STEAP2-directed T-cell adaptors and their compositions

Abstract

This invention relates to STEAP2-directed T-cell adaptors and compositions thereof. T-cell adaptor molecules are provided that bind antigens on non-immune cells and antigens on immune cells. The T-cell adaptor molecules can bind STEAP2 on cancer cells and, for example, CD3 on T cells. The T-cell adaptor molecules can also bind STEAP2 on cancer cells and, for example, CD8 on T cells. Alternatively, the T-cell adaptor molecules can bind STEAP2 on cancer cells and CD3 and CD8 on T cells.

Description

[0001] This application is a divisional application of Chinese patent application 202480025162.2, "STEAP2-oriented T-cell adaptor and composition thereof," filed on April 10, 2024.

[0002] Cross-references to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 495,547, filed April 11, 2023, the entire contents of which are incorporated herein by reference.

[0003] Reference to the sequence listing submitted electronically The contents of the sequence listing (name: STEAP2TED-100-WO-PCT_ST26.xml; size: 182,496 bytes; creation date: April 10, 2024), which was submitted electronically with this application, are incorporated herein by reference in their entirety. Background Technology

[0004] Identifying multivalently binding molecules with two or more distinct epitopes is of interest in diagnostic and therapeutic applications. However, their generation presents challenges. Mixed pairing of heavy and light chains expressed in a single cell can lead to the production of several different molecules, with only one pairing being the desired one, while the remaining pairings produce nonfunctional or single-specific molecules.

[0005] Various strategies have been developed to attempt to overcome this problem and facilitate the correct assembly of desired multivalent molecules. However, with the introduction of each additional binding arm, the likelihood of mismatch increases.

[0006] Therefore, additional mechanisms are needed to improve the pairing of polypeptide chains in multivalent molecules and promote their efficient production. Summary of the Invention

[0007] A T-cell adaptor molecule is provided, comprising: (a) an antigen-binding arm that binds to an epitope on human prostatic six-transmembrane epithelial antigen 2 (STEAP2), and comprising a heavy chain including a heavy chain variable domain (VH) and a heavy chain CH1 domain, the heavy chain variable domain (VH) including a variable heavy chain complementarity-determining region 1 (VH-CDR1) selected from SEQ ID NO: 1, 9, 17, 103, 111, 127, 135 and 143; and VH-CDR2 selected from SEQ ID NO: 2, 10, 18, 104, 112, 128, 136 and 144; and VH-CDR2 selected from SEQ ID NO: 1, 9, 17, 103, 111, 127, 135 and 143. VH-CDR3 selected from SEQ ID NO: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137, and 145; and a light chain comprising a light chain variable structural domain (VL) and a light chain constant structural domain, the light chain variable structural domain (VL) comprising a variable light chain complementarity determination region 1 (VL-CDR1) selected from SEQ ID NO: 4, 12, 20, 100, 108, 130, 138, and 146; VL-CDR2 selected from SEQ ID NO: 5, 13, 21, 101, 109, 131, 139, and 147; selected from SEQ ID NO: (a) VL-CDR3 of SEQ ID NO: 6, 14, 22, 102, 110, 132, 140, and 148; (b) a first T cell binding arm comprising a heavy chain containing a heavy chain variable domain (VH) and a heavy chain CH1 domain, the heavy chain variable domain (VH) comprising VH-CDR1 selected from SEQ ID NO: 36, 40, and 44; VH-CDR2 selected from SEQ ID NO: 37, 41, and 45; VH-CDR3 selected from SEQ ID NO: 38, 42, and 46; and a light chain containing a light chain variable domain (VL) and a light chain constant domain, the light chain variable domain (VL) comprising VL-CDR1 selected from SEQ ID NO: 27 and 31; VL-CDR2 selected from SEQ ID NO: 28 and 32; VL-CDR3 selected from SEQ ID NO: 29 and 33; and (c) The Fc domain comprises a first Fc region and a second Fc region, each Fc region comprising a CH2 domain and a CH3 domain; the Fc domain further comprises at least one modification to promote heterodimerization.

[0008] In some aspects, a trivalent T cell adaptor molecule is provided, comprising the T cell adaptor molecule as described, and further comprising: (d) a second T cell binding arm that binds to differentiation cluster 8 (CD8) and comprising a heavy chain comprising a heavy chain variable domain (VH) and a heavy chain CH1 domain, the heavy chain variable domain (VH) comprising VH-CDR1 shown in SEQ ID NO: 48, VH-CDR2 shown in SEQ ID NO: 49, and VH-CDR3 shown in SEQ ID NO: 50; and a light chain comprising a light chain variable domain (VL) and a light chain constant domain, the light chain variable domain (VL) comprising VL-CDR1 shown in SEQ ID NO: 51, VL-CDR2 shown in SEQ ID NO: 52, and VL-CDR3 shown in SEQ ID NO: 53.

[0009] In some respects, the heavy chain of the second T cell binding arm attaches to the heavy chain of the first T cell binding arm via a connector.

[0010] In some respects, the heavy chain of the second T cell binding arm attaches to the heavy chain of the antigen binding arm via a connector.

[0011] In some respects, the adapter contains the amino acid sequence of SEQ ID NO: 89.

[0012] In some respects, the connector contains 1 to approximately 10 copies of SEQ ID NO: 89.

[0013] In some respects, the connector contains two copies of SEQ ID NO: 89.

[0014] In some respects, one of the two CH3 domains of a T-cell adaptor molecule or a trivalent T-cell adaptor molecule contains a club-shaped mutation, and the other of the two CH3 domains contains a mortar-shaped mutation.

[0015] In some respects, the CH1 domain and light chain constant domain of one or more of each of (a) the antigen-binding arm, (b) the first T-cell binding arm and (d) the second T-cell binding arm also contain charge-pair substitutions comprising a first charged amino acid substitution in the CH1 domain and a second charged amino acid substitution in the light chain constant domain, wherein the first charged amino acid substitution and the second charged amino acid substitution have opposite charges.

[0016] In some respects, one or more of the light chain constant domains of each of (a), (b), and (d) of the T cell adaptor molecule or the trivalent T cell adaptor molecule are λ light chain constant domains (CLλ), and wherein the charge pair is a λ charge pair comprising a positively charged amino acid residue selected from arginine, lysine, or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine, wherein the numbering is based on the EU index, and wherein the charged amino acid of the λ charge pair is located at one or more of the following positions: (i) Position 117 in CLλ and position 141 in the CH1 structural domain; (ii) Position 117 in CLλ and position 185 in the CH1 structural domain; (iii) Position 119 in CLλ and position 128 in the CH1 structural domain; (iv) Position 134 in CLλ and position 128 in the CH1 structural domain; (v) Position 134 in CLλ and position 145 in the CH1 structural domain; (vi) Position 134 in CLλ and position 183 in the CH1 structural domain; (vii) Position 136 in CLλ and position 185 in the CH1 structural domain; (viii) Position 178 in CLλ and position 173 in the CH1 structural domain; and / or (ix) Position 117 in CLλ and position 187 in the CH1 structural domain.

[0017] In some respects, the charged amino acids of the λ charge pair of T cell adaptor molecules or trivalent T cell adaptor molecules are as follows: (i) The charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is threonine. (ii) The charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is threonine; (iii) The charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is threonine; (iv) The charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is threonine; (v) The charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is threonine. (vi) The charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is threonine. (vii) The charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is threonine; (viii) The charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is serine; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is threonine; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is serine; or the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is threonine; and / or (ix) The charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is threonine.

[0018] In some respects, one or more of the light chain constant domains in each of (a), (b) and (d) are κ light chain constant domains (CLκ), and wherein the charge pair is a κ charge pair comprising a positively charged amino acid residue selected from arginine, lysine or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine or threonine, wherein the numbering is based on the EU index, and wherein the κ charge pair is located at position 133 in the CLκ and position 183 in the CH1 domain.

[0019] In some respects, the charged amino acids of the κ charge pair of the T cell adaptor molecule or the trivalent T cell adaptor molecule are as follows: (i) the charged amino acid at position 133 is glutamic acid and the charged amino acid at position 183 is lysine; or (ii) the charged amino acid at position 133 is lysine and the charged amino acid at position 183 is glutamic acid.

[0020] In some respects, the light chain constant domain of one of (a), (b) and (d) is CLλ, and the charge pair is a λ charge pair; the light chain constant domain of the second and third of (a), (b) and (d) is CLλ or CLκ, and the charge pair is a λ or κ charge pair; and the second of (a), (b) and (d) contains a charged amino acid in the CH1 domain with the same charge as the charged amino acid in the CH1 domain of the third of (a), (b) and (d).

[0021] In some respects, the light chain constant domain of one of (a), (b) and (d) is CLλ, and the charge pair is λ charge pair; the light chain constant domain of the second and third of (a), (b) and (d) is CLκ, and the charge pair is κ charge pair; and the second of (a), (b) and (d) contains a charged amino acid in the CH1 domain with the same charge as the charged amino acid in the CH1 domain of the third of (a), (b) and (d).

[0022] In some respects, the CH1 domains of (a), (b), and / or (d) of T-cell adaptor molecules or trivalent T-cell adaptor molecules can be linked to light chain constant domains via engineered disulfide bonds.

[0023] In some respects, the CH1 domains of (b) and (d) of the T-cell adaptor molecule or the trivalent T-cell adaptor molecule can be linked to the light chain constant domain via engineered disulfide bonds, and the CH1 domain of (a) is linked to the light chain constant domain via a natural disulfide bond.

[0024] In some respects, the CH1 domain of T-cell adaptor molecules or trivalent T-cell adaptor molecules that can be linked to light chain constant domains via engineered disulfide bonds includes: (i) Substitution of natural cysteine ​​to non-cysteine ​​amino acids, (ii) Substitution of natural noncysteine ​​amino acids to cysteine; Furthermore, the light chain constant structural domain contains (i) Substitution of natural cysteine ​​to non-cysteine ​​amino acids, (ii) Substitution of natural noncysteine ​​amino acids to cysteine; and The substituted cysteine ​​in the light chain constant domain and the substituted cysteine ​​in the CH1 domain can form disulfide bonds.

[0025] In some respects, the CH1 domain of T-cell adaptor molecules or trivalent T-cell adaptor molecules that can be linked to light chain constant domains via engineered disulfide bonds includes: (i) Substitution of native cysteine ​​to non-cysteine ​​amino acid at position 220, (ii) Substitution of natural noncysteine ​​amino acid to cysteine ​​at position 126; Furthermore, the light chain constant structure domain contains: (i) Substitution of native cysteine ​​to non-cysteine ​​amino acid at position 212, (ii) Substitution of a natural non-cysteine ​​amino acid to cysteine ​​at position 122. The cysteine ​​residue at position 126 of the CH1 domain and the cysteine ​​residue at position 122 of the light chain constant domain can form a disulfide bond; the numbering is based on the EU index.

[0026] In some respects, the CH1 domain of T-cell adaptor molecules or trivalent T-cell adaptor molecules that can be linked to light chain constant domains via engineered disulfide bonds includes: (i) Substitution of native cysteine ​​to non-cysteine ​​amino acid at position 220, (ii) Substitution of natural noncysteine ​​amino acid to cysteine ​​at position 126; Furthermore, the light chain constant structure domain contains: (i) Substitution of native cysteine ​​to non-cysteine ​​amino acid at position 214, (ii) Substitution of a natural non-cysteine ​​amino acid to cysteine ​​at position 121 The cysteine ​​residue at position 126 of the CH1 domain and the cysteine ​​residue at position 121 of the light chain constant domain can form a disulfide bond; the numbering is based on the EU index.

[0027] In some aspects, the antigen-binding arm of a T-cell adaptor molecule or a trivalent T-cell adaptor molecule includes a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 7, 15, 23, 93, 95, 97, 106, 114, 133, 141, and 149.

[0028] In some aspects, the antigen-binding arm of a T-cell adaptor molecule or a trivalent T-cell adaptor molecule is attached to an Fc domain and includes a heavy chain constant region containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 66, 67, 69, 72, 73, and 76.

[0029] In some respects, the antigen-binding arm of a T-cell adaptor molecule or a trivalent T-cell adaptor molecule includes a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 8, 16, 24, 107, 115, 134, 142, and 150.

[0030] In some respects, the antigen-binding arm of a T-cell adaptor molecule or a trivalent T-cell adaptor molecule includes a light chain constant domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 63.

[0031] In some aspects, the VL of the T cell binding arm of the T cell adaptor molecule or the VL of the first or second T cell binding arm of the trivalent T cell adaptor molecule comprises VL-CDR1 selected from SEQ ID NO: 27 and 31; VL-CDR2 selected from SEQ ID NO: 28 and 32; and VL-CDR3 selected from SEQ ID NO: 29 and 33; and the VH comprises VH-CDR1 selected from SEQ ID NO: 36, 40 and 44; VH-CDR2 selected from SEQ ID NO: 37, 41 and 45; and VH-CDR3 selected from SEQ ID NO: 38, 42 and 46.

[0032] In some respects, the VL of the T-cell binding arm of a T-cell adaptor molecule or the VL of the first or second T-cell binding arm of a trivalent T-cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30 or 34.

[0033] In some respects, the light chain constant domain of the T cell adaptor molecule or the light chain constant domain of the first or second T cell binding arm of the trivalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 60 or 61.

[0034] In some respects, the VH of the T-cell binding arm of a T-cell adaptor molecule or the VH of the first or second T-cell binding arm of a trivalent T-cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, 43, or 47.

[0035] In some aspects, the heavy chain of the T-cell binding arm of the T-cell adaptor molecule or the first or second T-cell binding arm of the trivalent T-cell adaptor molecule is attached to the Fc domain and includes a heavy chain constant region containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 66, 67, 69, 74, 75, 77, 78, and 79.

[0036] In some respects, the VH of the second T cell binding arm of the trivalent T cell adaptor molecule comprises VH-CDR1 shown in SEQ ID NO: 48; VH-CDR2 shown in SEQ ID NO: 49; VH-CDR3 shown in SEQ ID NO: 50; and the VL comprises VL-CDR1 shown in SEQ ID NO: 51; VL-CDR2 shown in SEQ ID NO: 52; and VL-CDR3 shown in SEQ ID NO: 53.

[0037] In some respects, the VL of the second T cell binding arm of the trivalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 54.

[0038] In some respects, the constant light chain domain of the second T cell binding arm of the trivalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 64.

[0039] In some respects, the VH of the second T cell binding arm of the trivalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56.

[0040] In some respects, the CH1 domain of the second T cell binding arm of the trivalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 57, 66, 67, 69, 74, 75, 77, 78, and 79.

[0041] In some respects, the antigen-binding arm of a T-cell adaptor molecule or a trivalent T-cell adaptor molecule binds to an epitope on the extracellular loop of STEAP2.

[0042] In some respects, T-cell adaptor molecules include SEQ ID NOs: 25, 26, 35, 81, and 92. In some respects, trivalent T-cell adaptor molecules include SEQ ID NOs: 25, 26, 35, 55, and 58.

[0043] In some aspects, a trivalent T-cell adaptor molecule is provided, comprising: (a) a first antigen-binding arm and a second antigen-binding arm, each binding to an epitope on human prostatic six-transmembrane epithelial antigen 2 (STEAP2), and each comprising a heavy chain including a heavy chain variable domain (VH) and a heavy chain CH1 domain, the heavy chain variable domain (VH) including a variable heavy chain complementarity-determining region 1 (VH-CDR1) selected from SEQ ID NO: 1, 9, 17, 103, 111, 127, 135 and 143; VH-CDR2 selected from SEQ ID NO: 2, 10, 18, 104, 112, 128, 136 and 144; selected from SEQ ID NO: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137 and 145 of VH-CDR3; and a light chain comprising a light chain variable domain (VL) and a light chain constant domain, the light chain variable domain (VL) comprising a variable light chain complementarity-determining region 1 (VL-CDR1) selected from SEQ ID NO: 4, 12, 20, 100, 108, 130, 138 and 146; VL-CDR2 selected from SEQ ID NO: 5, 13 and 21; VL-CDR3 selected from SEQ ID NO: 6, 14, 22, 102, 110, 132, 140 and 148; (b) a first T cell binding arm that binds to differentiation cluster 3 (CD3) and comprises a heavy chain comprising a VH and a heavy chain CH1 domain, the VH comprising a variable light chain complementarity-determining region 1 (VL-CDR1) selected from SEQ ID NO: 4, 12, 20, 100, 108, 130, 138 and 146 of VH-CDR3; (b) a first T cell binding arm that binds to differentiation cluster 3 (CD3) and comprises a heavy chain comprising a VH and a heavy chain CH1 domain, the VH comprising a variable light chain complementarity-determining region 1 (VL-CDR1) selected from SEQ ID NO: 4, 12, 20, 100, 108, 130, 138 and 146 of VH-CDR3; VH-CDR1 selected from SEQ ID NO: 36, 40 and 44; VH-CDR2 selected from SEQ ID NO: 37, 41 and 45; VH-CDR3 selected from SEQ ID NO: 38, 42 and 46; and a light chain comprising a VL and a light chain constant domain, the VL comprising VL-CDR1 selected from SEQ ID NO: 27 and 31; VL-CDR2 selected from SEQ ID NO: 28 and 32; VL-CDR3 selected from SEQ ID NO: 29 and 33; and (c) an Fc domain comprising a first Fc region and a second Fc region, each Fc region comprising a CH2 domain and a CH3 domain, and further comprising at least one modification to promote heterodimerization; and wherein the heavy chain of the first antigen-binding arm and the heavy chain of the first T cell-binding arm are attached to the Fc domain, and the heavy chain of the second antigen-binding arm is attached to the heavy chain of the first T cell-binding arm.

