than 3, or less than 2). In some embodiments, one or more mutations include deletion of a glutamate residue, insertion of a glutamate residue, substitution of a glutamate residue, substitution with a glutamate residue, or any combination thereof. In some embodiments, if the ELNN sequence is different from any one of sequence numbers 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, then any difference of at least 80%, at least 90%, at least 95%, at least 97%, or about 100% between the ELNN sequence and the corresponding sequence in Table 3b includes deletion of glutamate residues, insertion of glutamate residues, substitution of glutamate residues, substitution with glutamate residues, 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 difference between the ELNN sequence and the corresponding sequence in Table 3b includes substitutions of glutamate residues, or substitutions with glutamate residues, or both.

[0396] As used herein, “substitution of the first amino acid” refers to the replacement of a first amino acid residue with a second amino acid residue, where the second amino acid residue occupies the position of the substitution in the resulting sequence. For example, “substitution of glutamate” refers to the replacement of a glutamate (E) residue with a non-glutamate 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, amino acid mutations are performed on ELNNs of intermediate length to those in Table 3b, as well as on ELNNs longer than those in Table 3b, such as those in which one or more dodecamer motifs from Table 1 are added to the N-terminus or C-terminus of the general-purpose ELNNs in Table 3b, in order to construct the barcoded ELNN sequences.

[0410] Additional examples of existing ELNNs that can be used in accordance with this disclosure include 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 Disclosed in Nos. 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 into the polypeptide chain adjacent to the N-terminus of a polypeptide chain ("N-terminal ELNN") may be bound to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate the purification of the fused polypeptide. In some embodiments, a barcoded ELNN fused into the polypeptide chain at the C-terminus of a polypeptide chain ("C-terminal ELNN") may contain or be bound to the sequence EPEA at the C-terminus to facilitate the purification of the fused polypeptide. In some embodiments, the fusion polypeptide comprises both an N-terminal barcoded ELNN and a C-terminal barcoded ELNN, wherein the N-terminal barcoded ELNN is bound at its N-terminus to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49), and the C-terminal barcoded ELNN is bound at its C-terminus to the sequence EPEA, thereby facilitating the purification of the fusion polypeptide to, for example, a purity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% by chromatographic methods known in the art, including, but not limited to, IMAC chromatography, C-tagXL affinity matrices, and other such methods.

[0412] The barcode fragments described herein may be fused in a cleavable manner within an ELNN and may be released from the ELNN during protease digestion of the polypeptide (i.e., may be configured to be released). In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves at the C-terminal side of glutamic acid residues that are not followed by proline. In some embodiments, the barcoded ELNN (an ELNN containing a barcode fragment) 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 during Glu-C digestion. Therefore, 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, if a dipeptide Glu-Pro(EP) sequence is present in the fused polypeptide, it may not be cleaved by the Glu-C protease during the barcode release process.

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

[0414] In some embodiments, the fusion protein includes a C-terminal ELNN and optionally a linker between the ELNN and TCE (such as those listed in Table C, for example). In some embodiments, such a fusion protein is represented by formula I (shown from the N-terminus to the C-terminus): (TCE)-(linker)-(ELNN)(I) It can be represented by the formula, where TCE is as described herein, linker is a linker sequence (such as those described herein, for example, in Table C) comprising 1 to about 50 amino acid residues which may optionally include a TCE release segment (as described herein), and ELNN may be any ELNN as described herein.

[0415] In some embodiments, the fusion protein includes an N-terminal ELNN and optionally a linker between the ELNN and the TCE (such as those listed in Table C, for example). In some embodiments, such a fusion protein is represented by formula II (shown from the N-terminus to the C-terminus): (ELNN)-(linker)-(TCE)(II) It can be represented by the formula, where TCE is as described herein, linker is a linker sequence (such as those described herein, for example, in Table C) comprising 1 to about 50 amino acid residues which may optionally include a TCE release segment (as described herein), and ELNN may be any ELNN as described herein.

[0416] In some embodiments, the fusion protein includes both an N-terminal ELNN and a C-terminal ELNN. In some embodiments, such a fusion protein is given by formula III: (ELNN)-(linker)-(TCE)-(linker)-(ELNN)(III) It can be represented by the formula, where TCE is as described herein, each linker is a linker sequence (such as those described in Table C herein) having 1 to about 50 amino acid residues which may individually and optionally include a TCE release segment (such as those described herein), and each ELNN may individually be any ELNN described herein.

