STEAP2-directed T cell engager and composition thereof

Abstract

T cell recruitment molecules are provided that bind to antigens on non-immune cells and antigens on immune cells. The T cell recruitment molecules can bind to STEAP2 on cancer cells and, for example, CD3 on T cells. The T cell recruitment molecules can also bind to STEAP2 on cancer cells and, for example, CD8 on T cells. Alternatively, the T cell recruitment molecules can bind to STEAP2 on cancer cells, as well as both CD3 and CD8 on T cells.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 495,547, filed on 11 April 2023, which is incorporated herein by reference in its entirety.

[0002] References to electronically submitted sequence listings The electronically submitted sequence listing (name: STEAP2TED-100-WO-PCT_ST26.xml, size: 182,496 bytes, creation date: April 10, 2024) is incorporated herein by reference in its entirety. [Background technology]

[0003] Polyvalent binding molecules that recognize two or more different epitopes are of interest in diagnostic and therapeutic approaches. However, their generation presents challenges. Indiscriminate pairing of heavy and light chains expressed in a single cell can result in the production of several different molecules, with only one pairing being desirable and the remaining pairings resulting in non-functional or monospecific molecules.

[0004] Various strategies have been developed in attempts to overcome this problem and promote the precise assembly of desired polyvalent molecules. However, the introduction of each additional binding arm increases the possibility of mispairing.

[0005] Therefore, additional mechanisms are needed to improve polypeptide chain pairing in polyvalent molecules and promote their efficient production. [Overview of the project]

[0006] (a) Heavy chain variable domains that bind to an epitope on the six transmembrane epithelial antigen of prostate-2 (STEAP2) of human prostate and include a variable heavy chain complementarity determining region 1 (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. A heavy chain containing the heavy chain CH1 domain (VH), a variable light chain complementarity determining region 1 (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 a light chain variable domain (VL-CDR3) selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148.(b) an antigen-binding arm comprising a light chain including a light chain constant domain (VL), and a heavy chain comprising a heavy chain variable domain (VH) including VH-CDR1 selected from SEQ ID NOs: 36, 40, and 44, VH-CDR2 selected from SEQ ID NOs: 37, 41, and 45, VH-CDR3 selected from SEQ ID NOs: 38, 42, and 46, and a heavy chain including a heavy chain CH1 domain, and VL-CDR1 selected from SEQ ID NOs: 27 and 31, and selected from SEQ ID NOs: 28 and 32 A T cell mobilization molecule is provided, comprising: a first T cell binding arm comprising a light chain including a variable light chain domain (VL) comprising VL-CDR2, VL-CDR3 selected from SEQ ID NOs. 29 and 33, and a light chain comprising a constant light chain domain; and (c) an Fc domain comprising a first Fc region and a second Fc region, wherein each Fc region comprises a CH2 domain and a CH3 domain, and the Fc domain further comprises at least one modification to promote heterodimerization.

[0007] In some embodiments, a trivalent T cell mobilization molecule is provided, further comprising (d) a second T cell binding arm comprising a heavy chain comprising a heavy chain variable domain (VH) containing VH-CDR1 described in SEQ ID NO: 48, VH-CDR2 described in SEQ ID NO: 49, and VH-CDR3 described in SEQ ID NO: 50, and a heavy chain comprising a heavy chain CH1 domain, and a light chain comprising a light chain variable domain (VL) containing VL-CDR1 described in SEQ ID NO: 51, VL-CDR2 described in SEQ ID NO: 52, and VL-CDR3 described in SEQ ID NO: 53, and a light chain comprising a light chain constant domain.

[0008] In some embodiments, 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 linker.

[0009] In some embodiments, the heavy chain of the second T cell-binding arm is attached to the heavy chain of the antigen-binding arm via a linker.

[0010] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 89.

[0011] In some embodiments, the linker includes one to ten copies of sequence number 89.

[0012] In one embodiment, the linker includes two copies of sequence number 89.

[0013] In some embodiments, one of the two CH3 domains of the T cell mobilization molecule or trivalent T cell mobilization molecule contains a knob mutation, and the other of the two CH3 domains contains a hole mutation.

[0014] In some embodiments, one or more CH1 domains and light chain constant domains of each of (a) an antigen-binding arm, (b) a first T cell-binding arm, and (d) a second T cell-binding arm further include 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 and second charged amino acid substitutions have opposite charges.