[0044] In some aspects, a tetravalent T cell adaptor molecule is provided, comprising the trivalent T cell adaptor molecule described above, and further comprising (d) a second T cell binding arm that binds to differentiation cluster 8 (CD8), and comprising a heavy chain containing a variable restructure domain (VHH), the variable restructure domain comprising VH-CDR1 shown in SEQ ID NO: 84; VH-CDR2 shown in SEQ ID NO: 85; and VH-CDR3 shown in SEQ ID NO: 86.

[0045] In some respects, the heavy chain of the second T cell binding arm of the tetravalent T cell adaptor molecule attaches to the heavy chain of the first antigen binding arm.

[0046] In some respects, the heavy chain of the second antigen-binding arm of a trivalent or quadrivalent T-cell adaptor molecule attaches to the heavy chain of the first T-cell-binding arm via a linker.

[0047] In some respects, the heavy chain of the second T cell binding arm of the tetravalent T cell adaptor molecule attaches to the heavy chain of the first antigen binding arm via a connector.

[0048] In some respects, the adapter contains the amino acid sequence of SEQ ID NO: 89.

[0049] In some respects, the connector contains 1 to approximately 10 copies of SEQ ID NO: 89.

[0050] In some respects, the connector contains two copies of SEQ ID NO: 89.

[0051] In some respects, one of the two CH3 domains of the trivalent or tetravalent T cell adaptor molecule contains a club-shaped mutation, and the other of the two CH3 domains contains a mortar-shaped mutation.

[0052] In some respects, one or more of the CH1 domains and light chain constant domains of (a) the first antigen-binding arm or the second antigen-binding arm and (b) the first T cell-binding arm contain charge-pair substitutions comprising a first charged amino acid substitution in the CH1 domain and a second charged amino acid substitution in the light chain constant domain, wherein the first charged amino acid substitution and the second charged amino acid substitution have opposite charges.

[0053] In some respects, one or more of the light chain constant domains of (a) the first antigen-binding arm or the second antigen-binding arm and (b) the first T cell-binding arm are λ light chain constant domains (CLλ), and the charge pair is a λ charge pair containing a positively charged amino acid residue selected from arginine, lysine, or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine, wherein the numbering is based on the EU index, and wherein the λ charge pair is located at one or more of the following positions: (i) Position 117 in CLλ and position 141 in the CH1 structural domain; (ii) Position 117 in CLλ and position 185 in the CH1 structural domain; (iii) Position 119 in CLλ and position 128 in the CH1 structural domain; (iv) Position 134 in CLλ and position 128 in the CH1 structural domain; (v) Position 134 in CLλ and position 145 in the CH1 structural domain; (vi) Position 134 in CLλ and position 183 in the CH1 structural domain; (vii) Position 136 in CLλ and position 185 in the CH1 structural domain; (viii) Position 178 in CLλ and position 173 in the CH1 structural domain; and (ix) Position 117 in CLλ and position 187 in the CH1 structural domain.

[0054] In some respects, the charged amino acids of the λ charge pairs of trivalent or tetravalent T cell adaptor molecules are as follows: (i) The charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 141 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 141 is threonine. (ii) The charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is threonine; (iii) The charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is threonine; (iv) The charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is threonine; (v) The charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is threonine. (vi) The charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is threonine. (vii) The charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is threonine; (viii) The charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is serine; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is threonine; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is serine; or the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is threonine; and / or (ix) The charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is threonine.

[0055] In some respects, one or more of the light chain constant domains of (a) the first antigen-binding arm or the second antigen-binding arm and (b) the first T cell-binding arm are κ light chain constant domains (CLκ), and the charge pair is a κ charge pair containing a positively charged amino acid residue selected from arginine, lysine or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine or threonine, wherein the numbering is based on the EU index, and wherein the κ charge pair is located at position 133 in CLκ and position 183 in CH1.

[0056] In some respects, the charged amino acids of the κ charge pair of trivalent or tetravalent T cell adaptor molecules are as follows: (i) the charged amino acid at position 133 is glutamic acid and the charged amino acid at position 183 is lysine; or (ii) the charged amino acid at position 133 is lysine and the charged amino acid at position 183 is glutamic acid.

[0057] In some respects, the light chain constant domain of one of (a), (b) and (d) is CLλ, and the charge pair is a λ charge pair; the light chain constant domain of the second and third of (a), (b) and (d) is CLλ or CLκ, and the charge pair is a λ or κ charge pair; and the second of (a), (b) and (d) contains a charged amino acid in the CH1 domain with the same charge as the charged amino acid in the CH1 domain of the third of (a), (b) and (d).

[0058] In some respects, the light chain constant domain of one of (a), (b) and (d) is CLλ, and the charge pair is λ charge pair; the light chain constant domain of the second and third of (a), (b) and (d) is CLκ, and the charge pair is κ charge pair; and the second of (a), (b) and (d) contains a charged amino acid in the CH1 domain with the same charge as the charged amino acid in the CH1 domain of the third of (a), (b) and (d).

[0059] In some respects, the CH1 domains of (a) and / or (b) of the trivalent T cell adaptor molecule or the CH1 domains of (a), (b) and / or (d) of the tetravalent T cell adaptor molecule can be connected to the light chain constant domains of (a) and / or (b) of the trivalent T cell adaptor molecule or the light chains of (a), (b) and / or (d) of the tetravalent T cell adaptor molecule via engineered disulfide bonds.

[0060] In some respects, the CH1 domain of (b) of trivalent or tetravalent T cell adaptor molecules can form a disulfide bond with the light chain constant domain via engineered disulfide bonds, while the CH1 domain of (a) is connected to the light chain constant domain via a natural disulfide bond.

[0061] In some respects, the CH1 domains of (b) and (d) of the tetravalent T cell adaptor molecule can be connected to the light chain constant domains of (b) and (d) via engineered disulfide bonds, and the CH1 domain of (a) is connected to the light chain constant domain of (a) via a natural disulfide bond.

[0062] In some respects, the CH1 domain of the first T cell binding arm of a trivalent or tetravalent T cell adaptor molecule contains (i) Substitution of natural cysteine ​​to non-cysteine ​​amino acids, (ii) Substitution of natural noncysteine ​​amino acids to cysteine; and The light chain invariant domain of the second T cell binding arm contains: (i) Substitution of natural cysteine ​​to non-cysteine ​​amino acids, (ii) Substitution of natural noncysteine ​​amino acids to cysteine; The substituted cysteine ​​residues in the CH1 domain of the first T cell binding arm and the substituted cysteine ​​residues in the light chain constant domain of the first T cell binding arm can form disulfide bonds.

[0063] In some respects, the CH1 domain of the first T cell binding arm of a trivalent or tetravalent T cell adaptor molecule contains: (i) Substitution of native cysteine ​​to non-cysteine ​​amino acid at position 220, (ii) Substitution of natural noncysteine ​​amino acid to cysteine ​​at position 126; Furthermore, the light chain constant structure domain contains: (i) Substitution of native cysteine ​​to non-cysteine ​​amino acid at position 212, (ii) Substitution of a natural non-cysteine ​​amino acid to cysteine ​​at position 122. The cysteine ​​residue at position 126 of the CH1 domain and the cysteine ​​residue at position 122 of the light chain constant domain can form a disulfide bond; the numbering is based on the EU index.

[0064] In some respects, the light chains of the first and second antigen-binding arms of trivalent or tetravalent T-cell adaptor molecules contain native cysteine ​​residues, and the CH1 domains of the first and second antigen-binding arms contain native cysteine ​​residues, wherein the native cysteine ​​residues in the CH1 domain of the first antigen-binding arm and the native cysteine ​​residues in the constant domain of the light chain of the first antigen-binding arm can form disulfide bonds, and the native cysteine ​​residues in the CH1 domain of the second antigen-binding arm and the native cysteine ​​residues in the constant domain of the light chain of the second antigen-binding arm can form disulfide bonds.

[0065] In some respects, the VH of the first antigen-binding arm of the trivalent or tetravalent T-cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0066] In some respects, CH1 of the first antigen-binding arm of a trivalent or tetravalent T-cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 67 and 93.

[0067] In some respects, the VL of the first antigen-binding arm of a trivalent or tetravalent T-cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 8, 16, 24, 107, 115, 134, 142, and 150.

[0068] In some respects, the light chain constant domain of the first antigen-binding arm of a trivalent or tetravalent T-cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 63.

[0069] In some aspects, the VL of the first T cell binding arm of the trivalent or tetravalent T cell adaptor molecule comprises VL-CDR1 as shown in SEQ ID NO: 27 or 31; VL-CDR2 as shown in SEQ ID NO: 28 or 32; VL-CDR3 as shown in SEQ ID NO: 29 or 33; and the VH of the first T cell binding arm comprises VH-CDR1 as shown in SEQ ID NO: 36, 40 or 44; VH-CDR2 as shown in SEQ ID NO: 37, 41 or 45; and VH-CDR3 as shown in SEQ ID NO: 38, 42 or 46.

[0070] In some respects, the VL of the first T cell binding arm of a trivalent or tetravalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30 or 34.

[0071] In some respects, the VH of the first T cell binding arm of a trivalent or tetravalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 39, 43, and 47.

[0072] In some respects, the VH of the second T cell binding arm of the tetravalent T cell adaptor molecule includes VH-CDR1 shown in SEQ ID NO: 84; VH-CDR2 shown in SEQ ID NO: 85; and VH-CDR3 shown in SEQ ID NO: 86.

[0073] In some respects, the VH of the second T cell binding arm of the tetravalent T cell adaptor molecule contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56 or 83.

[0074] In some aspects, the tetravalent T cell adaptor molecule comprising the trivalent T cell adaptor molecule further comprises (d) a second T cell binding arm. In some aspects, the second T cell binding arm comprises VL and VH, wherein the VL comprises VL-CDR1 shown in SEQ ID NO: 51; VL-CDR2 shown in SEQ ID NO: 52; and VL-CDR3 shown in SEQ ID NO: 53, and the VH comprises VH-CDR1 shown in SEQ ID NO: 48; VH-CDR2 shown in SEQ ID NO: 49; and VH-CDR3 shown in SEQ ID NO: 50. In some aspects, the VL of the second T cell binding arm comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 54, and wherein the VH of the second T cell binding arm comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56. In some aspects, the trivalent or tetravalent T cell adaptor molecule binds to an epitope on the extracellular loop of STEAP2.

[0075] In some aspects, the trivalent or tetravalent T-cell adaptor molecule comprises a heavy chain containing a heavy chain constant region of a second antigen-binding arm, and the first T-cell binding arm contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. In some aspects, the heavy chain containing the heavy chain constant region of the first antigen-binding arm and the second T-cell binding arm contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence selected from SEQ ID NO: 87. In some aspects, the trivalent T-cell adaptor molecule comprises SEQ ID NO: 25, 35, 81, 25, and 93. In some aspects, the tetravalent T-cell adaptor molecule comprises SEQ ID NO: 25, 35, 81, 25, and 87. In some respects, the tetravalent T-cell adaptor molecule comprises SEQ ID NO: 151, 152, 153, 154, and 152. In other respects, the tetravalent T-cell adaptor molecule comprises SEQ ID NO: 151, two copies of SEQ ID NO: 152, SEQ ID NO: 153, and SEQ ID NO: 154.

[0076] In some aspects, the T-cell adaptor molecule is encoded by one or more nucleic acids. In some aspects, the vector contains nucleic acids. In some aspects, the isolated host cell contains nucleic acids or a vector. In other aspects, the T-cell adaptor molecule is formulated together with a pharmaceutically acceptable carrier. This disclosure also relates to the treatment of diseases. In some aspects, this disclosure relates to a method of treating a patient in need of a disease, the method comprising administering an effective amount of the T-cell adaptor molecule to the patient. In some aspects, the disease is cancer. In some aspects, the disease is prostate cancer. In some aspects, the T-cell adaptor molecule is formulated for use as a drug. In some aspects, the T-cell adaptor molecule is used to treat cancer. In some aspects, the T-cell adaptor molecule is used to manufacture a drug for treating cancer. Attached Figure Description

[0077] Figure 1A A schematic diagram showing the T-cell adaptor molecule (TED). Figure 1B A schematic diagram showing the T-cell adaptor molecule (TED2). Figure 1C A schematic diagram showing the trivalent T cell adaptor molecule (TED3). Figure 1D A schematic diagram showing the tetravalent T cell adaptor molecule (TED4).

[0078] Figure 2 A banded diagram showing the interaction surface of the κ and CH1 chains of the binding arm of the T cell adaptor.

[0079] Figure 3 A band diagram showing the interaction surface of the λ and CH1 chains of the binding arm of the T cell adaptor.

[0080] Figure 4A The trivalent STEAP2 T cell adaptor molecules TED330D12 G96P / CD3 K29-SN75_3 and TED3 30D12 G96P / CD3 K29-SN75_V12 were shown to bind to C4-2 cells compared to the TED3 Nip228_V12 control. Figure 4B The CD8 binding of the STEAP2 trivalent T cell adaptor molecules TED3 30D12 G96P / CD3 K29-SN75_3 and TED3 30D12G96P / CD3 K29-SN75_V12 is shown. Figure 4C The binding of the tetravalent STEAP2 T cell adaptor molecule 40A3LO12-TED4-K17 / E75 to C4-2 cells was demonstrated. Figure 4D This demonstrates CD8 binding of the STEAP2 quadrivalent T cell adaptor molecule 40A3LO12-TED4-K17 / E7. Figure 4E The STEAP2 trivalent T cell adaptor molecules 30D12-TED3 K29 / SN75 and 30D12-TED3 K29 / SN75 V12 were shown to be cytotoxic compared with 30D12-TED3-control-CD3, 30D12-TED3-control-CD3 V12 and TED3NIP228 / control-CD3 / CD8 V12 controls. Figure 4F The trivalent STEAP2 T cell adaptor molecules 30D12-TED3 K29 / SN75 and 30D12-TED3 K29 / SN75 V12 showed activation of CD8+ T cells compared to 30D12-TED3-control-CD3, 30D12-TED3-control-CD3 V12 and TED3 NIP228 / control-CD3 / CD8 V12 controls. Figure 4G The trivalent STEAP2 T cell adaptor molecules 30D12-TED3 K29 / SN75 and 30D12-TED3 K29 / SN75 V12 showed activation of CD4+ T cells compared to 30D12-TED3 control-CD3, 30D12-TED3 control-CD3 V12 and TED3 NIP228 / control-CD3 / CD8 V12 control. Figure 4HThe trivalent STEAP2 T cell adaptor molecule 40A3LO12-TED2-K29 / SN75 showed high cytotoxicity against STEAP2-overexpressing LNCap, C4-2, VCaP, and 22Rv1 cells, and low cytotoxicity against STEAP2-underexpressing DU145 cells. Figure 4I The trivalent STEAP2 T cell adaptor molecule 30D12-TED3-K29 / SN75 was shown to exhibit high cytotoxicity against STEAP2-overexpressing LNCap, C4-2, VCaP, and 22Rv1 cells, and low cytotoxicity against STEAP2-underexpressing DU145 cells. Figure 4J The EC50 of STEAP2 T cell adaptor molecules and trivalent STEAP2 T cell adaptor molecules in cell lines with different STEAP2 expression levels is shown. Figure 4K This shows the density of STEAP2 receptors on different cell lines. Figure 4L The study showed cytotoxicity induced by STEAP2 T cell adaptor molecule 40A3LO12-TED2-K29 / SN75 and trivalent STEAP2 T cell adaptor molecule 30D12-TED3-K29 / SN75, as well as the release of IL-6 and TNFα cytokines in LNCaP, C4-2, and VCaP cell lines.

[0081] Figure 5A This demonstrates the activation of CD4 and CD8 T cells by STEAP2 T cell adaptor molecules (TED) and trivalent (TED3) STEAP2 CD8-guided T cell adaptor molecules. Figure 5B This demonstrates the activation of T cells by bivalent STEAP2 T cell adaptor molecules (TED2) and various tetravalent (TED4) STEAP2 CD8-guided T cell adaptor molecules. Figure 5C The study showed that, at similar cytotoxic levels, 30D12-TED3-K29 / SN75-triggered IL-6 release was reduced compared to the 30D12-TED-K29 / SN75 molecule. Figure 5D Compared to 40A3LO12-TED2-K29 / SN75, lower levels of IL-6 release were also observed for all variants of the TED4 T cell adaptor at similar levels of cytotoxicity.

[0082] Figure 6A The percentage of cell lysis inhibition by TED2 T cell adaptor and TED3 trivalent STEAP2 T cell adaptor at different Treg to T effector cell ratios is shown. Figure 6BThe image shows Treg activation in untreated cells and cells treated with T cell adaptor molecules (TED) and trivalent (TED3) STEAP2 CD8-guided T cell adaptor molecules.

[0083] Figure 7A The fluorescence of mKate-labeled C4-2 globular tumors over time is shown in the absence of (control) and in the presence of increased concentrations of the trivalent STEAP2 T cell adaptor molecule (TED3). Figure 7B The killing of tumor cells expressing mKate2 in C4-2 globules was also observed in all variants of the TED4 T cell adaptor, and was increased compared to 40A3LO12-TED2-K29 / SN75.

[0084] Figure 8A This shows the tumor volume in human PBMC transplanted mice implanted with LNCaP tumor cells and treated with either untreated or non-targeted trivalent T cell adaptor molecules (NIP228-TED3-K29 / SN75 V12) or trivalent (30D12-TED3-K29 / SN75 V12) STEAP2 CD8-guided T cell adaptor molecules. Figure 8B This shows the tumor volume in human PBMC transplanted mice implanted with C4-2 tumor cells and either untreated or treated with TED2 T cell adaptor molecules or trivalent STEAP2 CD8-guided T cell adaptor molecules. Figure 8C Bioluminescent images showing mice transplanted with human PBMCs and intravenously injected with C4-2 tumor cells expressing luciferase and untreated (PBS) or treated with trivalent STEAP2 CD8-guided T-cell adaptor molecules. Figure 8D show Figure 8C A diagram of bioluminescence in mice.