[0417] This disclosure provides BsAbs (e.g., TCEs) comprising one or more sequences of the disclosure in any one of Tables 6a to 6g of this specification.

[0418] Of particular interest are BsAbs (e.g., TCEs) for which increased pharmacokinetic parameters, increased solubility, increased stability, masking of activity, or some other improvement in pharmaceutical properties are desired, or BsAbs (e.g., TCEs) for which an increase in terminal phase half-life improves efficacy and / or safety. Therefore, paTCE fusion protein compositions are prepared with various objectives in mind, including, for example, improving the therapeutic efficacy of TCE by increasing in vivo exposure or increasing the length for which TCE remains within the therapeutic window when administered to a subject, compared to TCE not linked to any ELNN.

[0419] For example, it will be understood that variants can be created by making various amino acid substitutions (specifically, conserved amino acid substitutions) in the bispecific sequence without departing from the spirit of this disclosure with respect to the biological activity or pharmacological properties of TCE. Examples of conserved amino acid substitutions in polypeptide sequences are shown in Table 4. In addition, variants may also include polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the full-length native amino acid sequence of TCE, for example, while retaining 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 of activity compared to the corresponding original TCE sequence are considered suitable for inclusion in the target 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 to the sequences from Tables 3a-3b (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).

[0421] [Table 5]

[0422] This disclosure provides an ELLN-modified TCE (such as paTCE) that targets PSMA, wherein the TCE is a bispecific antibody (e.g., a bispecific TCE) that specifically binds to PSMA with a portion of the bispecific TCE and specifically binds to CD3 with the other portion of the bispecific TCE.

[0423] In some embodiments, the ELLN-modified TCE comprises (1) a first portion including a first binding domain and a second binding domain, (2) a second portion including an release segment, and (3) a third portion including an unstructured polypeptide mask (which may also be referred to herein as a masking portion).

[0424] In some embodiments, the ELLN-modified TCE has the stereochemistry of formula Ia (shown from the N-terminus to the C-terminus): (Part 1) - (Part 2) - (Part 3) (Ia) The formula comprises, wherein the first part is a bispecific antibody domain comprising the two antigen-binding domains described above, the first binding domain having a specific binding affinity to PSMA (e.g., expressed on cancer cells), the second binding domain having a specific binding affinity to CD3 (e.g., expressed on effector cells), the second part comprising a release segment (RS) that can be cleaved by a mammalian protease, and the third part being a masking portion that functions to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that can be cleaved by proteases present in the tumor microenvironment.

[0425] In some embodiments, the first part comprises a binding domain containing VHH and a binding domain containing VL and VH, in which the binding domain of the first part may be in the order (VL-VH)1-(VHH)2, or (VH-VL)1-(VHH)2, or (VHH)1-(VL-VH)2, or (VHH)1-(VH-VL)2 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein). In some embodiments, the first part comprises two binding domains, each containing VL and VH, the binding domains of the first part 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 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein).

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

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

[0428] In some embodiments, the fusion protein has the conformation of formula IIa (shown from the N-terminus to the C-terminus): (Part 3) - (Part 2) - (Part 1) (IIa) The formula comprises, wherein the first part is bispecific and comprises two antigen-binding domains, the first binding domain having a specific binding affinity to PSMA (e.g., expressed on cancer cells), the second binding domain having a specific binding affinity to CD3 (e.g., expressed on effector cells), the second part comprises a release segment (RS) that can be cleaved by a mammalian protease, and the third part is a masking portion that functions to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that can be universally cleaved in the tumor microenvironment.

[0429] In some embodiments, the first part comprises a binding domain containing VHH and a binding domain containing VL and VH, in which the binding domain of the first part may be in the order (VL-VH)1-(VHH)2, or (VH-VL)1-(VHH)2, or (VHH)1-(VL-VH)2, or (VHH)1-(VH-VL)2 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein). In some embodiments, the first part comprises two binding domains, each containing VL and VH, the binding domains of the first part 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 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein).