[0015] In some embodiments, one or more light chain constant domains in each of (a), (b), and (d) of the T cell mobilization molecule or trivalent T cell mobilization molecule are lambda light chain constant domains (CLλ), and the charge pair is a lambda 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, with numbering following the EU index, and the charged amino acids of the lambda charge pair are in the following positions: (i) Position 117 in CLλ and position 141 in the CH1 domain, (ii) Position 117 in CLλ and position 185 in the CH1 domain, (iii) Position 119 in CLλ and position 128 in the CH1 domain, (iv) Position 134 in CLλ and position 128 in the CH1 domain, (v) position 134 in CLλ and position 145 in the CH1 domain, (vi) position 134 in CLλ and position 183 in the CH1 domain, (vii) position 136 in CLλ and position 185 in the CH1 domain, (viii) position 178 in CLλ and position 173 in the CH1 domain, and / or (ix) is located at one or more of position 117 in CLλ and position 187 in the CH1 domain.

[0016] In some embodiments, the charged amino acids of the lambda charge pair of the T cell mobilizing molecule or the trivalent T cell mobilizing molecule are as follows. (i) The charged amino acid at position 117 is arginine, the charged amino acid at position 141 is aspartic acid, the charged amino acid at position 117 is arginine, the charged amino acid at position 141 is glutamic acid, the charged amino acid at position 117 is arginine, the charged amino acid at position 141 is serine, the charged amino acid at position 117 is arginine, the charged amino acid at position 141 is threonine, the charged amino acid at position 117 is lysine, the charged amino acid at position 141 is aspartic acid, the charged amino acid at position 117 is lysine, the charged amino acid at position 141 is glutamic acid, the charged amino acid at position 117 is lysine, 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.

[0017] In some embodiments, one or more light chain constant domains in (a), (b), and (d) are kappa light chain constant domains (CLκ), and the charge pair is a kappa 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, with numbering according to the EU index, and the kappa charge pair is located at position 133 in CLκ and position 183 in the CH1 domain.

[0018] In some embodiments, the charged amino acids of the kappa charge pair of the T cell mobilization molecule or trivalent T cell mobilization 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.

[0019] In some embodiments, one of the light chain constant domains of (a), (b), and (d) is CLλ and the charge pair is a lambda charge pair, the second and third light chain constant domains of (a), (b), and (d) are CLλ or CLκ and the charge pair is a lambda or kappa charge pair, and the second of (a), (b), and (d) contains a charged amino acid in the CH1 domain having the same charge as the charged amino acid in the third CH1 domain of (a), (b), and (d).

[0020] In some embodiments, one of the light chain constant domains of (a), (b), and (d) is CLλ, and the charge pair is a lambda charge pair; the second and third light chain constant domains of (a), (b), and (d) are CLκ, and the charge pair is a kappa 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 third CH1 domain of (a), (b), and (d).

[0021] In some embodiments, the CH1 domains of (a), (b), and / or (d) of the T cell mobilization molecule or trivalent T cell mobilization molecule can be linked to the light chain constant domain via an engineered disulfide linkage.

[0022] In some embodiments, the CH1 domains of (b) and (d) of the T cell mobilization molecule or trivalent T cell mobilization molecule can be linked to the light chain constant domain via an engineered disulfide linkage, while the CH1 domain of (a) is linked to the light chain constant domain via a native disulfide linkage.

[0023] In some embodiments, the CH1 domain of a T cell mobilization molecule or trivalent T cell mobilization molecule, which can be linked to a light chain constant domain via an manipulated disulfide linkage, (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of a natural non-cysteine ​​amino acid with cysteine, The light chain constant domain is (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of a natural non-cysteine ​​amino acid with cysteine, Substitutive cysteines in the light chain constant domain and the CH1 domain can form disulfide bonds.

[0024] In some embodiments, the CH1 domain of a T cell mobilization molecule or trivalent T cell mobilization molecule, which can be linked to a light chain constant domain via an manipulated disulfide linkage, (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 220, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, The light chain constant domain is (i) Substitution of the native cysteine ​​with a non-cysteine ​​amino acid at position 212, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 122, 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, and the numbering follows the EU index.