[0085] Figure 9 This shows the TNF-α levels in mice implanted with C4-2 tumor cells expressing luciferase and transplanted with untreated (PBS) or human PBMCs treated with STEAP2 T cell adaptor molecules with CD3 control binding arms (30D12-TED-CD3 control), STEAP2 T cell adaptor molecules with modified CD3 binding arms (30D12-TED-KN29 / SN75), or trivalent STEAP2 CD8-guided T cell adaptor molecules with modified CD3 binding arms (30D12-TED3-K29 / SN75 V12).

[0086] Figure 10This shows tumor volume in mice subcutaneously implanted with a mixture of human PBMCs and C4-2 tumor cells and untreated (PBS) or treated with 40A3LO12-TED2-K29 / SN75 or 30D12-TED3-K29 / SN75 V12 molecules.

[0087] Figure 11 This shows the tumor volume in mice with subcutaneously implanted C4-2 tumor cells, and in mice that were either untreated or treated with 30D12-TED3-K29 / SN75 V12 molecules. Figure 12 The correlation between EC50 of cell lysis of the tetravalent STEAP2 T cell adaptor molecule 40A3LO12-TED4-K17 / E75 and STEAP2 cell surface expression levels (antigen binding capacity, ABC) in eight tumor cell lines was shown.

[0088] Figure 13 Adding regulatory T cells (Tregs) to a coculture of effector T cells and tumor cells reduced granzyme B production in CD8 T cells responding to non-CD8-guided T cell adaptor molecules, but no effect was observed in Tregs responding to CD8-guided 40A3LO12-TED4-K17 / E75.

[0089] Figure 14 The study demonstrated sustained cytotoxicity of PBMCs after co-culturing with tumor cells for up to four rounds in the presence of 40A3LO12-TED4-K17 / E75.

[0090] Figure 15 The cytotoxicity of STEAP2-negative cells was shown to occur only when co-cultured with varying amounts of STEAP2-positive cells.

[0091] Figure 16A and Figure 16B Cytotoxicity of xenograft organoids derived from prostate cancer patients was demonstrated when co-cultured with T cells in the presence of 40A3LO12-TED4-K17 / E75. No cytotoxicity in response to the non-targeting NIP228-TED4-K17 / E75 molecule was observed. Figure 16C The results showed increased activation of CD8+ T cells compared to CD4+ T cells at the end of the assay.

[0092] Figure 17 Increased IFNg release was observed in primary prostate tumor slices treated with 40A3LO12-TED4-K17 / E75.

[0093] Figure 18AThis shows the tumor volume in human PBMC transplanted mice implanted with 22Rv1 tumor cells and treated with either untreated or non-targeted T-cell adaptor molecules (NIP228-TED4-K17 / E75) or tetravalent (40A3LO12-TED4-K17 / E75) STEAP2 CD8-guided T-cell adaptor molecules. Figure 18B The results showed that after receiving three doses of 40A3LO12-TED4-K17 / E75, the levels of CD69 and CD25 on T cells in the blood of these animals remained unchanged. Figure 18C The study showed that after receiving three doses of 40A3LO12-TED4-K17 / E75, the levels of CD69 and CD25 on CD8+ T cells but not CD4+ T cells in the tumors of these animals increased.

[0094] Figure 19 This shows the tumor burden (as measured by bioluminescence imaging) in human PBMC transplanted mice with C4-2 tumor cells expressing luciferase implanted in the tibia and untreated or treated with non-targeted T cell adaptor molecules (NIP228-TED4-K17 / E75) or tetravalent (40A3LO12-TED4-K17 / E75) STEAP2 CD8-guided T cell adaptor molecules.

[0095] Figure 20 This shows the tumor volume in human PBMC transplanted mice with fragments implanted from a prostate cancer patient-derived xenograft model and neither treated nor treated with the tetravalent (40A3LO12-TED4-K17 / E75) STEAP2 CD8-guided T-cell adaptor molecule. Detailed Implementation

[0096] T-cell adaptor molecules are provided that bind antigens on target cells (e.g., human prostatic six-transmembrane epithelial antigen-2 (STEAP2) on cancer cells) and T-cell antigens (e.g., differentiation cluster 3 (CD3) and / or CD8 protein). Methods for preparing and using the T-cell adaptor molecules are also provided. In some aspects, the T-cell adaptor molecules bind one or more antigens, such as one or more epitopes on STEAP2 of cancer cells, and one or more T-cell proteins, such as CD3 and / or CD8. Without wishing to be theoretically bound, it is assumed that the quantity and efficacy of target protein and T-cell interactions provided by the T-cell adaptor molecules described herein can increase target cell killing while reducing the risk of induced cytokine release syndrome.

[0097] i. Definition To facilitate understanding of this description, certain terms are first defined. Additional definitions are set forth throughout the specific implementation.

[0098] It should be noted that the terms “a” or “an” refer to one or more of the same entity; for example, “nucleotide sequence” should be understood to mean one or more nucleotide sequences. Therefore, the terms “a” (or “an”), “one or more”, and “at least one” are used interchangeably herein.

[0099] Furthermore, the term “and / or” as used herein is considered to refer to each of two specified features or components, whether or not they are specifically disclosed with the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following: 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).

[0100] It should be understood that wherever the term “comprise” is used to describe an aspect herein, other similar aspects described by the terms “comprising” and / or “substantially comprising” are also provided. As used herein, the terms “comprise” and “include” and their variations (e.g., “comprises”, “comprising”, “includes”, and “including”) will be understood to indicate that a component, feature, element, or step or group of components, features, elements, or steps stated therein is included, but does not exclude any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprise”, “substantially comprising”, and “comprising” may be replaced by any of the other two terms while retaining their ordinary meaning.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0102] Units, prefixes, and symbols are represented in their internationally recognized (SI) form. Numerical ranges include the values ​​that define that range. Unless otherwise indicated, nucleotide sequences are written from left to right with a 5' to 3' orientation. Amino acid sequences are written from left to right with an amino-to-carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to the entire specification. Therefore, the terms that are immediately defined below are more fully defined by reference to the entire specification.

[0103] The term “approximately” is used in this document to mean approximately, roughly, about, or in the range of… When the term “approximately” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical value. Generally, the term “approximately” can modify numerical values ​​above and below the stated value by varying upwards or downwards (higher or lower), for example, by 10%.

[0104] As used herein, the term "T-cell adaptor molecule" refers to any molecule that can target an antigen on a target cell (e.g., STEAP2 on cancer cells) and also bind to proteins expressed on T cells (e.g., CD3 and / or CD8 proteins). A T-cell adaptor molecule may comprise at least one antigen-binding moiety or antigen-binding arm, and at least one T-cell binding moiety or T-cell binding arm. The terms "antigen-binding moiety" and "antigen-binding arm" are used interchangeably. T-cell adaptor molecules may include portions of monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. For example, T-cell adaptor molecules may contain an antigen-binding portion or arm of an antibody (e.g., anti-STEAP2 antibody and / or anti-CD3 antibody and / or anti-CD8 antibody) and include (i) a Fab fragment (a fragment cleaved by papain) or a similar monovalent fragment consisting of VL, VH, LC, and CH1 domains; (ii) an F(ab')2 fragment (a fragment cleaved by pepsin) or a similar bivalent fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of an antibody single arm; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) a separate complementarity-determining region (CDR); (vii) a combination of two or more separate CDRs optionally linked by a synthetic linker; or (viii) a single-chain Fv (scFv). Obtaining antibody fragments using conventional techniques known to those skilled in the art, and screening these fragments in the same manner as intact antibodies, is ineffective. Antigen-binding moieties or arms can be generated via recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Banding diagrams of the κ and CH1 chain interaction surfaces of the binding arms of T cell adaptors and the λ and CH1 chain interaction surfaces of the binding arms of T cell adaptors are shown respectively. Figure 2 and Figure 3 middle.

[0105] As used herein, “antigen-binding arm” and “antigen-binding moiety” refer to the portion of a molecule that binds all or part of the target epitope and typically contain six complementarity-determining regions (CDRs): three in the VH region: HCDR1, HCDR2, and HCDR3, and three in the VL region: LCDR1, LCDR2, and LCDR3. The six CDRs together define the complementary site of the antigen-binding moiety or arm, which is the portion of the antigen-binding moiety or arm that binds the target epitope. An antigen-binding moiety or arm may also contain only three heavy chain CDRs. As used herein, the term “epitope” refers to the portion of the antigen bound by the antigen-binding arm. Monoclonal monospecific IgG antibody molecules contain two antigen-binding arms, each antigen-binding domain capable of binding the same epitope (i.e., it is bivalent for a single epitope).

[0106] Examples of bispecific antibody formulations incorporated into these modifications to improve the efficient production of these molecules are “DuetMab”, as described in Mazor 2015 and WO 2013 / 096291. The DuetMab antibody molecule uses a mortar and pestle technique for the heterodimerization of two different heavy chains and increases the efficiency of homologous heavy and light chain pairing by replacing the native disulfide bonds in one of the CH1-CL interfaces with engineered disulfide bonds.

[0107] As used herein, the term "valence" refers to the presence of a specific number of antigen-binding portions or arms in a T-cell adaptor molecule that bind to an epitope. For example, as used herein, the term "trivalent" refers to a T-cell adaptor molecule having three antigen-binding portions or arms. As used herein, the term "tetravalent" refers to a T-cell adaptor molecule having four antigen-binding portions or arms. The antigen-binding portions or arms of trivalent and / or tetravalent T-cell adaptor molecules can bind to the same antigenic molecule, can bind to the same epitope on the antigenic molecule, can bind to different epitopes on the same antigenic molecule, and / or can bind to different epitopes on different antigenic molecules. In some aspects of the bivalent molecules described herein, a single antigen-binding domain binds to CD3, and a second antigen-binding domain binds to another target, and is referred to as the bispecific T-cell adaptor DuetMab ("TED"; see also...). Figure 1A In some aspects of the trivalent molecule described herein, a single antigen-binding domain binds to CD3 in the trivalent antibody, and two additional antigen-binding domains bind to other targets, and this is referred to as the bispecific T-cell adaptor DuetMab (“TED2”; see also...). Figure 1B In some aspects of the trivalent molecule described herein, a single antigen-binding domain binds to CD3 in the trivalent antibody, a single antigen-binding domain binds to CD8, and a third antigen-binding domain binds to another target, referred to as the trispecific T-cell adaptor DuetMab (“TED3”; see also...). Figure 1C In some aspects of the tetravalent molecule described herein, a single antigen-binding domain binds to CD3 in the tetravalent molecule, a single antigen-binding domain binds to CD8, and the third and fourth antigen-binding domains each bind to another or the same target, and may be referred to as the four-specific T-cell adaptor DuetMab (“TED4”; see also...). Figure 1D In some respects, the TED4 form contains two antigen-binding domains capable of binding to the same target.

[0108] The natural evolution of bispecific antibodies has led to the introduction of trispecific antibodies, where, as used herein, the term "linker" refers to a peptide chain linking two polypeptide chains (e.g., the heavy chain of an antigen-binding arm and the heavy chain of a T-cell-binding arm) with at least two amino acids (e.g., glycine and / or serine). The linker may contain one or more glycine and / or serine amino acids. As used herein, the terms "link," "attachment," and "fusion" refer to the association of two or more molecules. Linkages can be covalent or non-covalent. Linkages can also be genetic (i.e., recombinant fusion). Such linkages can be achieved using a variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production. For example, the linker contains at least one "GGGGS".

[0109] In some aspects, the connection between the binding domain and the Fc domain includes a standard hinge region. In some aspects, the hinge region is an IgG1 hinge containing the sequence DKTHTCPPCPAPE (SEQ ID NO: 155) between the CH1 domain of the antigen-binding arm and the CH2 domain of the Fc region.

[0110] As used in this article, the phrase "capable of being linked by a disulfide bond" means that each of the two polypeptide chains contains at least one cysteine ​​residue at a position, such that when the two polypeptide chains are present in a T cell connective molecule, a disulfide bridge can be formed between the two polypeptide chains.

[0111] In some respects, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). In some antibodies (e.g., naturally occurring IgG antibodies), the heavy chain constant region consists of a hinge and three domains: CH1, CH2, and CH3. In some antibodies (e.g., naturally occurring IgG antibodies), each light chain consists of a light chain variable region (VL) and a light chain constant domain. The light chain constant domain consists of a single domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). The VH and VL regions contain framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region contains the following structure: N-terminus - [HFR1] - [HCDR1] - [HFR2] - [HCDR2] - [HFR3] - [HCDR3] - [HFR4] - C-terminus; and the VL region contains the following structure: N-terminus - [LFR1] - [LCDR1] - [LFR2] - [LCDR2] - [LFR3] - [LCDR3] - [LFR4] - C-terminus. Antibodies can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are subclassed in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Antibodies (e.g., IgG1) exist in several allotypes, which differ from each other by at most a few amino acids. Antibodies include, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and fully synthetic antibodies.

[0112] The variable regions of both the heavy and light chains contain binding domains (also known as complementary sites) that interact with antigen molecules (or epitopes on antigen molecules, such as epitopes on CD3 molecules). The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise stated herein, amino acids in the variable regions are numbered using the Kabat numbering system, and those in the constant regions are numbered using EU numbering (Edelman, 2007). The light chain associates with VH and CH1 in the heavy chain to form an “antigen-binding arm,” and the variable domains in the antigen-binding arm interact to form complementary sites for binding the antigen. The light chains in natural antibodies are “λ (lambda)” or “κ (kappa)” light chains, which differ in their amino acid sequences. The light chains of the trivalent and / or tetravalent T cell adaptor molecules described herein can also be chimeric light chains, such as those containing CLλ and VLκ.

[0113] Antibodies and their construction and use are well known in the art and described, for example, in Holliger and Hudson, Nature Biotechnology 23(9):1126-1136 (2005). Given the current technology associated with monoclonal antibody technology, antibodies can be prepared against most targets. It is possible to use monoclonal and other antibody molecules, and techniques using recombinant DNA technology, to generate other antibodies, chimeric molecules, and / or T-cell adaptor molecules. Such techniques may involve introducing a CDR or variable region of an antibody into different antibody molecules or attaching a CDR or variable region to an antibody molecule.

[0114] As used herein, the term “affinity” refers to a measure of the strength of binding between an antigen or target (such as an epitope) and its homologous binding domain (such as a complementary site). As used herein, the term “affinity” refers to the overall stability of the complex between a population of epitopes and complementary sites (i.e., antigens and antigen-binding arms).

[0115] The term "epitope" refers to a site on an antigen (e.g., STEAP2, CD3, or CD8) that a trivalent and / or tetravalent T-cell adaptor molecule can bind to. Epitopes can be formed from consecutive amino acids (typically linear epitopes) or from discontinuous amino acids juxtaposed through the ternary folding of a protein (typically conformational epitopes). Epitopes formed from consecutive amino acids are generally, but not always, preserved upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0116] The term "binds to the same epitope" for two or more antigen-binding moieties means that the antigen-binding moieties bind to the same amino acid residue segment. An antigen-binding moiety that "competes with another antibody to bind to the target" refers to an antigen-binding moiety that inhibits (partially or completely) the binding of another antibody to the target.

[0117] As used herein, the terms “specific binding,” “selective binding,” “selectively binding,” and “specific binding” refer to an antigen-binding portion or arm binding to an epitope on a predetermined antigen. Typically, an antigen-binding portion or arm: (i) when, for example, via BIACORE ® When using surface plasmon resonance (SPR) technology, or Scatchard assay of T-cell adaptor molecules binding to antigen-positive cells, with a predetermined antigen (e.g., human STEAP2, CD3, or CD8) as the analyte and T-cell adaptor molecules as ligands, in the 2000 instrument, the result is approximately less than 10. -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K) D (i) binds to the predetermined antigen with an affinity at least twice that of non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. Therefore, a T-cell adaptor molecule that "specifically binds to human STEAP2" refers to one that binds to the predetermined antigen with an affinity at least twice that of non-specific antigens (e.g., BSA, casein). -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower K D It binds to the antigen-binding portion or arm of human STEAP2.

[0118] As used herein, the term "peptide" is intended to cover both the singular and plural "peptide" and any one or more chains containing two or more amino acids. Therefore, as used herein, "peptide," "peptide subunit," "protein," "amino acid chain," "amino acid sequence," or any other term used to refer to one or more chains containing two or more amino acids is included in the definition of "peptide," even though each of these terms may have a more specific meaning. The term "peptide" may be used in place of any of these terms or used interchangeably with any of these terms. The term also includes peptides that have undergone post-translational or post-synthetic modifications, such as palmitoyl group conjugation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, disulfide bond formation, proteolytic cleavage, or modification by non-naturally occurring amino acids. As used herein, the term "peptide" covers full-length peptides and their fragments, variants, or derivatives. As used herein, a "peptide" can be a portion of a fusion peptide that includes additional components (such as, for example, albumin or PEG moieties) to increase its half-life. Peptides as used herein can also be derivatized in a variety of different ways. Peptides may contain modifications, including, for example, conjugations of palmitoyl groups.

[0119] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, a predicted non-essential amino acid residue in the antigen-binding arm can be substituted with another amino acid residue from the same side chain family.

[0120] The percentage of identity between two sequences is a function of the number of common positions in the sequences (i.e., percentage of homology = number of common positions / total number of positions × 100), taking into account the number of gaps required for optimal alignment of the two sequences and the length of each gap. As described in the following non-restrictive example, mathematical algorithms can be used to compare sequences and determine the percentage of identity between two sequences.