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

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

[0432] In some embodiments, the paTCE composition has the stereochemistry of formula IIIa (shown from the N-terminus to the C-terminus): (Part 5) - (Part 4) - (Part 1) - (Part 2) - (Part 3) (IIIa) The formula comprises, wherein the first part is bispecific and comprises two antigen-binding domains, the first binding domain having a specific binding affinity to PSMA (e.g., expressed on cancer cells), the second binding domain having a specific binding affinity to CD3 (e.g., expressed on effector cells), the second part comprises a release segment (RS) that can be cleaved by a mammalian protease, the third part is a masking portion that functions to mask the biological properties of the bispecific antibody domain, the fourth part comprises a release segment (RS) that can be cleaved by a mammalian protease which may be the same as or different from the second part, and the fifth part is a masking portion which may be the same as or different from the third part.

[0433] In some embodiments, the first part comprises a binding domain containing VHH and a binding domain containing VL and VH, in which the binding domain of the first part may be in the order (VL-VH)1-(VHH)2, or (VH-VL)1-(VHH)2, or (VHH)1-(VL-VH)2, or (VHH)1-(VH-VL)2 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein). In some embodiments, the first part comprises two binding domains, each containing VL and VH, the binding domains of the first part 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 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein).

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

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

[0436] Provided herein are compositions that advantageously provide a PSMA-targeted bispecific therapeutic agent having higher selectivity, a longer half-life, lower toxicity and fewer side effects when cleaved by proteases found in target tissue or disease-induced unhealthy tissue, and as a result the composition has an improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions are useful in the treatment of cancer. In some embodiments, when paTCE is in proximity to target tissue or cells carrying or secreting a protease capable of cleaving RS, the bispecific binding domain is released from ELNN by the action of the protease, removing the steric hindrance barrier and freeing TCE to exert its pharmacological effect. This property is particularly advantageous in treating immunologically cold tumors expressing PSMA. In some embodiments, the paTCE provided herein is activated in target tissue, where the target tissue is a solid tumor of an organ or system.

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

[0438] In some embodiments, single-chain binding domains are used that are capable of binding to effector cells and ligands or receptors associated with antigens of diseased tissues or cells, such as cancer, tumors, or other malignant tissues, 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.

[0439] In some embodiments, the binding domain is a bispecific antibody domain, which 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., scFv, or an ISVD such as VHH), and the second antigen-binding domain is a second antigen-binding fragment (e.g., scFv, or an ISVD such as VHH).

[0440] In some embodiments, the antigen-binding fragment (AF) (e.g., a first antigen-binding fragment (AF1) and / or a second antigen-binding fragment (AF2)) may be (each independently) a chimeric, humanized, or human antigen-binding fragment. The antigen-binding fragment (AF) (e.g., a first antigen-binding fragment (AF1) and / or a second antigen-binding fragment (AF2)) may be (each independently) Fv, Fab, Fab', Fab'-SH, a 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 having binding specificity to a cancer cell marker and a second binding domain having binding specificity to an effector cell antigen. In some embodiments, the binding domain to a tumor cell target is a variable domain of a T cell receptor engineered to bind to MHC, filled with a peptide fragment of a protein overexpressed by tumor cells.

[0442] In some embodiments, paTCEs are designed to provide a broad therapeutic window by considering the location of the target tissue protease, the presence of the same protease in healthy tissue not intended to be targeted, and the presence of the target ligand in healthy tissue but in greater abundance in unhealthy target tissue. The “therapeutic window” refers to the difference between the minimum effective dose and the maximum permissible dose for a given therapeutic composition. In some embodiments, to help achieve a broad therapeutic window for the TCE, the binding domain of the TCE is shielded by the proximity of a masking (e.g., ELNN) moiety such that the binding affinity of the intact composition to one or both ligands is reduced compared to the composition cleaved by the mammalian protease, thereby releasing the first portion from the shielding effect of the masking moiety.

[0443] In some embodiments, the 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, there are three complementarity-determining regions (CDRs) that interact to define the antigen-binding site on the surface of the VH-VL dimer, and the six CDRs of the binding domain confer antigen-binding specificity to the antibody or single-chain binding domain. Framework sequences adjacent to the CDRs have a tertiary structure that is mainly conserved in native immunoglobulins across species, and 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 the VH-VL interaction. In some embodiments, the binding site may be a pair of VH-VL, VH-VH, or VL-VL domains from the same or different immunoglobulins; however, it is generally preferred to construct the single-chain binding domain using the respective VH and VL chains from the parent antibody. In some embodiments, the order of the VH and VL domains within the polypeptide chain is not limited, as long as the VH and VL domains are positioned so that the antigen-binding site can be properly folded. Therefore, in some embodiments, a single-chain binding domain containing VH and VL (for example, in scFv) may have VH and VL arranged as VL-VH or VL-VH.