[0025] In some embodiments, the CH1 domain of a T cell mobilization molecule or trivalent T cell mobilization molecule, which can be linked to a light chain constant domain via an manipulated disulfide linkage, (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 220, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, The light chain constant domain is (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 214, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 121, 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, and the numbering follows the EU index.

[0026] In some embodiments, the antigen-binding arm of a T cell mobilization molecule or a trivalent T cell mobilization molecule includes a VH 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 to an amino acid sequence selected from SEQ ID NOs. 7, 15, 23, 93, 95, 97, 106, 114, 133, 141, and 149.

[0027] In some embodiments, the antigen-binding arm of a T cell mobilization molecule or a trivalent T cell mobilization molecule is attached to the Fc domain and includes a heavy chain constant region 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 to an amino acid sequence selected from SEQ ID NOs. 66, 67, 69, 72, 73, and 76.

[0028] In some embodiments, the antigen-binding arm of a T cell mobilization molecule or a trivalent T cell mobilization 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 to an amino acid sequence selected from SEQ ID NOs: 8, 16, 24, 107, 115, 134, 142, and 150.

[0029] In some embodiments, the antigen-binding arm of the T cell mobilization molecule or trivalent T cell mobilization molecule includes a 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 respect to the amino acid sequence described in SEQ ID NO: 63.

[0030] In some embodiments, the VL of the T cell binding arm of a T cell mobilization molecule or the VL of the first T cell binding arm or the second T cell binding arm of a trivalent T cell mobilization molecule includes 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, and the VH includes 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.

[0031] In some embodiments, the VL of the T cell binding arm of a T cell mobilization molecule or the VL of the first or second T cell binding arm of a trivalent T cell mobilization 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 described in SEQ ID NO: 30 or 34.

[0032] In some embodiments, the light chain constant domain of the T cell mobilization molecule or the light chain constant domain of the first T cell binding arm or the second T cell binding arm of the trivalent T cell mobilization 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 described in SEQ ID NO: 60 or 61.

[0033] In some embodiments, the VH of the T cell binding arm of a T cell mobilization molecule or the VH of the first or second T cell binding arm of a trivalent T cell mobilization 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 described in SEQ ID NO: 39, 43, or 47.

[0034] In some embodiments, the heavy chain of the T cell binding arm of a T cell mobilization molecule or the first or second T cell binding arm of a trivalent T cell mobilization 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 NOs. 66, 67, 69, 74, 75, 77, 78, and 79.

[0035] In some embodiments, the VH of the second T cell-binding arm of the trivalent T cell mobilization molecule includes VH-CDR1 described in SEQ ID NO: 48, VH-CDR2 described in SEQ ID NO: 49, and VH-CDR3 described in SEQ ID NO: 50, and the VL includes VL-CDR1 described in SEQ ID NO: 51, VL-CDR2 described in SEQ ID NO: 52, and VL-CDR3 described in SEQ ID NO: 53.

[0036] In some embodiments, the VL of the second T cell-binding arm of the trivalent T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 54.

[0037] In some embodiments, the light chain constant domain of the second T cell-binding arm of the trivalent T cell mobilization 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 described in SEQ ID NO: 64.

[0038] In some embodiments, the VH of the second T cell-binding arm of the trivalent T cell mobilization 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 described in SEQ ID NO: 56.

[0039] In some embodiments, the CH1 domain of the second T cell-binding arm of the trivalent T cell mobilization 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 to an amino acid sequence selected from SEQ ID NOs. 57, 66, 67, 69, 74, 75, 77, 78, and 79.

[0040] In some embodiments, the antigen-binding arm of a T cell mobilization molecule or a trivalent T cell mobilization molecule binds to an epitope on the extracellular loop of STEAP2.

[0041] In some embodiments, the T cell mobilization molecules include SEQ ID NOs: 25, 26, 35, 81, and 92. In some embodiments, the trivalent T cell mobilization molecules include SEQ ID NOs: 25, 26, 35, 55, and 58.