[0121] The percentage of identity between two nucleotide sequences can be determined using the GAP procedure in the GCG software package (available at worldwideweb.gcg.com) with the NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN procedure (version 2.0), using the PAM120 weighted residue table, vacancy length penalty of 12, and vacancy penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm in the GAP program, which has been incorporated into the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0122] The nucleic acid and protein sequences described herein can be further used as “query sequences” for searching public databases to, for example, identify relevant sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparative purposes, vacancy BLAST, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, can be utilized. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0123] As used herein, the term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0124] As used herein, the terms “polynucleotide” and “nucleic acid molecule” are intended to include both DNA and RNA molecules. Polynucleotide or nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0125] As used herein, the term "promoter" refers to a DNA sequence recognized by cellular mechanisms or introduced synthetic mechanisms required for the specific transcription of a gene. The term "promoter" is also intended to encompass those nucleic acid elements sufficient for use in controlling cell-type-specific, tissue-specific, or inducible expression of promoter-dependent genes through external signals or agents; such elements may be located in the 5' or 3' region of a natural gene. In some respects, a promoter can be a constitutively active promoter, a cell-type-specific promoter, or an inducible promoter.

[0126] As used herein, the term "IRES" refers to an element (such as ATG) that facilitates direct entry of the internal ribosome into the cistron (protein-coding region), thereby leading to cap-independent translation of the gene. See, for example, Jackson RJ et al. Trends Biochem Sci 15(12):477-83 (199); Jackson RJ and Kaminski, A. RNA 1(10):985-1000 (1995). Under IRES translation control, translation is performed in a hat-independent manner.

[0127] As used herein, the term "termination signal sequence" can refer to any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence. The polyadenylation signal sequence is a recognition region essential for endonuclease cleavage of the RNA transcript, followed by the polyadenylation concordance sequence AATAAA. The polyadenylation signal sequence provides a "polyA site," a site on the RNA transcript where adenine residues are added post-transcriptionally via polyadenylation.

[0128] As used herein, the terms “operably linked,” “operably inserted,” “operably positioned,” “under control,” or “under transcriptional control” mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. The term “operably linked” means that the DNA sequence and the regulatory sequence are linked in such a way that gene expression is permitted when an appropriate molecule (e.g., a transcription-activating protein) binds to the regulatory sequence. The term “operably inserted” means that the DNA of interest introduced into the cell is located near the DNA sequence that guides the transcription and translation of the introduced DNA (i.e., promotes, for example, the production of a polypeptide encoded by the DNA of interest).

[0129] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is the viral vector, in which the additional DNA segment can be linked to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and paraneoplastic mammalian vectors). Other vectors (e.g., non-paraneoplastic mammalian vectors) can integrate into the host cell genome after introduction into the host cell, thereby replicating along with the host genome. Moreover, some vectors can direct the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors that are practically useful in recombinant DNA technology are often in plasmid form. The terms "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors that serve an equivalent function are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0130] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing nucleic acids that are not naturally present in cells, and may be a cell in which a recombinant expression vector has been introduced. It should be understood that these terms refer not only to the specific test cell, but also to the progeny of such cells. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may not be identical to the parent cells, but are still included within the scope of the term "host cell" as used herein.

[0131] As used herein, the terms “subject,” “individual,” or “patient” mean any organism to which the compositions disclosed herein (e.g., T-cell adaptor molecules) may be administered, for example, for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Mammal subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, etc. Subjects may be persons or animals who seek or have a need for treatment, require treatment, are currently receiving treatment, will receive treatment in the future, or are being cared for by a trained professional for a specific disease or symptom.

[0132] As used herein, the term "treatment" when used in the context of cancer treatment refers to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treatment can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treatment also refers to alleviating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of symptoms and / or preventing or delaying the onset of the disease or illness.

[0133] The term "immune response," as understood in the art, generally refers to a biological response within a vertebrate against exogenous drugs or abnormal cells (e.g., cancer cells) that protects the organism against these drugs and the diseases they cause. Immune responses are mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation within the vertebrate body. Immune responses include, for example, T cells (e.g., effector T cells, Th cells, CD4+). + Cells, CD8 + The activation or suppression of T cells (or Treg cells), or the activation or suppression of any other cells of the immune system (e.g., NK cells).

[0134] As used herein, the term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting a disease or experiencing a relapse of a disease by means of methods including inducing, enhancing, inhibiting or otherwise altering the immune system or immune response.

[0135] As used herein, the term "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0136] Terms such as “effective amount,” “therapeutic effective amount,” and “sufficient amount” for T-cell adaptor molecules or compositions described herein refer to amounts sufficient to produce a beneficial or desired outcome, including: relieving symptoms; reducing the severity of a symptom, condition, or disease when administered to subjects, including humans; achieving a stable (i.e., non-worsening) state of the symptom, condition, or disease; delaying the onset of or slowing the progression of the symptom, condition, or disease; improving or alleviating the state of the symptom, condition, or disease (whether partial or complete), whether detectable or undetectable; improving at least one measurable bodily parameter, which is not necessarily identifiable by the patient; or enhancing or improving the symptom, condition, or disease. In some respects, treatment includes causing a clinically significant response without excessive levels of side effects. Therefore, “therapeutic effective amount” or its synonyms depend on the context in which they are applied. In some respects, a therapeutically effective amount of a drug (e.g., a T-cell adaptor molecule or composition described herein) is an amount that produces a beneficial or desired outcome in subjects compared to a control group that does not receive the drug. The amount of a given agent (e.g., a T-cell adaptor molecule or composition) will vary depending on various factors, such as the given agent, the drug formulation, the route of administration, the type of disease or condition, the identity of the subject (e.g., age, sex, and / or weight), or the host being treated.

[0137] As used herein, the term "preventive effective dose" refers to the amount of a drug (e.g., a T-cell adaptor molecule or composition) that delays, prevents, or blocks the onset, development, or progression of symptoms or disease for a period of time (including weeks, months, or years). Preventive effective doses can vary depending on the characteristics of the drug; how the drug is administered; the degree of risk for the disease; and the subject's medical history, age, weight, family history, genetic makeup; the type of prior or concomitant treatment (if any); and other individual characteristics of the patient to be treated.

[0138] As used in this article, the terms “ug” and “uM” are used interchangeably with “μg” and “μM”, respectively.

[0139] The various aspects described in this article are described in more detail in the following sections.

[0140] II. antigen-binding arm The antigen-binding arm of the T-cell adaptor molecule described herein binds to STEAP2. In some aspects, the T-cell adaptor molecule includes one STEAP2-binding arm. In other aspects, the T-cell adaptor molecule binds to two STEAP2 antigens.

[0141] In some respects, the antigen-binding arm of the T-cell adaptor molecule described herein can be of any type, including Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some respects, the antigen-binding arm comprises Fab.

[0142] In some respects, the antigen-binding arm comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein the VH comprises VH complementarity-determining region (CDR)1, VH-CDR2, and VH-CDR3; and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3.

[0143] In some aspects, the antigen-binding arm comprises VH-CDR1 containing an amino acid sequence selected from SEQ ID No: 1, 9, 17, 103, 111, 127, 135, and 143. In some aspects, the antigen-binding arm comprises VH-CDR2 containing an amino acid sequence selected from SEQ ID No: 2, 10, 18, 104, 112, 128, 136, and 144. In some aspects, the antigen-binding arm comprises VH-CDR3 containing an amino acid sequence selected from SEQ ID No: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137, and 145.

[0144] In some aspects, the antigen-binding arm comprises VL-CDR1 containing an amino acid sequence selected from SEQ ID No: 4, 12, 20, 100, 108, 130, 138, and 146. In some aspects, the antigen-binding arm comprises VL-CDR2 containing an amino acid sequence selected from SEQ ID No: 5, 13, 21, 101, 109, 131, 139, and 147. In some aspects, the antigen-binding arm comprises VL-CDR3 containing an amino acid sequence selected from SEQ ID No: 6, 14, 22, 102, 110, 132, 140, and 148.

[0145] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0146] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 9, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 10, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 11, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 12, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0147] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 17, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 18, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 19, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 20, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 22.

[0148] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 94, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0149] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 96, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0150] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 98, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0151] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 103, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 104, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 105, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 100, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 101, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 102.

[0152] In some aspects, the antigen-binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 111, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 112, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 113, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 108, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 109, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 110.

[0153] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 7.

[0154] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 15.

[0155] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 23.

[0156] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 95. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 95.

[0157] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 97. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 97.

[0158] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 99. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 99.

[0159] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 106. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 106.

[0160] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 114. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 114.

[0161] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 133. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 133.

[0162] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 141. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 141.

[0163] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 149. In some aspects, the antigen-binding arm comprises a VH containing the amino acid sequence shown in SEQ ID NO: 149.

[0164] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 8.

[0165] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 16.

[0166] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 24.

[0167] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 107. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 107.

[0168] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 115. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 115.

[0169] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 134. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 134.

[0170] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 142. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 142.

[0171] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 150. In some aspects, the antigen-binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 150.

[0172] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 7, and the VL contains the amino acid sequence shown in SEQ ID NO: 8.

[0173] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 15, and the VL contains the amino acid sequence shown in SEQ ID NO: 16.

[0174] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 23, and the VL contains the amino acid sequence shown in SEQ ID NO: 24.

[0175] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 95, and the VL contains the amino acid sequence shown in SEQ ID NO: 8.

[0176] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 97, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 97, and the VL contains the amino acid sequence shown in SEQ ID NO: 16.

[0177] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 99, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 99, and the VL contains the amino acid sequence shown in SEQ ID NO: 24.

[0178] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 106, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 107. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 106, and the VL comprises the amino acid sequence shown in SEQ ID NO: 107.

[0179] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 114, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 115. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 114, and the VL comprises the amino acid sequence shown in SEQ ID NO: 115.

[0180] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 133, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 134. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 133, and the VL comprises the amino acid sequence shown in SEQ ID NO: 134.

[0181] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 141, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 142. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 141, and the VL contains the amino acid sequence shown in SEQ ID NO: 142.

[0182] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 149, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 150. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 149, and the VL contains the amino acid sequence shown in SEQ ID NO: 150.

[0183] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 8, 16, 24, 107, 115, 134, 142, and 150. In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising VH and VL, wherein the VH comprises an amino acid sequence selected from SEQ ID No: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149, and the VL comprises an amino acid sequence selected from SEQ ID NO: 8, 16, 24, 107, 115, 134, 142, and 150.

[0184] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a light chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 59, 60, and 61. In some aspects, the antigen-binding arm includes a light chain constant domain comprising an amino acid sequence selected from SEQ ID Nos: 59, 60, and 61.

[0185] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a light chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 62, 63, and 64. In some aspects, the antigen-binding arm includes a light chain constant domain comprising an amino acid sequence selected from SEQ ID Nos: 62, 63, and 64.

[0186] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 8, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0187] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 8, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 8, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0188] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 16, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0189] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 16, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 16, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0190] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 24, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0191] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 24, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 24, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0192] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 107, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 107, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0193] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 107, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 107, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0194] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 115, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 115, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0195] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 115, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 115, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0196] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 115, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 115, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0197] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 115, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 115, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0198] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 115, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 115, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0199] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 134, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 134, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0200] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 142, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 142, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0201] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 150, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the antigen-binding arm comprises a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 150, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0202] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 8, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0203] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 16, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0204] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 24, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0205] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 107, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID NO: 107, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0206] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 115, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 115, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0207] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a volume linker (VL) and a light chain constant domain. The VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 134. The light chain constant domain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a volume linker (VL) and a light chain constant domain. The VL comprises the amino acid sequence shown in SEQ ID No: 134, and the light chain constant domain comprises an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0208] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 142, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 142, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0209] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a volume linker (VL) and a light chain constant domain. The VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 150. The light chain constant domain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a volume linker (VL) and a light chain constant domain. The VL comprises the amino acid sequence shown in SEQ ID No: 150. The light chain constant domain comprises an amino acid sequence selected from SEQ ID No: 60, 61, 63, and 64.

[0210] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a heavy chain constant region containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 65-80. In some aspects, the antigen-binding arm includes a heavy chain constant region containing an amino acid sequence selected from SEQ ID No: 65-80.

[0211] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0212] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0213] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 23, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0214] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 95, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0215] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 97, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 97, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0216] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 99, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 99, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0217] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 106, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 106, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0218] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 114, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 114, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0219] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 133, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 133, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0220] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 141, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 141, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0221] In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 149, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID NO: 65-80. In some aspects, the T-cell adaptor molecule comprises an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 149, and the heavy chain constant region comprises an amino acid sequence selected from SEQ ID NO: 65-80.

[0222] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0223] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0224] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0225] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0226] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 97, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0227] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 99, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0228] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 106, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0229] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 114, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0230] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 133, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0231] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 141, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0232] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 149, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0233] In some respects, T-cell adaptor molecules include a heavy chain containing a STEAP2 antigen-binding region fused to the CD8 binding region. In other respects, the antigen-binding arm also includes a light chain containing the STEAP2 binding region.

[0234] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 25. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having the amino acid sequence shown in SEQ ID NO: 25.

[0235] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 116. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having the amino acid sequence shown in SEQ ID NO: 116.

[0236] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 117. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having the amino acid sequence shown in SEQ ID NO: 117.

[0237] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 118. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having the amino acid sequence shown in SEQ ID NO: 118.

[0238] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 119. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a light chain having the amino acid sequence shown in SEQ ID NO: 119.

[0239] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 26. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 26.

[0240] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 120. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 120.

[0241] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 121. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 121.

[0242] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 122. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 122.

[0243] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 123. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 123.

[0244] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 124. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 124.

[0245] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 125. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 125.

[0246] In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 126. In some aspects, the T-cell adaptor molecule includes a STEAP2 binding arm comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 126.

[0247] In some aspects, the T-cell adaptor molecule comprises a STEAP2 binding arm comprising a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 26, and the light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 25. In some aspects, the T-cell adaptor molecule comprises a STEAP2 binding arm comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 26, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 25.

[0248] III. T Cell-binding arm The T-cell adaptor molecule described herein comprises at least one T-cell binding arm. In some aspects, the T-cell adaptor molecule described herein comprises two T-cell binding arms. In some aspects, the T-cell adaptor molecule comprises at least one T-cell binding arm for binding CD3 antigen. In some aspects, the T-cell adaptor molecule comprises at least one T-cell binding arm for binding CD8 antigen. In some aspects, the T-cell adaptor molecule comprises at least one T-cell binding arm for binding CD3 antigen and at least one T-cell binding arm for binding CD8 antigen. In some aspects, the T-cell binding arm comprises a heavy chain variable domain and a light chain variable domain. In some aspects, the T-cell binding arm comprises only a heavy chain variable domain.

[0249] In some respects, the T-cell binding arm of the T-cell adaptor molecule described herein can be of any type, including Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some respects, the antigen-binding arm comprises Fab. In some respects, the antigen-binding arm comprises VHH.

[0250] In some respects, the T-cell binding arm comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein the VH contains VH-CDR1, VH-CDR2, and VH-CDR3; and wherein the VL contains VL-CDR1, VL-CDR2, and VL-CDR3.

[0251] In some respects, the T-cell binding arm of a T-cell adaptor molecule can bind CD3. CD3 (Cluster 3 of Differentiation) is a protein complex composed of four subunits (CD3γ chain, CD3δ chain, and two CD3ε chains). CD3 associates with the T-cell receptor and the ζ chain to generate activation signals in T lymphocytes. T-cell adaptor molecules targeting CD3 and target cell antigens (or multiple antigens) (e.g., STEAP2) can force a transient interaction between target cells (or multiple cell types) (e.g., cancer cells expressing STEAP2) and T cells, thereby inducing cross-linking, T-cell activation, and subsequent antigen-dependent T-cell killing of the target cell. In some respects, the T-cell adaptor molecule binds monovalently to the CD3 protein, and the T-cell receptor cross-links and activates only upon binding to the target cell.

[0252] In some aspects, the T-cell binding arm comprises VH-CDR1 containing an amino acid sequence selected from SEQ ID No: 36, 40, and 44. In some aspects, the T-cell binding arm comprises VH-CDR2 containing an amino acid sequence selected from SEQ ID No: 37, 41, and 45. In some aspects, the T-cell binding arm comprises VH-CDR3 containing an amino acid sequence selected from SEQ ID No: 38, 42, and 46.

[0253] In some aspects, the T-cell binding arm comprises VL-CDR1 containing an amino acid sequence selected from SEQ ID No: 27 and 31. In some aspects, the T-cell binding arm comprises VL-CDR2 containing an amino acid sequence selected from SEQ ID No: 28 and 32. In some aspects, the T-cell binding arm comprises VL-CDR3 containing an amino acid sequence selected from SEQ ID No: 29 and 33.

[0254] In some respects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 36, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 37, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 38, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 33.

[0255] In some respects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 36, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 37, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 38, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 27, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 29.

[0256] In some respects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 40, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 41, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 42, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 33.

[0257] In some respects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 40, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 41, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 42, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 27, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 29.

[0258] In some respects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 44, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 46, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 33.

[0259] In some respects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 44, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 45, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 46, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 27, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 28, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 29.

[0260] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39. In some aspects, the T-cell binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 39.

[0261] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 43. In some aspects, the T-cell binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 43.

[0262] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 47. In some aspects, the T-cell binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 47.

[0263] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34. In some aspects, the T-cell binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 34.

[0264] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30. In some aspects, the T-cell binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 30.

[0265] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34.

[0266] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30.

[0267] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34.

[0268] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising VH and VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30.

[0269] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34.

[0270] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30.

[0271] In some respects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 in the VH region having the amino acid sequence shown in SEQ ID NO: 39; and VL-CDR1, VL-CDR2, and VL-CDR3 in the VL region having the amino acid sequence shown in SEQ ID NO: 34.