[0444] In some embodiments, the arrangement of the V chains 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 following sequence is used: 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) The following combinations are possible: (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, “at the N-terminus” or “at the C-terminus,” and their grammatical variations, refer to relative positions within the primary amino acid sequence, rather than absolute N-terminus or C-terminus placement of a bispecific single-chain antibody. Therefore, as a non-limiting example, “the first binding domain located at the C-terminus relative to the second binding domain” indicates that the first binding is located on the carboxyl side of the second binding domain within the bispecific single-chain antibody, without prejudice that additional sequences, such as a linker and / or another compound, including an ELNN, His tag, or radioisotope, may be located at the C-terminus of the bispecific single-chain antibody.

[0447] In some embodiments, paTCE comprises a first portion containing a first binding domain and a second binding domain, each of which is an scFv, and each scFv contains one VL and one VH. In some embodiments, the paTCE composition comprises a first portion containing a first binding domain and a second binding domain, one of which is an scFV and the other binding domain is a VHH. In some embodiments, the CD3 binding domain may be an scFV (including, for example, the sequence shown in any of Tables 6a-e), and the second binding domain is a PSMA-binding VHH. In some embodiments, paTCE comprises a first portion containing a first binding domain and a second binding domain, where the binding domains are in a diabody configuration, one domain containing one VHH region and the other domain containing one VL region and one VH region. An example of a PSMA-binding VHH binding domain is shown in Table 6f. In some embodiments, paTCE comprises a first portion including a first binding domain and a second binding domain, wherein the binding domains are in a diabody configuration, and each domain includes one VL region and one VH region. Exemplary PSMA-binding VH and VL regions may be derived from the sequences shown in Table 6g.

[0448] In non-limiting examples, TCEs may include sequences exhibiting at least about 80% sequence identity to the antibody sequences identified herein, or alternatively, sequences exhibiting 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the TCE comprises a bispecific sequence (e.g., BsAb) comprising a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity to a tumor-specific marker or cancer cell antigen, and has at least about 80% sequence identity to the paired VHH sequence of the anti-PSMA antibody disclosed in Table 6f herein, or the paired VL and VH sequences of the anti-PSMA antibody disclosed in Table 6g herein, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, The second binding domain has specific binding affinity to effector cells and exhibits at least about 80% sequence identity to the paired VL and VH sequences of the anti-CD3 antibodies disclosed in any of Tables 6a to 6e herein, 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.

[0449] In some embodiments, the TCE may include a binding domain (e.g., 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 the 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 those of pasotaxizumab (also known as AMG-212) and acapatamab (also known as AMG-160).

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

[0451] In some embodiments, TCE is pasotaxizumab. In some embodiments, TCE is akapatamab.

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

[0453] In some embodiments, ISVD is VHH.

[0454] Also provided are nucleic acid molecules encoding the ISVD or polypeptide of this disclosure, or vectors containing nucleic acids.

[0455] This disclosure also relates to non-human host or host cells transformed or transfected with nucleic acids or vectors encoding the ISVD or polypeptide disclosed herein.

[0456] This disclosure further relates to compositions such as pharmaceutical compositions, comprising the ISVD or polypeptide disclosed herein.

[0457] This specification includes methods for producing ISVDs or polypeptides disclosed herein, the methods being a. The step of expressing a nucleic acid sequence encoding ISVD or polypeptide in a host cell or host organism, or in another expression system, and thereafter optionally, b. A method comprising the step of isolating and / or purifying ISVD or polypeptide.

[0458] Provided herein are compositions and polypeptides containing ISVD for use as pharmaceuticals. In some embodiments, the polypeptide or composition is for use in the treatment of proliferative disorders. In some embodiments, the proliferative disorder is cancer.

[0459] This disclosure also provides a method of treatment comprising the step of administering a composition or polypeptide comprising ISVD to a subject in need of treatment. In some embodiments, the method of treatment is for treating a proliferative disorder. In some embodiments, the proliferative disorder is cancer.