[0272] In some respects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 in the VH region having the amino acid sequence shown in SEQ ID NO: 39; and VL-CDR1, VL-CDR2, and VL-CDR3 in the VL region having the amino acid sequence shown in SEQ ID NO: 30.

[0273] In some respects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 in the VH region having the amino acid sequence shown in SEQ ID NO: 43; and VL-CDR1, VL-CDR2, and VL-CDR3 in the VL region having the amino acid sequence shown in SEQ ID NO: 34.

[0274] In some respects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 in the VH region having the amino acid sequence shown in SEQ ID NO: 43; and VL-CDR1, VL-CDR2, and VL-CDR3 in the VL region having the amino acid sequence shown in SEQ ID NO: 30.

[0275] In some respects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 in the VH region having the amino acid sequence shown in SEQ ID NO: 47; and VL-CDR1, VL-CDR2, and VL-CDR3 in the VL region having the amino acid sequence shown in SEQ ID NO: 34.

[0276] In some respects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 in the VH region having the amino acid sequence shown in SEQ ID NO: 47; and VL-CDR1, VL-CDR2, and VL-CDR3 in the VL region having the amino acid sequence shown in SEQ ID NO: 30.

[0277] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a light chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 59, 60, and 61. In some aspects, the T-cell binding arm includes a light chain constant domain comprising an amino acid sequence selected from SEQ ID Nos: 59, 60, and 61.

[0278] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a light chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 62, 63, and 64. In some aspects, the T-cell binding arm includes a light chain constant domain comprising an amino acid sequence selected from SEQ ID Nos: 62, 63, and 64.

[0279] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61.

[0280] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64.

[0281] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61.

[0282] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64.

[0283] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64.

[0284] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 60, 61, 63, and 64.

[0285] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a heavy chain constant region containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 65-80. In some aspects, the T-cell binding arm includes a heavy chain constant region comprising an amino acid sequence selected from SEQ ID No: 65-80.

[0286] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 65-80.

[0287] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 65-80.

[0288] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 65-80.

[0289] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, and 80.

[0290] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, and 80.

[0291] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a heavy chain constant region, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, and the heavy chain constant region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, and 80.

[0292] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a heavy chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81 or 82. In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a heavy chain having the amino acid sequence shown in SEQ ID NO: 81 or 82.

[0293] In some respects, one of the T-cell binding arms can bind CD8. CD8 (differentiation cluster 8) is a dimer composed of a pair of CD8 chains. The most common form of CD8 consists of CD8-α and CD8-β chains. CD8 acts as a co-receptor on MCHI I-restricted T cells and is used to enhance the antigen sensitivity of CD8+ T cells by binding to the essentially invariant region of MCHI at sites different from where the T-cell receptor binds.

[0294] In some implementations, the T-cell adaptor molecule includes a T-cell binding arm capable of binding CD3 and a T-cell binding arm capable of binding CD8.

[0295] In some aspects, the T-cell binding arm comprises VH-CDR1 containing an amino acid sequence selected from SEQ ID No: 48 and 84. In some aspects, the T-cell binding arm comprises VH-CDR2 containing an amino acid sequence selected from SEQ ID No: 49 and 85. In some aspects, the T-cell binding arm comprises VH-CDR3 containing an amino acid sequence selected from SEQ ID No: 50 and 86.

[0296] In some aspects, the T-cell binding arm comprises VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 51. In some aspects, the T-cell binding arm comprises VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 52. In some aspects, the T-cell binding arm comprises VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 53.

[0297] In some respects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 48, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 49, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 50, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 51, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 52, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 53.

[0298] In some aspects, the T-cell binding arm comprises VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 84, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 85, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 86. In some aspects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 present in the VHH region having the amino acid sequence shown in SEQ ID NO: 83.

[0299] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm containing a VH, the VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56. In some aspects, the T-cell binding arm includes a VH containing the amino acid sequence shown in SEQ ID NO: 56.

[0300] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VL containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 54. In some aspects, the T-cell binding arm comprises a VL containing the amino acid sequence shown in SEQ ID NO: 54.

[0301] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a VH and a VL, wherein the VH contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56, and the VL contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 54.

[0302] In some respects, the T-cell binding arm comprises VH-CDR1, VH-CDR2, and VH-CDR3 in the VH region having the amino acid sequence shown in SEQ ID NO: 56; and VL-CDR1, VL-CDR2, and VL-CDR3 in the VL region having the amino acid sequence shown in SEQ ID NO: 54.

[0303] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a light chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 62, 63, and 64. In some aspects, the T-cell binding arm includes a light chain constant domain comprising an amino acid sequence selected from SEQ ID Nos: 62, 63, and 64.

[0304] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a light chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 59, 60, and 61. In some aspects, the T-cell binding arm includes a light chain constant domain comprising an amino acid sequence selected from SEQ ID Nos: 59, 60, and 61.

[0305] In some aspects, the T-cell adaptor molecule comprises a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 54, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 62, 63, and 64. In some aspects, the T-cell adaptor molecule comprises a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 54, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 62, 63, and 64.

[0306] In some aspects, the T-cell adaptor molecule comprises a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID No: 54, and the light chain constant domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequences selected from SEQ ID No: 59, 60, and 61. In some aspects, the T-cell adaptor molecule comprises a T-cell binding arm comprising a VL and a light chain constant domain, the VL comprising the amino acid sequence shown in SEQ ID No: 54, and the light chain constant domain comprising an amino acid sequence selected from SEQ ID No: 59, 60, and 61.

[0307] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm comprising a heavy chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 57, 65, 66, and 67. In some aspects, the T-cell binding arm includes a heavy chain constant region comprising an amino acid sequence selected from SEQ ID Nos: 57, 65, 66, and 67.

[0308] In some aspects, the T-cell adaptor molecule includes a T-cell binding arm containing a VH, the VH containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 83. In some aspects, the T-cell binding arm includes a VH containing the amino acid sequence shown in SEQ ID NO: 83.

[0309] IV. charge pairs The terms “charge pair” and “charge mutation” are used interchangeably herein and refer to amino acids with opposite charges, such as a positively charged amino acid residue and a negatively charged amino acid residue, one located in the light chain region (e.g., the constant light chain domain) of the antigen and / or T cell binding arm and the other in the heavy chain region (e.g., the constant heavy chain region 1 (CH1)), located at positions designed to promote association between the light and heavy chains. The term “λ charge pair” refers to a charge pair in which the positively or negatively charged amino acid residues are located in the λ light chain (e.g., CLλ). The term “κ charge pair” refers to a charge pair in which the positively or negatively charged amino acid residues in the light chain are located in the κ light chain (e.g., CLκ).

[0310] Without being bound by theory, it is believed that amino acid residues with opposite charges in charge pairs increase the attraction of the heavy chain to the light chain in immunoglobulin and / or T cell binding arms, thereby promoting the formation of immunoglobulin and / or T cell binding arms with the correct heavy and light chains.

[0311] At least one amino acid residue in the charge pair may be engineered into the immunoglobulin and / or T cell binding arm (i.e., at least one amino acid residue in the pair is not a wild-type amino acid residue). In some aspects, both amino acid residues in the charge pair are engineered into the immunoglobulin and / or T cell binding arm (i.e., neither amino acid residue in the pair is a wild-type amino acid residue).

[0312] In some embodiments, the positively charged amino acid residue in the charge pair is located on the light chain, and the negatively charged amino acid residue in the charge pair is located on the corresponding heavy chain. In other embodiments, the negatively charged amino acid residue is located on the light chain, and the positively charged amino acid residue in the charge pair is located on the corresponding heavy chain.

[0313] The amino acid residues in the charge pairs are typically naturally occurring. Naturally occurring positively charged amino acid residues according to this disclosure include arginine, lysine, and histidine. Naturally occurring negatively charged amino acid residues according to this disclosure include glutamic acid, serine, threonine, and aspartic acid. Although serine and threonine are often described in the art as “uncharged,” they have isoelectric points below 6 and are therefore partially negatively charged at neutral pH. For the purposes of the charge pairs disclosed herein, serine and threonine are examples of negatively charged amino acid residues (along with glutamic acid and aspartic acid).

[0314] In some respects, a charge pair comprises a positively charged amino acid residue selected from arginine, lysine, or histidine at one position in the charge pair and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine at the other position in the charge pair. For example, a charge pair may comprise any one of the following amino acid residue pairs: Arginine and aspartic acid; Arginine and glutamic acid; Arginine and serine; Arginine and threonine; Lysine and aspartic acid; Lysine and glutamic acid; Lysine and serine; Lysine and threonine; Histidine and aspartic acid; Histidine and glutamic acid; Histidine and serine; and Histidine and threonine.

[0315] λ charge pairs As illustrated in this article, λ charge pairs can be introduced at several locations to improve the correct pairing of light and heavy chains in the antigen and / or T cell binding arm.

[0316] In some respects, λ charge pairs comprise positively or negatively charged amino acid residues at positions 117, 119, 134, 136, or 178 of the constant light chain λ region (CLλ). In other respects, λ charge pairs comprise positively or negatively charged amino acid residues at positions 141, 185, 128, 145, 183, 185, 173, or 187 of the CH1 domain. Elsewhere, the numbering is based on EU numbering.

[0317] In some respects, the λ charge pair is located at one or more of the following position pairs: (iv) Position 117 in CLλ and position 141 in CH1; (ii) Position 117 in CLλ and position 185 in CH1; (iii) Position 119 in CLλ and position 128 in CH1; (iv) Position 134 in CLλ and position 128 in CH1; (v) Position 134 in CLλ and position 145 in CH1; (vi) Position 134 in CLλ and position 183 in CH1; (vii) Position 136 in CLλ and position 185 in CH1; (viii) Position 178 in CLλ and position 173 in CH1; and (ix) Position 117 in CLλ and position 187 in CH1.

[0318] In some respects, the λ charge pairs are located at position 117 in CLλ and position 141 in CH1. For example, the λ charge pairs can be selected from the following list: Arginine at position 117 of CLλ and aspartic acid at position 141 of CH1; Arginine at position 117 of CLλ and glutamic acid at position 141 of CH1; Arginine at position 117 of CLλ and serine at position 141 of CH1; Arginine at position 117 of CLλ and threonine at position 141 of CH1; Lysine at position 117 of CLλ and aspartic acid at position 141 of CH1; Lysine at position 117 of CLλ and glutamic acid at position 141 of CH1; The lysine at position 117 of CLλ and the serine at position 141 of CH1; and Lysine at position 117 of CLλ and threonine at position 141 of CH1.

[0319] In some aspects, the λ charge pair is selected from any of a. to f. in the list above. In some aspects, the λ charge pair is selected from any of a. to e. in the list above. In some aspects, the λ charge pair is selected from any of a., b., and e. in the list above. In some aspects, the λ charge pair is a.

[0320] In some respects, the λ charge pairs are located at position 117 in CLλ and position 185 in CH1. For example, the λ charge pairs can be selected from the following list: Arginine at position 117 of CLλ and aspartic acid at position 185 of CH1; Arginine at position 117 of CLλ and glutamic acid at position 185 of CH1; Arginine at position 117 of CLλ and serine at position 185 of CH1; Arginine at position 117 of CLλ and threonine at position 185 of CH1; Lysine at position 117 of CLλ and aspartic acid at position 185 of CH1; Lysine at position 117 of CLλ and glutamic acid at position 185 of CH1; The lysine at position 117 of CLλ and the serine at position 185 of CH1; and Lysine at position 117 of CLλ and threonine at position 185 of CH1.

[0321] In some respects, the λ charge pairs are located at position 119 in the CLλ domain and position 128 in the CH1 structural domain. For example, the λ charge pairs can be selected from the following list: Arginine at position 119 of CLλ and aspartic acid at position 128 of CH1; Arginine at position 119 of CLλ and glutamic acid at position 128 of CH1; Arginine at position 119 of CLλ and serine at position 128 of CH1; Arginine at position 119 of CLλ and threonine at position 128 of CH1; Lysine at position 119 of CLλ and aspartic acid at position 128 of CH1; Lysine at position 119 of CLλ and glutamic acid at position 128 of CH1; The lysine at position 119 of CLλ and the serine at position 128 of CH1; and Lysine at position 119 of CLλ and threonine at position 128 of CH1.

[0322] In some respects, the λ charge pairs are located at position 134 in the CLλ domain and position 128 in the CH1 structural domain. For example, the λ charge pairs can be selected from the following list: Arginine at position 134 of CLλ and aspartic acid at position 128 of CH1; Arginine at position 134 of CLλ and glutamic acid at position 128 of CH1; Arginine at position 134 of CLλ and serine at position 128 of CH1; Arginine at position 134 of CLλ and threonine at position 128 of CH1; Lysine at position 134 of CLλ and aspartic acid at position 128 of CH1; Lysine at position 134 of CLλ and glutamic acid at position 128 of CH1; The lysine at position 134 of CLλ and the serine at position 128 of CH1; and Lysine at position 134 of CLλ and threonine at position 128 of CH1.

[0323] In some respects, the λ charge pairs are located at position 134 in the CLλ domain and position 145 in the CH1 structural domain. For example, the λ charge pairs can be selected from the following list: Arginine at position 134 of CLλ and aspartic acid at position 145 of CH1; Arginine at position 134 of CLλ and glutamic acid at position 145 of CH1; Arginine at position 134 of CLλ and serine at position 145 of CH1; Arginine at position 134 of CLλ and threonine at position 145 of CH1; Lysine at position 134 of CLλ and aspartic acid at position 145 of CH1; Lysine at position 134 of CLλ and glutamic acid at position 145 of CH1; The lysine at position 134 of CLλ and the serine at position 145 of CH1; and Lysine at position 134 of CLλ and threonine at position 145 of CH1.

[0324] In some respects, the λ charge pairs are located at position 134 in the CLλ domain and position 183 in the CH1 structural domain. For example, the λ charge pairs can be selected from the following list: Arginine at position 134 of CLλ and aspartic acid at position 183 of CH1; Arginine at position 134 of CLλ and glutamic acid at position 183 of CH1; Arginine at position 134 of CLλ and serine at position 183 of CH1; Arginine at position 134 of CLλ and threonine at position 183 of CH1; Lysine at position 134 of CLλ and aspartic acid at position 183 of CH1; Lysine at position 134 of CLλ and glutamic acid at position 183 of CH1; The lysine at position 134 of CLλ and the serine at position 183 of CH1; and Lysine at position 134 of CLλ and threonine at position 183 of CH1.

[0325] In some respects, the λ charge pair is lysine at position 134 of the CLλ domain and either aspartic acid or serine at position 183 of the CH1 domain.

[0326] In some respects, the λ charge pairs are located at position 136 in the CLλ domain and position 185 in the CH1 structural domain. For example, the λ charge pairs can be selected from the following list: Arginine at position 136 of CLλ and aspartic acid at position 185 of CH1; Arginine at position 136 of CLλ and glutamic acid at position 185 of CH1; Arginine at position 136 of CLλ and serine at position 185 of CH1; Arginine at position 136 of CLλ and threonine at position 185 of CH1; Lysine at position 136 of CLλ and aspartic acid at position 185 of CH1; Lysine at position 136 of CLλ and glutamic acid at position 185 of CH1; The lysine at position 136 of CLλ and the serine at position 185 of CH1; and Lysine at position 136 of CLλ and threonine at position 185 of CH1.

[0327] In some respects, the λ charge pairs are located at position 178 in the CLλ domain and position 173 in the CH1 structural domain. For example, the λ charge pairs can be selected from the following list: Arginine at position 178 of CLλ and aspartic acid at position 173 of CH1; Arginine at position 178 of CLλ and glutamic acid at position 173 of CH1; Arginine at position 178 of CLλ and serine at position 173 of CH1; Arginine at position 178 of CLλ and threonine at position 173 of CH1; Lysine at position 178 of CLλ and aspartic acid at position 173 of CH1; Lysine at position 178 of CLλ and glutamic acid at position 173 of CH1; The lysine at position 178 of CLλ and the serine at position 173 of CH1; and Lysine at position 178 of CLλ and threonine at position 173 of CH1.

[0328] In some respects, the antigen and / or T-cell binding arm containing a λ charge pair contains more than one λ charge pair. For example, the first antigen and / or T-cell binding arm may contain two, three, four, five, six, seven, eight, or nine λ charge pairs at the positions (i) to (ix) above.

[0329] In some respects, the first antigen and / or T cell binding arm contains a κ charge pair, and the second antigen binding arm and / or the second T cell binding arm both contain a λ charge pair, wherein the charged amino acid residues located on the second antigen binding CH1 domain and CLλ have opposite charges to the charged amino acid residues located on the second T cell binding CH1 domain and CLλ.

[0330] In some exemplary aspects, the positively charged amino acid residues in the λ charge pair are located on the light chain, and the negatively charged amino acid residues in the λ charge pair are located on the heavy chain. In some aspects, the negatively charged amino acid residues are located on the light chain, and the positively charged amino acid residues in the λ charge pair are located on the heavy chain.

[0331] κ charge pairs In the T-cell adaptor molecules described herein, at least one antigen and / or T-cell binding arm contains a κ charge pair. As described above, a κ charge pair refers to a positively charged amino acid residue and a negatively charged amino acid residue, one of which is located in the κ light chain (e.g., CLκ) of the antigen and / or T-cell binding arm and the other in the heavy chain (e.g., CH1) of the antigen and / or T-cell binding arm, located at sites designed to facilitate association between the light chain of a second antigen and / or T-cell binding arm and CH1.

[0332] In some aspects, the antigen and / or T-cell binding arm containing CLκ includes a κ charge pair located at position 133 in CLκ and position 183 in the second CH1. In some aspects, the negatively charged amino acid residue in the κ charge pair is at position 133 in CLκ, and the positively charged amino acid residue in the κ charge pair is at position 183 in the second CH1. In some aspects, the positively charged amino acid residue in the κ charge pair is at position 133 in CLκ, and the negatively charged amino acid residue in the κ charge pair is at position 183 in the second CH1. In some aspects, the negatively charged amino acid residue (e.g., at position 133 in CLκ) is glutamic acid, and the positively charged amino acid residue (e.g., at position 183 in the second CH1) is lysine. As submitted, the numbering is based on EU numbering.

[0333] In some respects, both the second antigen-binding arm and the second T-cell binding arm contain κ charge pairs. In some respects, the first antigen-binding arm contains κ charge pairs, and the second antigen-binding arm and the second T-cell binding arm contain λ charge pairs.

[0334] In some aspects, in the second antigen-binding arm, the positively charged amino acid residues of the κ charge pair are located on the second CH1, and the negatively charged amino acid residues are located on the second light chain; and in the second T cell-binding arm, the positively charged amino acid residues of the κ charge pair are located on the third light chain, and the negatively charged amino acid residues are located on the third CH1.

[0335] In some aspects, the first antigen-binding arm includes a λ charge pair containing a positively charged amino acid residue and a negatively charged amino acid residue at the interface between the first CH1 and CLλ; the second antigen-binding arm includes a κ charge pair containing a positively charged amino acid residue and a negatively charged amino acid residue at the interface between the second CH1 and CLκ of the second light chain; and the second T cell-binding arm includes a κ charge pair containing a positively charged amino acid residue and a negatively charged amino acid residue at the interface between the third CH1 and CLκ of the third light chain, wherein the charged amino acid residues on the third CH1 and CLκ of the third light chain have opposite charges to the charged amino acid residues on the second CH1 and CLκ of the second light chain.

[0336] In other aspects, the first antigen-binding arm contains a κ charge pair, and the second antigen-binding arm and the second T-cell binding arm contain λ charge pairs with opposite charges. In some aspects, the first antigen-binding arm contains a κ charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue at the interface between the first CH1 and CLκ; the second antigen-binding arm contains a λ charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue at the interface between the third CH1 and CLλ of the third light chain; and the second T-cell binding arm contains a λ charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue at the interface between the third CH1 and CLλ of the third light chain, wherein the charged amino acid residues at the third CH1 and CLλ of the third light chain have opposite charges to the charged amino acid residues at the second CH1 and CLλ of the second light chain.

[0337] For example, the charge pair in the second antigen-binding arm can be formed by positively charged amino acid residues in the second CH1 and negatively charged amino acid residues in the second light chain, and the κ charge pair in the second T cell-binding arm can be formed by negatively charged amino acid residues in the third CH1 and positively charged amino acid residues in the third light chain.

[0338] Alternatively, the charge pair in the second antigen-binding arm may be formed by negatively charged amino acid residues in the second CH1 and positively charged amino acid residues in the second light chain, and the κ charge pair in the second T cell-binding arm may be formed by positively charged amino acid residues in the third CH1 and negatively charged amino acid residues in the third light chain.

[0339] As described in the examples, several methods are known to be used to determine the correct light chain pairings. These include mass spectrometry-based methods that can be used to establish the correct heavy chain / light chain associations. In some aspects, when T cell adaptor molecules contain a mixture of κ and λ light chains, the ratio of κ to λ light chains in the assembled T cell adaptor molecules can be determined using microfluidic-based electrophoresis as a reading of the correct light chain ratio.

[0340] Therefore, in some respects, T cell adaptor molecules containing λ charge pairs exhibit improved correct light chain pairing compared to equivalent T cell adaptor molecules lacking λ charge pairs. In some respects, optionally after purification of the T cell adaptor molecules using light chain affinity purification, trivalent antibodies containing λ charge pairs exhibit correct light chain ratios greater than 90%, 95%, 96%, 97%, 98%, or 99% (e.g., as determined using microfluidic-based electrophoresis methods).

[0341] As described herein, techniques such as light chain affinity chromatography using affinity resins specific for CLκ or CLλ can be used to selectively purify T-cell adaptor molecules based on their light chains. Examples of such affinity resins include LambdaFabSelect and KappaSelect resins from GE Healthcare. Such methods can be used to selectively purify T-cell adaptor molecules containing both CLκ and CLλ, and therefore can be used to improve the production of T-cell adaptor molecules.

[0342] V. Engineered disulfide bonds In some respects, T-cell adaptor molecules contain engineered disulfide bonds in addition to charge pairs. An "engineered disulfide bond" means that the native interchain disulfide bond at the CH1-CL interface (e.g., at 220 of CH1 and 212 of LC) of at least one of the first antigen-binding arms, the second antigen-binding arm, the first T-cell binding arm, or the second T-cell binding arm has been replaced by an engineered (non-natural) interchain disulfide bond, while one or more other binding arms contain a native interchain disulfide bond at the CH1-CL interface. Engineered disulfide bonds are typically formed by engineering cysteine ​​residues into the CL domain of the light chain and the corresponding CH1 domain of the heavy chain, replacing the cysteine ​​residues that would normally form interchain disulfide bonds. Publications relating to the introduction of engineered disulfide bonds into antibodies to promote heterodimerization can be found, for example, in U.S. Patent Nos. 9,527,927 and Mazor, 2015, which are incorporated herein by reference in their entirety.

[0343] The formation of disulfide bonds between cysteine ​​residues occurs during the folding of many proteins entering the secretory pathway. When a polypeptide chain folds, adjacent cysteine ​​residues can form a covalent bond during a process catalyzed by members of the protein disulfide isomerase family. As used herein, the term "disulfide bond linkage" or "disulfide-linked" refers to a single covalent bond formed by the coupling of thiol groups, particularly cysteine ​​residues. In some respects, the covalent bond between two cysteine ​​residues is between the two sulfur atoms of each residue. However, depending on the environment, not all protein species may always have disulfide bonds present, for example, in the case of disulfide bond reduction. Therefore, in some respects, the term "disulfide bond linkage" or "disulfide-linked" (whether natural or engineered) also refers to the presence of two cysteine ​​residues capable of forming a disulfide bond linkage, regardless of whether they are actually linked at that single point in time.

[0344] In some respects, the disulfide bond connection between the light chain and CH1 in at least one of the antigen and / or T-cell binding arms can be formed between the light chain engineered to the antigen and / or T-cell binding arm and the cysteine ​​pair in the CH1 domain. In other respects, the disulfide bond connection between the light chain and CH1 in both binding arms (e.g., a first antigen binding arm and a second antigen binding arm, a first T-cell binding arm and a second T-cell binding arm, a first antigen binding arm and a first T-cell binding arm, or a second antigen binding arm and a second T-cell binding arm) can be formed between the light chain engineered to those two binding arms and the cysteine ​​pair in CH1.

[0345] In some aspects, the disulfide bond connection between the first light chain and CH1 can be formed between a cysteine ​​pair engineered into the first light chain and the first CH1. In some aspects, the disulfide bond connection between the third light chain and the third CH1 can be formed between a cysteine ​​pair engineered into the third light chain and the third CH1. As described above, the light chain may contain CLλ or CLκ. In some aspects, the cysteine ​​pair engineered into CLλ and CH1 is located at position 122 of CLλ and position 126 of CH1, wherein the same CLλ contains a non-cysteine ​​residue at position 212, and the same CH1 contains a non-cysteine ​​residue at position 220. In some aspects, the non-cysteine ​​residue is valine.

[0346] In some aspects, cysteine ​​pairs engineered into the constant light chain κ region (CLκ) and CH1 are located at position 121 of CLκ and position 126 of CH1, wherein the same CLκ contains a non-cysteine ​​residue at position 214, and the same CH1 contains a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0347] In some respects, the disulfide bond connection between the first light chain and the first CH1 can be formed between cysteine ​​pairs engineered into the first light chain and the first CH1, the disulfide bond connection formed between the second light chain and the second CH1 can be formed between native cysteine ​​pairs, and the disulfide bond connection formed between the third light chain and the third CH1 can be formed between native cysteine ​​pairs; or can be formed between cysteine ​​pairs engineered into the third light chain polypeptide and the third heavy chain polypeptide, wherein the cysteine ​​pairs inserted into the third light chain and the heavy chain polypeptide are located at different amino acid residue positions than the cysteine ​​pairs inserted into the first light chain and the heavy chain polypeptide.

[0348] In some respects, the disulfide bond connection between the first light chain and the first CH1 can be formed between a cysteine ​​pair engineered to position 122 of CLλ and position 126 of the first CH1, wherein CLλ contains a non-cysteine ​​residue at position 212 and the first CH1 contains a non-cysteine ​​residue at position 220; the disulfide bond connection formed between the second light chain and the second CH1 is formed between native cysteine ​​pairs; and the disulfide bond connection that can be formed between the third light chain and the third CH1 can be formed between a cysteine ​​pair engineered to position 121 of CLκ and position 126 of the first CH1, wherein CLκ contains a non-cysteine ​​residue at position 214 and the first CH1 contains a non-cysteine ​​residue at position 220.

[0349] In some respects, T-cell adaptor molecules comprise a first antigen-binding arm having a λ charge pair and engineered disulfide bond as described above, a second antigen-binding arm having a κ charge pair and native disulfide bond as described above, and a first T-cell binding arm having a κ charge pair and engineered disulfide bond as described above.

[0350] In some aspects, T-cell adaptor molecules contain other combinations of charge pairs and engineered disulfide bonds. In some aspects, the first antigen-binding arm contains a λ charge pair and a native disulfide bond, and the first T-cell binding arm and the second T-cell binding arm contain a κ charge pair and an engineered disulfide bond. In some aspects, the first T-cell binding arm contains a κ charge pair and an engineered disulfide bond, and the first and second antigen-binding arms contain a λ charge pair and a native disulfide bond. In some aspects, the second antigen-binding arm contains a κ charge pair and a native disulfide bond, and the first and second T-cell binding arms contain a λ charge pair and an engineered disulfide bond.

[0351] VI. Fc Area Modification In some respects, the first antigen-binding arm and the second antigen-binding arm also include a first Fc region and a second Fc region (i.e., also include the CH2 and CH3 regions of the heavy chain).

[0352] In some aspects, T-cell adaptor molecules contain one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote the formation of multivalent T-cell adaptor molecules by facilitating the formation of the first Fc region and the second Fc region. In some aspects, T-cell adaptor molecules contain a mortar-and-stick (KiH) Fc modification based on the substitution of a single amino acid in the CH3 domain, which promotes heavy chain heterodimerization, as described in Ridgway, 1996. The mortar-and-stick variant has a small amino acid that has been replaced by a larger amino acid, thereby creating a protrusion (mortar) on the surface of the CH3 domain, and the mortar-and-stick variant has a large amino acid that has been replaced by a smaller amino acid, thereby creating a cavity (mortar) on the surface of the CH3 domain. Further modifications may also be introduced to stabilize association between heavy chains.

[0353] In some respects, CH3 modifications that enhance heterodimerization include, for example, the "mortar" mutation Y407V / T366S / L368A on one Fc region and the "pounder" mutation T366W on another Fc region. In some respects, T cell adaptor molecules may also include a stable cystine mutation Y349C (e.g., on an Fc region with a "mortar" mutation) and a stable S354C mutation on another Fc region (e.g., on an Fc region with a "pounder" mutation).

[0354] In some respects, the substitution that produces the mordant is the substitution of tryptophan at position 366, and the substitution that produces the mordant is one or more of the following substitutions: iv) Substitution of valine at position 407; ii) Substitution of serine at position 366; and iii) Substitution of alanine at position 368.

[0355] In some respects, T-cell adaptor molecules comprise a "groove" on a first antigen-binding arm containing a λ charge pair and a mortar on a second antigen-binding arm containing one of a κ charge pair. However, the reverse arrangement is also specifically envisioned, wherein the "mortar" is located at CH3 of the first antigen and / or T-cell binding arm, and the "groove" is located at CH3 of the second antigen and / or T-cell binding arm.

[0356] Other examples of CH3 modifications that enhance heterodimerization are described, for example, in Table 1 of Brinkmann and Kontermann, 2017 MABS 9(2), 182-212, which is incorporated herein by reference.

[0357] For example, an Fc region can contain modifications to allow fractionation elution by protein A chromatography, as described in Tustian, 2016. In short, one of the Fc regions can contain modifications to remove binding to protein A (referred to as Fc*), thereby allowing selective purification of the heterodimer FcFc* multivalent product. Examples of suitable modifications for generating the Fc* region include replacing H435 with arginine and Y436 with phenylalanine.

[0358] Besides Fc modifications used to enhance heterodimerization, other Fc modifications that can be used are those that reduce or eliminate the binding of T cell adaptor molecules to one or more Fcγ receptors (such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII) and / or to complement. Such mutations reduce or eliminate Fc effector function. Mutations used to reduce or eliminate binding to one or more Fcγ receptors and complement are known, including, for example, the “triple mutation” or “TM” of L234F / L235E / P331S described in Organesyan, 2008.

[0359] In some aspects, the first antigen-binding arm comprises a λ charge pair and a first Fc region, wherein the disulfide bond link between the first light chain and the first CH1 can be formed between cysteine ​​pairs engineered into the first light chain and the first CH1, and the first Fc region contains a "grossroots" mutation; the second antigen-binding arm comprises a κ charge pair and a second Fc region, wherein the disulfide bond link formed between the second light chain and the second CH1 is formed between native cysteine ​​pairs, and the second Fc region contains a "grossroots" mutation; and the T-cell binding arm comprises a κ charge pair, wherein the charged amino acid residues located on the third CH1 and CLκ of the third light chain have opposite charges to those charged amino acid residues located on the second CH1 and CLκ of the second light chain, and wherein the disulfide bond link formed between the third light chain and the third CH1 is formed between native cysteine ​​pairs; or can be formed between cysteine ​​pairs engineered into the third light chain polypeptide and the third heavy chain polypeptide, wherein the cysteine ​​pairs inserted into the third light chain and the heavy chain polypeptide are located at amino acid residue positions different from those inserted into the first light chain and the heavy chain polypeptide.

[0360] The examples provide non-restrictive examples of multivalent T cell adaptor molecules that include λ charge pairs, κ charge pairs, engineered disulfide bonds, and modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0361] In some respects, T-cell adaptor molecules contain amino acid modifications. In other respects, modifications involve replacing amino acid residues with any other naturally occurring or non-naturally occurring amino acid residue.

[0362] Naturally occurring residues can be categorized based on shared side-chain characteristics as follows: 1) Nonpolar, aliphatic: glycine (G), methionine (M), alanine (A), valine (V), leucine (L), isoleucine (I); 2) Polarity: Cysteine ​​I, Asparagine (N), Glutamine (Q), Proline (P); 3) Polar, partially negatively charged: serine (S), threonine (T); 4) Acidic (negatively charged): Aspartic acid (D), glutamic acid (acI) (E); 5) Basic (positively charged): Histidine (H), Lysine (K), Arginine I; 6) Aromatic compounds: tryptophan (W), tyrosine (Y), phenylalanine (F).

[0363] As described above, serine (S) and threonine (T) have isoelectric points below 6 and are partially negatively charged at neutral pH, so they are classified here as "polar, partially negatively charged".

[0364] Amino acid substitution can be a conserved amino acid substitution. A conserved amino acid substitution can involve the exchange of one member of these classes with another member of the same class. For example, a conserved amino acid substitution can be the substitution of the acidic amino acid glutamic acid (E) for the acidic amino acid aspartic acid (D).

[0365] In one aspect, the antibody or its antigen-binding fragment does not have one or more effector functions. For example, in one aspect, the antibody or its antigen-binding fragment does not have antibody-dependent cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In one aspect, the antibody or its antigen-binding fragment does not bind to an Fc receptor and / or complement factor. In one aspect, the antibody or its antigen-binding fragment does not have effector function. In one aspect, the antibody or its antigen-binding fragment contains an Fc region with a triple mutation (TM), which has reduced antibody-dependent cytotoxicity (ADCC) compared to an antibody with a wild-type Fc region. In one aspect, the antibody or its antigen-binding fragment has an Fc region containing a triple mutation (TM) of L234F / L235E / P331S. In one aspect, the antibody or its antigen-binding fragment does not have effector function or... In one aspect, the antibody or its antigen-binding fragment has an Fc region containing E233P / L234V / L235A / G236del / S267K. In one respect, E233P / L234V / L235A / G236del / S267K is referred to as "invalid" or "Fc invalid".

[0366] VII. T Cell-connecting molecules This disclosure provides T-cell adaptor molecules. T-cell adaptor molecules as described herein are capable of binding to two different epitopes on the same or different antigens in some respects. In some aspects, the T-cell adaptor molecule comprises at least one antigen-binding arm and at least one T-cell binding arm. According to this disclosure, an "antigen-binding arm" comprises a heavy chain containing a VH domain and a CH1 domain and a light chain containing a VL domain and a light chain constant domain, wherein the light chain constant domain is connected to the CH1 domain by a disulfide bond, and wherein the antigen-binding arm binds the antigen of interest. In some aspects, a "T-cell binding arm" comprises a heavy chain containing a VH domain and a CH1 domain and a light chain containing a VL domain and a light chain constant domain, wherein the light chain constant domain is connected to the CH1 domain by a disulfide bond, and wherein the T-cell binding arm binds a T-cell antigen.

[0367] In some respects, the antigen-binding arm and / or T-cell-binding arm in the T-cell adaptor molecule also includes an Fc region. In some respects, the antigen-binding arm and / or T-cell-binding arm in the T-cell adaptor molecule includes a complete heavy chain (i.e., VH domain, CH1 domain, hinge region, CH2 domain, and CH3 domain).