[0460] This specification includes compositions and polypeptides containing ISVD for use in the preparation of pharmaceuticals. In some embodiments, the pharmaceuticals are used in the treatment of proliferative disorders. In some embodiments, the proliferative disorder is cancer.

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

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

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

[0464] Therefore, a single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof, insofar as it is possible to form a single antigen-binding unit (i.e., a functional antigen-binding unit that is primarily derived from a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit); or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof.

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

[0466] For example, an immunoglobulin monovariate domain may be a monodomain antibody (or an amino acid sequence suitable for use as a monodomain antibody), a "dAb", or a dAb (or an amino acid sequence suitable for use as a dAb); another monovariate 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] The "VHH domain," also known as VHH, VHH region, VHH antibody fragment, and VHH antibody, was originally described as the antigen-binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., "antibodies lacking a light chain," 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 domain," "VH region," and "VH") and the light chain variable domains present in conventional four-chain antibodies (referred to herein as "VL domain," "VL region," and "VL"). For further description of VHH, see the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

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

[0470] The generation of immunoglobulin sequences is extensively described in various publications, particularly in International Publication No. 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001. These methods involve immunizing camelid animals with a target antigen to induce an immune response to it. The repertoire of VHHs obtained from immunization is then further screened for VHHs that bind to the target antigen.

[0471] In these cases, antibody production requires purified antigens for immunization and / or screening. Antigens can be obtained from natural sources or purified during recombinant production processes.

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

[0473] This technology can utilize immunoglobulin sequences of different origins, including those from mouse, rat, rabbit, donkey, human, and camelid. The technology also includes fully human, humanized, or chimeric sequences. For example, the technology includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelid domain antibodies, such as camelid dAbs described by Ward et al. (see, e.g., International Publication No. 94 / 04678 and Davies and Riechmann (1994 and 1996)). In some embodiments, the technology also uses fused immunoglobulin sequences (polyvalent and multispecific polypeptides containing one or more VHH domains, and for their preparation, see Conrath et al., J. Biol. Chem., Vol. 276, 10.7346-7350, 2001, and also see, for example, International Publications 96 / 34103 and 99 / 23221) that form polyvalent and / or multispecific constructs, and immunoglobulin sequences containing tags or other functional parts that can be derived from the immunoglobulin sequences of the Technology, such as toxins, labels, radiochemicals, etc.

[0474] "Humanized VHH" includes an amino acid sequence that corresponds 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 (particularly in the framework sequence) are "humanized," that is, humanized by replacing them with one or more amino acid residues located at the corresponding positions in the VH domain from a conventional (e.g., shown above) four-chain antibody from humans. This can be carried out in essentially known ways, which will be apparent to those skilled in the art, for example, based on further description herein and prior art (e.g., International Publication No. 2008 / 020079). Again, it should be noted that such humanized VHH can be obtained in any suitable way that is essentially known, and is therefore not strictly limited to polypeptides obtained using polypeptides containing a naturally occurring VHH domain as starting material.

[0475] "Camelized VH" includes an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but is "camelized," that is, it is camelized by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain from a conventional four-chain antibody with one or more amino acid residues present at the corresponding positions in the VHH domain of a heavy-chain antibody. This can be carried out in essentially known ways, which will be apparent to those skilled in the art, for example, based on further description herein and prior art (e.g., International Publication No. 2008 / 020079). Such "camelized" substitutions are preferably inserted into amino acid positions that form and / or are present at the VH-VL interface, and / or into so-called camelid hallmark residues as defined herein (e.g., International Publication No. 94 / 04678 and Davies and Riechmann (1994 and 1996), see above). In some embodiments, the VH sequence used as a starting material or starting point for generating or designing camelid VH is preferably a VH sequence from a mammal, such as a human VH sequence like the VH3 sequence. However, it should be noted that such camelid VH can be obtained in essentially known and suitable ways, and is therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting materials.

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

[0477] As further described in paragraphs q) on pages 58 and 59 of International Publication No. 08 / 020079, the amino acid residues of a single variable immunoglobulin domain 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), which applies to VHH domains from camelids in the literature Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195, see, for example, Figure 2 of this publication). As is well known in the art with respect to the VH and VHH domains, it should be noted that the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions indicated by Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number permitted by Kabat numbering). This generally means that 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 is in the range of 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 area can also be done according to different methods.