[0368] In some aspects, the T-cell adaptor molecule comprises: (i) a first antigen-binding arm comprising a heavy chain containing a VH and a heavy chain constant region containing CH1, CH2, and CH3 domains, and a light chain containing a VL and a light chain constant region; (ii) a T-cell binding arm comprising a heavy chain containing a VH and a heavy chain constant region containing CH1, CH2, and CH3 domains, and a light chain containing a VL and a light chain constant region; and an Fc region wherein the heavy chain of the antigen-binding arm and the heavy chain of the T-cell binding arm are attached to the Fc region. An exemplary T-cell adaptor molecule is shown in Figure 1A middle.

[0369] In some aspects, the T-cell adaptor molecule comprises: (i) a first antigen-binding arm comprising a heavy chain containing a VH domain and a heavy chain constant region containing CH1, CH2, and CH3 domains, and a light chain containing a VL domain and a light chain constant region; (ii) a T-cell adaptor arm comprising a heavy chain containing a VH domain and a heavy chain constant region containing CH1, CH2, and CH3 domains, and a light chain containing a VL domain and a light chain constant region; and (iii) a second antigen-binding arm comprising a heavy chain containing a VH and CH1 domain and a light chain containing a VL domain and a light chain constant region; wherein the heavy chain of the second antigen-binding arm is attached to the heavy chain of the T-cell adaptor arm via a peptide linker. An exemplary T-cell adaptor molecule is shown in Figures 1A to 1D middle.

[0370] In some aspects, the light chain of the antigen-binding arm is linked to the heavy chain disulfide bond of the antigen-binding arm in the T-cell adaptor molecule via a natural interchain disulfide bond (e.g., the disulfide bond present in IgG antibodies). In some aspects, the light chain of the antigen-binding arm is linked to the heavy chain disulfide bond of the antigen-binding arm in the T-cell adaptor molecule via an engineered disulfide bond. In some aspects, the light chain of the T-cell binding arm is linked to the heavy chain disulfide bond of the T-cell binding arm in the T-cell adaptor molecule via a natural interchain disulfide bond. In some aspects, the light chain of the T-cell binding arm is linked to the heavy chain disulfide bond of the T-cell binding arm in the T-cell adaptor molecule via an engineered disulfide bond.

[0371] In some aspects, the heavy chain of an antigen-binding arm is attached to the heavy chain of another antigen-binding arm via a peptide linker. In some aspects, the heavy chain of an antigen-binding arm is attached to a T-cell binding arm via a peptide linker. In some aspects, the peptide linker consists of 5 to 100 amino acids, 5 to 50 amino acids, 5 to 25 amino acids, or 5 to 15 amino acids. In some aspects, the peptide linker is primarily composed of glycine and serine amino acid residues, and in some aspects includes the amino acid sequence GGGGS or SGGGGS. In some aspects, the peptide linker contains or consists of SEQ ID NO: 89. In some aspects, the linker contains 1 to about 10 copies of SEQ ID NO: 89. In some aspects, the linker contains 2 copies of SEQ ID NO: 89.

[0372] In some aspects, the T-cell adaptor molecule comprises (a) at least one antigen-binding arm that binds STEAP2 and comprises a heavy chain containing a heavy chain variable domain (VH) and a heavy chain constant domain (CH1); and a light chain containing a light chain variable domain (VL) and a light chain constant domain (CL); (b) a T-cell binding arm comprising a heavy chain containing a VH domain and a CH1 domain; a light chain containing a VL domain and a light chain constant domain; and (c) an Fc domain comprising a first Fc region and a second Fc region, each Fc region containing a CH2 domain and a CH3 domain, said Fc region further comprising at least one modification to promote heterodimerization.

[0373] In some aspects, the T-cell adaptor molecule includes an antigen-binding arm that binds STEAP2 and includes a heavy chain comprising a VH domain and a CH1 domain, wherein the VH domain comprises VH-CDR1 selected from SEQ ID Nos: 1, 9, 17, 103, 111, 127, 135 and 143; VH-CDR2 selected from SEQ ID Nos: 2, 10, 18, 104 and 112; and VH-CDR3 selected from SEQ ID Nos: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137 and 145. In some respects, the antigen-binding arm of the T-cell adaptor molecule also includes a light chain comprising a VL domain and a light chain constant domain, wherein the VL domain comprises VL-CDR1 selected from SEQ ID No: 4, 12, 20, 100, 108, 130, 138 and 146; VL-CDR2 selected from SEQ ID No: 5, 13, 21, 101, 109, 131, 139 and 147; and VL-CDR3 selected from SEQ ID No: 6, 14, 22, 102, 110, 132, 140 and 148.

[0374] In some aspects, the T-cell adaptor molecule further comprises a T-cell binding arm that binds CD3 and includes a heavy chain comprising a VH domain and a CH1 domain, wherein the VH domain comprises VH-CDR1 selected from SEQ ID Nos: 36, 40, and 44; VH-CDR2 selected from SEQ ID Nos: 37, 41, and 45; and VH-CDR3 selected from SEQ ID Nos: 38, 42, and 46. In some aspects, the T-cell binding arm further comprises a light chain comprising a VL and a light chain constant domain, wherein the VL comprises VL-CDR1 selected from SEQ ID Nos: 27 and 31; VL-CDR2 selected from SEQ ID Nos: 28 and 32; and VL-CDR3 selected from SEQ ID Nos: 29 and 33. In some respects, the T-cell adaptor molecule also contains an Fc domain comprising a first Fc region and a second Fc region, each Fc region containing a CH2 domain and a CH3 domain; it also contains at least one modification to promote heterodimerization.

[0375] In some respects, the T-cell adaptor molecule also includes a second antigen-binding arm that binds STEAP2 and a heavy chain comprising a VH domain and a CH1 domain, wherein the VH domain comprises VH-CDR1 selected from SEQ ID Nos: 1, 9, 17, 103, 111, 127, 135 and 143; VH-CDR2 selected from SEQ ID Nos: 2, 10, 18, 104, 112, 128, 136 and 144; and VH-CDR3 selected from SEQ ID Nos: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137 and 145. In some respects, the second antigen-binding arm of the T-cell adaptor molecule also includes a light chain comprising a VL and a light chain constant domain, wherein the VL comprises VL-CDR1 selected from SEQ ID Nos: 4, 12, 20, 100, 108, 130, 138 and 146; VL-CDR2 selected from SEQ ID Nos: 5, 13, 21, 101, 109, 131, 139 and 147; and VL-CDR3 selected from SEQ ID Nos: 6, 14, 22, 102, 110, 132, 140 and 148.

[0376] In some respects, one of the CH3 domains in the Fc region of the T cell adaptor molecule contains a club-shaped mutation, and the other CH3 domain contains a mortar-shaped mutation.

[0377] In some respects, the CH1 domain and light chain constant domain of one or more of the antigen-binding arm and T-cell-binding arm of the T-cell adaptor molecule also contain charge-pair substitutions, which include a first charged amino acid substitution in the CH1 domain and a second charged amino acid substitution in the light chain constant domain, wherein the first charged amino acid substitution and the second charged amino acid substitution have opposite charges.

[0378] In some aspects, one or more of the light chain constant domains of each of the antigen-binding arm and the T-cell-binding arm are λ light chain constant domains (CLλ), and the charge pair is a λ charge pair containing a positively charged amino acid residue selected from arginine, lysine, or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine. In some aspects, the charged amino acid of the λ charge pair is located at one or more of the following positions: (i) Position 117 in CLλ and position 141 in the CH1 structural domain; (ii) Position 117 in CLλ and position 185 in the CH1 structural domain; (iii) Position 119 in CLλ and position 128 in the CH1 structural domain; (iv) Position 134 in CLλ and position 128 in the CH1 structural domain; (v) Position 134 in CLλ and position 145 in the CH1 structural domain; (vi) Position 134 in CLλ and position 183 in the CH1 structural domain; (vii) Position 136 in CLλ and position 185 in the CH1 structural domain; (viii) Position 178 in CLλ and position 173 in the CH1 structural domain; and / or (ix) Position 117 in CLλ and position 187 in the CH1 structural domain, where the numbering is based on the EU index.

[0379] In some respects, one or more of the light chain constant domains of each of the antigen-binding arm and the T-cell-binding arm are λ light chain constant domains (CLλ), and the charge pairs are λ charge pairs, wherein: (c) The charged amino acid at position 117 is arginine and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is arginine and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is arginine and the charged amino acid at position 141 is serine; the charged amino acid at position 117 is arginine and the charged amino acid at position 141 is threonine; the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is serine; or the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is threonine. (ii) The charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is threonine; (iii) The charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is threonine; (iv) The charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is threonine; (v) The charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is threonine. (vi) The charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is threonine. (vii) The charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is threonine; (viii) The charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is serine; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is threonine; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is serine; or the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is threonine; and / or (ix) The charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is threonine.

[0380] In some respects, one or more of the light chain constant domains in each antigen-binding arm and T-cell-binding arm of the T adaptor molecule are κ light chain constant domains (CLκ), and the charge pair is a κ charge pair containing a positively charged amino acid residue selected from arginine, lysine, or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine, wherein the κ charge pair is located at position 133 in CLκ and position 183 in CH1.

[0381] In some respects, one or more of the light chain constant domains in each antigen-binding arm and T-cell-binding arm of the T adaptor molecule are κ light chain constant domains (CLκ), and the charge pairs are κ charge pairs, wherein the charged amino acid at position 133 is glutamate and the charged amino acid at position 183 is lysine; or the charged amino acid at position 133 is lysine and the charged amino acid at position 183 is glutamate.

[0382] In some respects, the light chain constant domain of the T cell binding arm is CLλ, and the charge pair is a λ charge pair as described herein, and the light chain constant domain of the first antigen-binding domain and / or the second antigen-binding domain is a λ or κ charge pair, and the first antigen-binding arm contains a charged amino acid in CH1 with the same charge as the charged amino acid in the CH1 domain of the second antigen-binding arm.

[0383] In some respects, the light chain constant domain of the T cell binding arm is CLλ, and the charge pair is the λ charge pair as described herein, and the light chain constant domains of the first and second antigen binding arms are κ charge pairs, and the charged amino acids in the CH1 domain of the first antigen binding arm have the same charge as the charged amino acids in the CH1 domain of the second antigen binding arm.

[0384] In some respects, the CH1 domain of an antigen-binding arm or a T-cell-binding arm can be linked to a light chain constant domain via engineered disulfide bonds.

[0385] In some respects, the CH1 domain, which can be linked to the light chain constant domain via engineered disulfide bonds, comprises (i) substitution of native cysteine ​​to non-cysteine ​​amino acids and (ii) substitution of native non-cysteine ​​amino acids to cysteine; and the light chain constant domain comprises (i) substitution of native cysteine ​​to non-cysteine ​​amino acids and (ii) substitution of native non-cysteine ​​amino acids to cysteine; wherein the substituted cysteine ​​in the light chain constant domain and the substituted cysteine ​​in the CH1 domain can form disulfide bonds.

[0386] In some respects, the CH1 domain, which can be linked to the light chain constant domain via engineered disulfide bonds, comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 220, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 126; and the light chain constant domain comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 212, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 122, wherein the cysteine ​​at position 126 of the CH1 domain and the cysteine ​​at position 122 of the light chain constant domain can form a disulfide bond; wherein the numbering is based on the EU index.

[0387] In some respects, the CH1 domain, which can be linked to the light chain constant domain via engineered disulfide bonds, comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 220, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 126; and the light chain constant domain comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 214, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 121, wherein the cysteine ​​at position 126 of the CH1 domain and the cysteine ​​at position 121 of the light chain constant domain can form a disulfide bond; wherein the numbering is based on the EU index.

[0388] In some respects, the antigen-binding arm of the T-cell adaptor molecule includes a VH domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0389] In some respects, the antigen-binding arm of the T-cell adaptor molecule includes a heavy chain constant domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 90.

[0390] In some aspects, the antigen-binding arm of the T-cell adaptor molecule comprises a heavy chain containing a VH domain and a CH1 domain. The VH domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149. The CH1 domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence shown in SEQ ID NO: 90.

[0391] In some respects, the antigen-binding arm of the T-cell adaptor molecule includes a VL domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 8, 16, 24, 107, 115, 134, 142, and 150.

[0392] In some respects, the antigen-binding arm of the T-cell adaptor molecule includes a light chain constant domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 63.

[0393] In some aspects, the antigen-binding arm of the T-cell adaptor molecule comprises a light chain containing a VL domain and a light chain constant domain. The VL domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 8, 16, 24, 107, 115, 134, 142, and 150. The light chain constant domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence shown in SEQ ID NO: 63.

[0394] In some respects, the antigen-binding arm of the T-cell adaptor molecule includes a VH domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0395] In some respects, the antigen-binding arm of the T-cell adaptor molecule includes a heavy chain constant domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 88.

[0396] In some aspects, the antigen-binding arm of the T-cell adaptor molecule comprises a heavy chain containing a VH domain and a CH1 domain. The VH domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149. The CH1 domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence shown in SEQ ID NO: 88.

[0397] In some respects, the T-cell binding arm of the T-cell adaptor molecule includes a VH domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 39, 43, and 47.

[0398] In some respects, the T-cell binding arm of the T-cell adaptor molecule includes a heavy chain constant domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79.

[0399] In some aspects, the T-cell binding arm of the T-cell adaptor molecule comprises a heavy chain containing a VH domain and a heavy chain constant domain. The VH domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 39. The heavy chain constant domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence shown in SEQ ID NO: 79.

[0400] In some respects, the T-cell binding arm of the T-cell adaptor molecule includes a VL domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID No: 30 and 34.

[0401] In some respects, the T-cell binding arm of the T-cell adaptor molecule includes a light chain constant domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 61.

[0402] In some aspects, the T-cell binding arm of the T-cell adaptor molecule comprises a light chain containing a VL domain and a light chain constant domain. The VL domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 30 and 34. The light chain constant domain contains an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence shown in SEQ ID NO: 61.

[0403] In some aspects, the T-cell adaptor molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 25. In some aspects, the T-cell adaptor molecule comprises a light chain that comprises the amino acid sequence shown in SEQ ID NO: 25.

[0404] In some aspects, the T-cell adaptor molecule comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 116. In some aspects, the T-cell adaptor molecule comprises a light chain having the amino acid sequence shown in SEQ ID NO: 116.

[0405] In some aspects, the T-cell adaptor molecule comprises a light chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 117. In some aspects, the T-cell adaptor molecule comprises a light chain containing the amino acid sequence shown in SEQ ID NO: 117.

[0406] In some aspects, the T-cell adaptor molecule comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 118. In some aspects, the T-cell adaptor molecule comprises a light chain having the amino acid sequence shown in SEQ ID NO: 118.

[0407] In some aspects, the T-cell adaptor molecule comprises a light chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 119. In some aspects, the T-cell adaptor molecule comprises a light chain having the amino acid sequence shown in SEQ ID NO: 119.

[0408] In some aspects, the T-cell adaptor molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 35. In some aspects, the T-cell adaptor molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 81. In some aspects, the T-cell adaptor molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 88.

[0409] In some aspects, the T-cell adaptor molecule comprises the amino acid sequence shown in SEQ ID NO: 25. In some aspects, the T-cell adaptor molecule comprises the amino acid sequence shown in SEQ ID NO: 35. In some aspects, the T-cell adaptor molecule comprises the amino acid sequence shown in SEQ ID NO: 81. In some aspects, the T-cell adaptor molecule comprises the amino acid sequence shown in SEQ ID NO: 88.

[0410] In some respects, the T-cell adaptor molecule contains the amino acid sequences shown in SEQ ID NO: 25, 26, 35, 81 and 92.

[0411] VIII. Trivalent trispecific T Cell-connecting molecules This disclosure provides a trivalent, trispecific T-cell adaptor molecule. As described herein, the trivalent T-cell adaptor molecule is capable of binding to three different epitopes on the same or some respectably different antigens. In some aspects, the trivalent T-cell binding molecule comprises at least one antigen-binding arm and at least two different T-cell binding arms. In some aspects, the antigen-binding arm is referred to herein as a "first antigen-binding arm," and the T-cell binding arms are referred to herein as a "first T-cell binding arm" and a "second T-cell binding arm." According to this disclosure, the "antigen-binding arm" comprises a heavy chain containing a VH domain and a CH1 domain, and a light chain containing a VL domain and a light chain constant domain, wherein the light chain constant domain is connected to the CH1 domain by a disulfide bond. In some aspects, the "T-cell binding arm" comprises a heavy chain containing a VH domain and a CH1 domain, and a light chain containing a VL domain and a light chain constant domain, wherein the light chain constant domain is connected to the CH1 domain by a disulfide bond. In some aspects, the T-cell binding arm comprises only a VH domain.

[0412] In some aspects, the antigen-binding arm and / or at least one T-cell binding arm in the trivalent T-cell adaptor molecule also includes an Fc region. In some aspects, the antigen-binding arm and / or T-cell binding arm in the trivalent T-cell adaptor molecule includes a complete heavy chain (i.e., a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain).

[0413] In some aspects, the trivalent T-cell adaptor molecule comprises: (i) an antigen-binding arm comprising a heavy chain containing a VH domain and a heavy chain constant region containing CH1, CH2, and CH3 domains, and a light chain containing a VL domain and a light chain constant region; (ii) a first T-cell binding arm comprising a heavy chain containing a VH domain and a heavy chain constant region containing CH1, CH2, and CH3 domains, and a light chain containing a VL domain and a light chain constant region; and (iii) a second T-cell binding arm comprising a heavy chain containing a VH and CH1 domain and a light chain containing a VL domain and a light chain constant region; wherein the heavy chain of the second T-cell binding arm is attached to the heavy chain of the first T-cell binding arm via a peptide linker. An exemplary trivalent T-cell adaptor molecule is shown in Figure 1C middle.

[0414] In some aspects, the light chain of the antigen-binding arm is linked to the heavy chain disulfide bond of the antigen-binding arm in the trivalent T-cell adaptor molecule via a natural interchain disulfide bond (e.g., a disulfide bond present in IgG antibodies). In some aspects, the light chain of the antigen-binding arm is linked to the heavy chain disulfide bond of the antigen-binding arm in the trivalent T-cell adaptor molecule via an engineered disulfide bond. In some aspects, the light chains of the first T-cell binding arm and / or the second T-cell binding arm are linked to the heavy chain disulfide bond of the first T-cell binding arm and / or the second T-cell binding arm in the trivalent T-cell adaptor molecule via a natural interchain disulfide bond. In some aspects, the light chains of the first T-cell binding arm and / or the second T-cell binding arm are linked to the heavy chain disulfide bond of the first T-cell binding arm and / or the second T-cell binding arm in the T-cell adaptor molecule via an engineered disulfide bond.

[0415] In some aspects, the heavy chain of an antigen-binding arm is attached to the heavy chain of another antigen-binding arm via a peptide linker. In some aspects, the heavy chain of an antigen-binding arm is attached to a T-cell binding arm via a peptide linker. In some aspects, the peptide linker consists of 5 to 100 amino acids, 5 to 50 amino acids, 5 to 25 amino acids, or 5 to 15 amino acids. In some aspects, the peptide linker is primarily composed of glycine and serine amino acid residues, and in some aspects includes the amino acid sequence GGGGS or SGGGGS. In some aspects, the peptide linker contains or consists of SEQ ID NO: 89. In some aspects, the linker contains 1 to about 10 copies of SEQ ID NO: 89. In some aspects, the linker contains 2 copies of SEQ ID NO: 89.

[0416] In some respects, the trivalent T-cell adaptor molecule comprises (a) at least one antigen-binding arm that binds STEAP2 and comprises a heavy chain containing a VH domain and a CH1 domain; and a light chain containing a VL domain and a light chain constant domain; (b) at least one T-cell binding arm that comprises a heavy chain containing a VH domain and a CH1 domain; a light chain containing a VL domain and a light chain constant domain; and (c) an Fc domain comprising a first Fc region and a second Fc region, each Fc region comprising a CH2 domain and a CH3 domain; and also comprises at least one modification to promote heterodimerization.

[0417] In some respects, the trivalent T-cell adaptor molecule comprises (i) at least one antigen-binding arm that binds to an epitope on STEAP2, (ii) at least one T-cell-binding arm that binds to CD3, and (iii) at least one T-cell-binding arm that binds to CD8.

[0418] In some respects, one of the CH3 domains in the Fc region of the trivalent T cell adaptor molecule contains a club-shaped mutation, and the other CH3 domain contains a mortar-shaped mutation.

[0419] In some respects, the CH1 domain and light chain constant domain of one or more of each of the antigen-binding arm and T-cell-binding arm of the trivalent T-cell adaptor molecule also contain charge-pair substitutions comprising a first charged amino acid substitution in the CH1 domain and a second charged amino acid substitution in the light chain constant domain, wherein the first charged amino acid substitution and the second charged amino acid substitution have opposite charges.

[0420] In some aspects, the light chain constant domain of one or more of the antigen-binding arm and T-cell-binding arm of the trivalent T adaptor molecule is a λ light chain constant domain (CLλ), and the charge pair is a λ charge pair containing a positively charged amino acid residue selected from arginine, lysine, or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine. In some aspects, the charged amino acid of the λ charge pair is located at one or more of the following positions: (i) Position 117 in CLλ and position 141 in the CH1 structural domain; (ii) Position 117 in CLλ and position 185 in the CH1 structural domain; (iii) Position 119 in CLλ and position 128 in the CH1 structural domain; (iv) Position 134 in CLλ and position 128 in the CH1 structural domain; (v) Position 134 in CLλ and position 145 in the CH1 structural domain; (vi) Position 134 in CLλ and position 183 in the CH1 structural domain; (vii) Position 136 in CLλ and position 185 in the CH1 structural domain; (viii) Position 178 in CLλ and position 173 in the CH1 structural domain; and / or (ix) Position 117 in CLλ and position 187 in the CH1 structural domain, where the numbering is based on the EU index.

[0421] In some respects, one or more of the light chain constant domains in each of the antigen-binding arm and T cell-binding arm of the trivalent T adaptor molecule are λ light chain constant domains (CLλ), and the charge pairs are λ charge pairs, wherein: (c) The charged amino acid at position 117 is arginine and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is arginine and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is arginine and the charged amino acid at position 141 is serine; the charged amino acid at position 117 is arginine and the charged amino acid at position 141 is threonine; the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is aspartic acid; the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is glutamic acid; the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is serine; or the charged amino acid at position 117 is lysine and the charged amino acid at position 141 is threonine. (ii) The charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 185 is threonine; (iii) The charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 119 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 119 is lysine, and the charged amino acid at position 128 is threonine; (iv) The charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 128 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 128 is threonine; (v) The charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 145 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 145 is threonine. (vi) The charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is serine; the charged amino acid at position 134 is arginine, and the charged amino acid at position 183 is threonine; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is aspartic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is glutamic acid; the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is serine; or the charged amino acid at position 134 is lysine, and the charged amino acid at position 183 is threonine. (vii) The charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is serine; the charged amino acid at position 136 is arginine, and the charged amino acid at position 185 is threonine; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is aspartic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is glutamic acid; the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is serine; or the charged amino acid at position 136 is lysine, and the charged amino acid at position 185 is threonine; (viii) The charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is serine; the charged amino acid at position 178 is arginine, and the charged amino acid at position 173 is threonine; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is aspartic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is glutamic acid; the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is serine; or the charged amino acid at position 178 is lysine, and the charged amino acid at position 173 is threonine; and / or (ix) The charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is serine; the charged amino acid at position 117 is arginine, and the charged amino acid at position 187 is threonine; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is aspartic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is glutamic acid; the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is serine; or the charged amino acid at position 117 is lysine, and the charged amino acid at position 187 is threonine.

[0422] In some respects, the light chain constant domain of one or more of the antigen-binding arm and T-cell-binding arm of the trivalent T adaptor molecule is a κ light chain constant domain (CLκ), and the charge pair is a κ charge pair containing a positively charged amino acid residue selected from arginine, lysine, or histidine and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine, wherein the κ charge pair is located at position 133 in CLκ and position 183 in CH1.

[0423] In some respects, one or more of the light chain constant domains in each antigen-binding arm and T-cell-binding arm of the trivalent T adaptor molecule are κ light chain constant domains (CLκ), and the charge pairs are κ charge pairs, wherein the charged amino acid at position 133 is glutamate and the charged amino acid at position 183 is lysine; or the charged amino acid at position 133 is lysine and the charged amino acid at position 183 is glutamate.

[0424] In some respects, the light chain constant domain of the T cell binding arm of the trivalent T adaptor molecule is CLλ, and the charge pair is the λ charge pair as described herein, and the light chain constant domain of the antigen binding arm and the second T cell binding arm of the trivalent T adaptor molecule is the λ or κ charge pair, and the antigen binding arm contains a charged amino acid in the CH1 domain with the same charge as the charged amino acid in the CH1 domain of the second T cell binding arm.

[0425] In some respects, the light chain constant domain of the T cell binding arm is CLλ, and the charge pair is the λ charge pair as described herein, and the light chain constant domain of the antigen binding domain and the second T cell binding arm is the κ charge pair, and the charged amino acid in the CH1 domain of the antigen binding arm has the same charge as the charged amino acid in the CH1 domain of the second T cell binding arm.

[0426] In some respects, the antigen-binding arm or the CH1 domain of the T-cell binding arm of a trivalent T-cell adaptor molecule can be linked to a light chain constant domain via engineered disulfide bonds.

[0427] In some respects, the CH1 domain, which can be linked to the light chain constant domain via engineered disulfide bonds, comprises (i) substitution of native cysteine ​​to non-cysteine ​​amino acids and (ii) substitution of native non-cysteine ​​amino acids to cysteine; and the light chain constant domain comprises (i) substitution of native cysteine ​​to non-cysteine ​​amino acids and (ii) substitution of native non-cysteine ​​amino acids to cysteine; wherein the substituted cysteine ​​in the light chain constant domain and the substituted cysteine ​​in the CH1 domain can form disulfide bonds.

[0428] In some respects, the CH1 domain, which can be linked to the light chain constant domain via engineered disulfide bonds, comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 220, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 126; and the light chain constant domain comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 212, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 122, wherein the cysteine ​​at position 126 of the CH1 domain and the cysteine ​​at position 122 of the light chain constant domain can form a disulfide bond; wherein the numbering is based on the EU index.

[0429] In some respects, the CH1 domain, which can be linked to the light chain constant domain via engineered disulfide bonds, comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 220, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 126; and the light chain constant domain comprises (i) a substitution of native cysteine ​​to a non-cysteine ​​amino acid at position 214, and (ii) a substitution of native non-cysteine ​​amino acid to cysteine ​​at position 121, wherein the cysteine ​​at position 126 of the CH1 domain and the cysteine ​​at position 121 of the light chain constant domain can form a disulfide bond; wherein the numbering is based on the EU index.

[0430] In some respects, the trivalent T-cell adaptor molecule includes an antigen-binding arm that binds STEAP2 and includes a heavy chain comprising a VH domain and a CH1 domain, wherein the VH domain comprises VH-CDR1 selected from SEQ ID Nos: 1, 9, 17, 103, 111, 127, 135 and 143; VH-CDR2 selected from SEQ ID Nos: 2, 10, 18, 104, 112, 128, 136 and 144; and VH-CDR3 selected from SEQ ID Nos: 3, 11, 19, 94, 96, 98, 105 and 113. In some respects, the antigen-binding arm of the trivalent T-cell adaptor molecule also includes a light chain comprising a VL domain and a light chain constant domain, wherein the VL domain comprises VL-CDR1 selected from SEQ ID Nos: 4, 12, 20, 100, 108, 130, 138 and 146; VL-CDR2 selected from SEQ ID Nos: 5, 13, 21, 101, 109, 131, 139 and 147; and VL-CDR3 selected from SEQ ID Nos: 6, 14, 22, 102, 110, 132, 140 and 148.

[0431] In some aspects, the trivalent T-cell adaptor molecule includes an antigen-binding arm that binds STEAP2 and a heavy chain containing a VH domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID Nos: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149. In some aspects, the trivalent T-cell adaptor molecule includes an antigen-binding arm that binds STEAP2 and a heavy chain containing a VH domain comprising an amino acid sequence selected from SEQ ID Nos: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0432] In some aspects, the trivalent T-cell adaptor molecule includes an antigen-binding arm that binds STEAP2 and a heavy chain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 91. In some aspects, the trivalent T-cell adaptor molecule includes an antigen-binding arm that binds S...

Claims

1. A T-cell adaptor molecule, said T-cell adaptor molecule comprising: (a) An antigen-binding arm that binds to an epitope on human prostatic six-transmembrane epithelial antigen 2 (STEAP2) and comprises a heavy chain containing a heavy chain variable domain (VH) and a heavy chain CH1 domain, wherein the heavy chain variable domain (VH) contains a variable heavy chain complementarity-determining region 1 (VH-CDR1) selected from SEQ ID NO: 1, 9, 17, 103, 111, 127, 135 and 143; a VH-CDR2 selected from SEQ ID NO: 2, 10, 18, 104, 112, 128, 136 and 144; and a light chain containing a light chain variable domain (VL) and a light chain constant domain, wherein the light chain variable domain (VL) contains a variable heavy chain complementarity-determining region 1 (VH-CDR1) selected from SEQ ID NO: 1, 9, 17, 103, 111, 127, 135 and 143; a variable heavy chain complementarity-determining region 1 selected from SEQ ID NO: 1, 9, 17, 103, 111, 127, 135 and 145; and a light chain containing a light chain variable domain (VL) and a light chain constant domain, wherein the light chain variable domain (VL) contains ... Variable light chain complementarity-determining region 1 (VL-CDR1) selected from SEQ ID NO: 4, 12, 20, 100, 108, 130, 138 and 146; VL-CDR2 selected from SEQ ID NO: 5, 13, 21, 101, 109, 131, 139 and 147; VL-CDR3 selected from SEQ ID NO: 6, 14, 22, 102, 110, 132, 140 and 148; (b) A first T cell binding arm that binds to differentiation cluster 3 (CD3), the differentiation cluster 3 (CD3) comprising a heavy chain comprising a heavy chain variable domain (VH) and a heavy chain CH1 domain, the heavy chain variable domain (VH) comprising VH-CDR1 selected from SEQ ID NO: 36, 40 and 44; VH-CDR2 selected from SEQ ID NO: 37, 41 and 45; VH-CDR3 selected from SEQ ID NO: 38, 42 and 46; and a light chain comprising a light chain variable domain (VL) and a light chain constant domain, the light chain variable domain (VL) comprising VL-CDR1 selected from SEQ ID NO: 27 and 31; VL-CDR2 selected from SEQ ID NO: 28 and 32; VL-CDR3 selected from SEQ ID NO: 29 and 33; and (c) An Fc domain comprising a first Fc region and a second Fc region, each Fc region comprising a CH2 domain and a CH3 domain; the Fc domain further comprising at least one modification to promote heterodimerization.

2. A trivalent T cell adaptor molecule comprising the T cell adaptor molecule according to claim 1, the trivalent T cell adaptor molecule further comprising: (d) a second T cell binding arm, the second T cell binding arm binding to differentiation cluster 8 (CD8) and comprising a heavy chain comprising a heavy chain variable domain (VH) and a heavy chain CH1 domain, the heavy chain variable domain (VH) comprising VH-CDR1 shown in SEQ ID NO: 48, VH-CDR2 shown in SEQ ID NO: 49, and VH-CDR3 shown in SEQ ID NO: 50; and a light chain comprising a light chain variable domain (VL) and a light chain constant domain, the light chain variable domain (VL) comprising VL-CDR1 shown in SEQ ID NO: 51, VL-CDR2 shown in SEQ ID NO: 52, and VL-CDR3 shown in SEQ ID NO:

53.

3. A trivalent T cell adaptor molecule, said trivalent T cell adaptor molecule comprising: (a) A first antigen-binding arm and a second antigen-binding arm, each binding to an epitope on human prostatic six-transmembrane epithelial antigen 2 (STEAP2) and each comprising a heavy chain, the heavy chain comprising a heavy chain variable domain (VH) and a heavy chain CH1 domain, the heavy chain variable domain (VH) comprising a variable heavy chain complementarity-determining region 1 (VH-CDR1) selected from SEQ ID NO: 1, 9, 17, 103, 111, 127, 135 and 143; VH-CDR2 selected from SEQ ID NO: 2, 10, 18, 104, 112, 128, 136 and 144; selected from SEQ ID NO: VH-CDR3 of SEQ ID NO: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137 and 145; and a light chain comprising a light chain variable structural domain (VL) and a light chain constant structural domain, wherein the light chain variable structural domain (VL) comprises a variable light chain complementarity determination region 1 (VL-CDR1) selected from SEQ ID NO: 4, 12, 20, 100, 108, 130, 138 and 146; VL-CDR2 selected from SEQ ID NO: 5, 13, 21, 101, 109, 131, 139 and 147; and VL-CDR3 selected from SEQ ID NO: 6, 14, 22, 102, 110, 132, 140 and 148; (b) A first T-cell binding arm, the first T-cell binding arm binding to differentiation cluster 3 (CD3) and comprising a heavy chain comprising a VH and a heavy chain CH1 domain, the VH comprising VH-CDR1 selected from SEQ ID NO: 36, 40 and 44; VH-CDR2 selected from SEQ ID NO: 37, 41 and 45; VH-CDR3 selected from SEQ ID NO: 38, 42 and 46; and a light chain comprising a VL and a light chain constant domain, the VL comprising VL-CDR1 selected from SEQ ID NO: 27 and 31; VL-CDR2 selected from SEQ ID NO: 28 and 32; VL-CDR3 selected from SEQ ID NO: 29 and 33; and (c) An Fc domain comprising a first Fc region and a second Fc region, each Fc region comprising a CH2 domain and a CH3 domain; the Fc domain further comprising at least one modification to promote heterodimerization. The heavy chain of the first antigen-binding arm and the heavy chain of the first T-cell binding arm are attached to the Fc domain, and the heavy chain of the second antigen-binding arm is attached to the heavy chain of the first T-cell binding arm.

4. A tetravalent T cell adaptor molecule comprising the trivalent T cell adaptor molecule according to claim 3, the tetravalent T cell adaptor molecule further comprising: (d) a second T cell binding arm, the second T cell binding arm binding to differentiation cluster 8 (CD8) and comprising a heavy chain, the heavy chain comprising a variable restructure domain (VHH), the variable restructure domain (VHH) comprising VH-CDR1 shown in SEQ ID NO: 84; VH-CDR2 shown in SEQ ID NO: 85; and VH-CDR3 shown in SEQ ID NO:

86.

5. One or more nucleic acids, said one or more nucleic acids encoding a T-cell adaptor molecule according to any one of claims 1 to 4.

6. A vector comprising the nucleic acid according to claim 5.

7. An isolated host cell, said isolated host cell comprising the nucleic acid according to claim 5 or the vector according to claim 6.

8. A pharmaceutical composition comprising a T-cell adaptor molecule according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

9. A method of treating a disease in a patient in need, the method comprising administering to the patient an effective amount of a T-cell adaptor molecule according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 8.

10. The T-cell adaptor molecule according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 8, used as a drug or for treating cancer.

11. Use of the T-cell adaptor molecule according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 8 for the manufacture of a medicament for treating cancer.

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