[0479] In some embodiments, the VHH CDR sequence was 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 contains amino acid residues from positions 1 to 25, CDR1 contains amino acid residues from positions 26 to 35, FR2 contains amino acids from positions 36 to 49, CDR2 contains amino acid residues from positions 50 to 58, FR3 contains amino acid residues from positions 59 to 94, CDR3 contains amino acid residues from positions 95 to 102, and FR4 contains amino acid residues from positions 103 to 113.

[0480] In some embodiments, the CDR sequence is 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 monovariable domain contains amino acid residues from positions 1 to 30, CDR1 of the immunoglobulin monovariable domain contains amino acid residues from positions 31 to 35, FR2 of the immunoglobulin monovariable domain contains amino acids from positions 36 to 49, CDR2 of the immunoglobulin monovariable domain contains amino acid residues from positions 50 to 65, FR3 of the immunoglobulin monovariable domain contains amino acid residues from positions 66 to 94, CDR3 of the immunoglobulin monovariable domain contains amino acid residues from positions 95 to 102, and FR4 of the immunoglobulin monovariable domain contains amino acid residues from positions 103 to 113.

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

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

[0483] In such immunoglobulin sequences, the framework sequence can be any suitable framework sequence, and examples of suitable framework sequences will be apparent to those skilled in the art, for example, based on standard handbooks and further disclosures and references referred herein.

[0484] In some embodiments, the framework sequence is a suitable combination of an immunoglobulin framework sequence or a framework sequence derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence may be a framework sequence 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 sequence is either a framework sequence derived from a VHH sequence (the framework sequence may optionally be partially or fully humanized) or a camelized conventional VH sequence (as defined herein).

[0485] In some embodiments, the framework sequences present in the ISVD sequence used in the technique may contain one or more of the hallmark residues (as defined herein) such that the ISVD sequence is a VHH containing humanized VHH or camelid VHH. Some non-limiting examples of such framework sequences (or preferred combinations thereof) will become apparent from further disclosures herein.

[0486] Again, it is also possible to use any of the aforementioned preferred fragments (or combinations of fragments), such as a fragment containing one or more CDR sequences that are preferably adjacent to and / or linked thereto to one or more framework sequences (for example, these CDRs and framework sequences in the same order in which they may be present in the full-size immunoglobulin sequence from which the fragment originates), as is generally described herein with respect to immunoglobulin sequences.

[0487] However, it should be noted that the technology is not limited in terms of the origin of the ISVD sequence (or the nucleotide sequence used to express it) or the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or generated or obtained). Therefore, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In certain but non-limiting embodiments, an ISVD sequence is a synthetic or semi-synthetic sequence, including, but not limited to, naturally occurring sequences (from any suitable species), “humanized” immunoglobulin sequences (partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized VHH sequences, etc.), “camelized” immunoglobulin sequences (disclosed herein), and immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from a synthetic, random, or naturally occurring immunoglobulin sequence), CDR transplantation, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences known to those skilled in the art; or any preferred combination of any of the foregoing.

[0488] Similarly, nucleotide sequences may be naturally occurring nucleotide sequences, or synthetic or semi-synthetic sequences, and may, for example, be sequences isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from cells), nucleotide sequences isolated from a library (in particular, an expression library), nucleotide sequences prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique that is essentially known, such as mismatch PCR), nucleotide sequences prepared by PCR using duplicate primers, or nucleotide sequences prepared using techniques for DNA synthesis that are essentially known.

[0489] As described above, an ISVD may be an ISVD or a preferred fragment thereof. For a general description of an ISVD, 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 mainly describe so-called "VH3 class" ISVDs (i.e., ISVDs having a high degree of sequence homology to VH3 class human germline sequences such as DP-47, DP-51, or DP-29). However, it should be noted that the art in its broadest sense can generally use any type of ISVD, and that ISVDs belonging to the so-called "VH4 class" (i.e., ISVDs having a high degree of sequence homology to VH4 class human germline sequences such as DP-78), for example, as described in International Publication No. 2007 / 118670, may also be used.

[0490] In general, ISVDs (in particular VHH sequences including (partially) humanized VHH sequences 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, further described herein). Therefore, in general, ISVDs are (general) structural FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It may be defined as an immunoglobulin sequence containing, where FR1-FR4 refer to framework regions 1-4, respectively, 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 is the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 The immunoglobulin sequence may include FR1-FR4, where FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity-determining regions 1-3, respectively, with the framework sequence being as further defined herein.

[0492] In some embodiments, the ISVD is the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 The immunoglobulin sequence may include FR1-FR4, where FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity-determining regions 1-3, respectively, and one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108, as assigned by Kabat, are selected from the Hallmark residues mentioned in Table 5 below.

[0493] [Table 6]

[0494] In some embodiments, the techniques provided herein utilize ISVDs capable of binding to PSMA. In the context of this technique, "binding to a particular target molecule" has the usual meaning in the art as understood in the context of antibodies and their respective antigens.

[0495] In some embodiments, ISVD (e.g., VHH) or multispecific polyvalent polypeptides exhibit reduced binding by existing antibodies in human serum. For this purpose, in some embodiments, the polypeptide exhibits valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in ISVD. For example, the following sequence:

[0496] [Table 7] The following sequence:

[0497] [Table 8] It can be modified to become [this].

[0498] In some embodiments, the polypeptide exhibits an elongation of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) elongation, at the C-terminus of the ISVD (e.g., the C-terminal ISVD of a fusion protein or an ISVD 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 (Sequence ID 549) The following sequence:

[0499] [Table 9] It can be modified to become any one of the following.

[0500] In some embodiments, the polypeptide exhibits 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] The following sequence:

[0503] [Table 11] It can be modified to become any one of the following.

[0504] In some embodiments, ISVD exhibits lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) at least on the ISVD. In these embodiments, the C-terminus of 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), and as a result, 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 valine (V) at amino acid position 11 (Kabat numbering) and leucine (L) at amino acid position 89 in at least the C-terminal ISVD, and optionally lysine (K) or glutamine (Q) at position 110 (Kabat numbering) in at least one ISVD, and exhibits elongations of (preferably naturally occurring) amino acids, such as 1 to 5 single alanine (A) elongations, at the C-terminus of the C-terminal ISVD (resulting in the C-terminus of the polypeptide being, for example, 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 on this, see, for example, International Publication Nos. 2012 / 175741 and 2015 / 173325.

[0506] As will be apparent from the above and further descriptions herein, the ISVDs of the Art can be used as “building blocks” for forming polypeptides of the Art, which combine one or more desirable properties or biological functions within a single molecule, for example, by suitably combining them with other groups, residues, moieties, or binding units to form the compounds or fusion proteins of the Art described herein (including, but not limited to, divalent / trivalent / tetravalent / polyvalent and double / triple / quadrivalent / multispecific polypeptides of the Art described herein). Polypeptides having multiple ISVDs are also referred to herein as “ISVD constructs” or “ISVD formats.”

[0507] The terms “specificity,” “specific 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 ISVD (e.g., VHH) or scFv, can bind with sufficiently high affinity (see below). “Specificity,” “specific binding,” or “specific binding” are used herein interchangeably with “selectivity,” “selective binding,” or “selective binding.” Binding units such as VHH and scFv preferably bind specifically to their designated targets.

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

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

[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 K in moles / liter D It then binds to those targets.

[0511] In some embodiments, 10 -4 K for moles / liter D The value is considered nonspecific. In some embodiments, 10 -4 K less than moles / liter D The value is considered unique.

[0512] K is a biological interaction that is considered specific, such as the binding of antibody sequences to antigens. D Typically, this range is between 10,000 nM or 10 μM and 0.001 nM or 1 pM or less.

[0513] Therefore, specific / selective binding can be measured using the same method, e.g., SPR, where the binding unit (or polypeptide containing it) is 10 -5 ~10 -12 K is less than or equal to moles / liter D Combine the values ​​into PSMA, 10 -4 K for moles / liter D This could mean binding to different targets based on their values.

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

[0515] Specific binding from a particular species to a particular target does not preclude the binding unit from also specifically binding to similar targets from different species. For example, specific binding to human PSMA does not preclude the binding unit (or polypeptide containing it) from also specifically binding to PSMA from cynomolgus monkeys.

[0516] The specific binding of the binding unit to the specified target can be determined in any preferred manner that is essentially known, including, for example, Scatchard analysis, and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay, as well as different variations thereof that are essentially known in the art; and other techniques referred to herein.

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

[0518] The affinity of molecular interactions between two molecules can be measured through various techniques that are essentially known, 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 enables real-time analysis of 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 k on , k off Measured value, therefore K DThe substance passes over immobilized molecules under flowing conditions that yield the desired value. This can be carried out, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further information, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnson et al. (1991, Anal. Biochem. 198:268-277).

[0519] Another well-known biosensor technique for determining the affinity of biomolecular interactions is bio-layer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the terms “bio-layer interferometry” or “BLI” refer to an unlabeled optical technique that analyzes the interference patterns of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). A change in the number of molecules bound to the biosensor chip causes 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. Since the interactions can be measured in real time, the association and dissociation rates, as well as affinity, can 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 the 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 equilibrium solution of the antibody / antigen complex is passed through a column containing beads pre-coated with the antigen (or antibody) to bind the free antibody (or antigen) to the coated molecule. Detection of the thus captured antibody (or antigen) 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 is measured by, for example, SPR, such as being performed with a ProteOn XPR36 instrument at 25°, and is at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , and at least about 10 5 M -1 s -1 The binding rate (on-rate) constant (k) for binding to human PSMA, selected from the group consisting of the following: on ) has.

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

[0524] In some embodiments, the ISVD provided herein is measured by, for example, SPR, using a ProteOn XPR36 instrument, preferably at 25°C, and is measured at least about 10 -2 s -1 at most about 10 -3 s -1 , and at most about 10 -4 s -1 k of the group consisting of the following regarding binding to human PSMA off It has.

[0525] In some embodiments, the ISVD provided herein is measured by SPR, for example, using a ProteOn XPR36 instrument, for example, at 25°C, and is measured at most about 10 -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 of non-human primate binding to PSMA selected from the group consisting of the above off It has.

[0526] In some embodiments, the ISVD provided herein is measured by SPR, for example, using a ProteOn XPR36 instrument, for example, at 25°C, and is measured at most about 10 -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 the following: D ) has.

[0527] In some embodiments, the ISVD provided herein is measured by SPR, for example, using a ProteOn XPR36 instrument, for example, at 25°C, and is measured at most about 10 -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-coupled ISVD of this technology has the same or lower off-rate constant (k) compared to QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549). off ) binds to human PSMA. In some embodiments, the ISVD of this technology has the same or lower k values ​​compared to the ISVD of QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSS (SEQ ID NO: 549). off It binds to non-human primate PSMA.

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

[0530] In some embodiments, paTCE comprises a first binding domain which is an scFv and a second binding domain which is also an scFv. In some embodiments, the scFv comprises VL and VH domains, respectively, derived from a monoclonal antibody having binding specificity to a tumor-specific marker or cancer cell antigen and an effector cell antigen. In some embodiments, the first and second binding domains each comprise six CDRs, respectively, derived from a cancer cell marker such as a tumor-specific marker and a monoclonal antibody having binding specificity to an effector cell antigen. In some embodiments, the first and second binding domains of the first portion of the composition in question may have three, four, five, or six CDRs within each binding domain. In some embodiments, 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 cancer cell antigen and an effector cell antigen, respectively.

[0531] In some embodiments, the second binding domain includes VH and VL regions derived from a monoclonal antibody capable of binding to human CD3. In some embodiments, the second binding domain includes an scFv containing the VH and VL regions, each VH and VL region exhibiting or being identical to at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or...

Claims

1. A polypeptide containing the amino acid sequence of SEQ ID NO: 1000.

2. The polypeptide according to claim 1, comprising the amino acid sequence of sequence number 1000.

3. A pharmaceutical composition comprising the polypeptide described in claim 1 and one or more pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is for treating cancer, and the cancer is characterized by the expression of prostate-specific membrane antigen (PSMA).

5. The pharmaceutical composition according to claim 4, wherein the cancer is prostate cancer.

6. A polynucleotide encoding the polypeptide described in claim 1.

7. A vector comprising a polynucleotide according to claim 6 and a regulatory sequence operably linked to the polynucleotide.

8. A host cell comprising the vector according to claim 7.

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