[0042] In some embodiments, (a) a heavy chain comprising a variable heavy chain complementarity determining region 1 (VH-CDR1) each 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, each binding to an epitope on six-transmembrane epithelial antigen 2 (STEAP2) of the human prostate, and each comprising a variable heavy chain complementarity determining region 1 (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. An antigen-binding arm comprising a heavy chain containing a variable domain (VH) and a heavy chain CH1 domain, a variable light chain complementarity determining region 1 (VL-CDR1) selected from SEQ ID NOs: 4, 12, 20, 100, 108, 130, 138, and 146, a variable light chain domain (VL) containing a variable light chain complementarity determining region 1 (VL-CDR1) selected from SEQ ID NOs: 5, 13, and 21, and a VL-CDR3 selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148, and a light chain containing a constant light chain domain, and (b) a cluster of differentiation antigens 3 3, CD3) is bound to a heavy chain containing a heavy chain CH1 domain, a VH containing 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, and a light chain containing a light chain constant domain. A trivalent T cell mobilization molecule is provided, comprising: (c) a first T cell binding arm, 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, wherein the heavy chains of the first antigen binding arm and 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.

[0043] In some embodiments, a tetravalent T cell mobilization molecule is provided, comprising the above-described trivalent T cell mobilization molecule, and further comprising (d) a second T cell binding arm comprising a heavy chain that binds to differentiation antigen group 8 (CD8) and includes variable heavy chain domains (VHH) comprising VH-CDR1 as described in SEQ ID NO: 84, VH-CDR2 as described in SEQ ID NO: 85, and VH-CDR3 as described in SEQ ID NO: 86.

[0044] In some embodiments, the heavy chain of the second T cell-binding arm of the tetravalent T cell mobilization molecule is attached to the heavy chain of the first antigen-binding arm.

[0045] In some embodiments, the heavy chain of the second antigen-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule is attached to the heavy chain of the first T cell-binding arm via a linker.

[0046] In some embodiments, the heavy chain of the second T cell-binding arm of the tetravalent T cell mobilization molecule is attached to the heavy chain of the first antigen-binding arm via a linker.

[0047] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 89.

[0048] In some embodiments, the linker includes one to ten copies of sequence number 89.

[0049] In one embodiment, the linker includes two copies of sequence number 89.

[0050] In some embodiments, one of the two CH3 domains of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule contains a knob mutation, and the other of the two CH3 domains contains a hole mutation.

[0051] In some embodiments, (a) one or more CH1 domains and light chain constant domains of each of the first antigen-binding arm or the second antigen-binding arm, and (b) the first T cell-binding arm, include 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 and second charged amino acid substitutions have opposite charges.

[0052] In some embodiments, one or more light chain constant domains of (a) a first antigen-binding arm or a second antigen-binding arm, and (b) a first T cell-binding arm, are lambda light chain constant domains (CLλ), and the charge pair is a lambda 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, numbering according to the EU index, and the lambda charge pair is located at the following positions: (i) Position 117 in CLλ and position 141 in the CH1 domain, (ii) Position 117 in CLλ and position 185 in the CH1 domain, (iii) Position 119 in CLλ and position 128 in the CH1 domain, (iv) Position 134 in CLλ and position 128 in the CH1 domain, (v) Position 134 in CLλ and position 145 in the CH1 domain, (vi) Position 134 in CLλ and position 183 in the CH1 domain, (vii) Position 136 in CLλ and position 185 in the CH1 domain, (viii) Position 178 in CLλ and position 173 in the CH1 domain, and (ix) Located at one or more of the following positions: (ix) Position 117 in CLλ and position 187 in the CH1 domain.

[0053] In some embodiments, the charged amino acids of the lambda charge pair of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule 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.

[0054] In some embodiments, one or more light chain constant domains of (a) a first antigen-binding arm or a second antigen-binding arm, and (b) a first T cell-binding arm, are kappa light chain constant domains (CLκ), and the charge pair is a kappa 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, with numbering according to the EU index, and the kappa charge pair is located at position 133 in CLκ and position 183 in CH1.

[0055] In some embodiments, the charged amino acids of the kappa charge pair of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization 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.

[0056] In some embodiments, one of the light chain constant domains of (a), (b), and (d) is CLλ and the charge pair is a lambda charge pair, the second and third light chain constant domains of (a), (b), and (d) are CLλ or CLκ and the charge pair is a lambda or kappa charge pair, and the second of (a), (b), and (d) contains a charged amino acid in the CH1 domain having the same charge as the charged amino acid in the third CH1 domain of (a), (b), and (d).

[0057] In some embodiments, one of the light chain constant domains of (a), (b), and (d) is CLλ, and the charge pair is a lambda charge pair; the second and third light chain constant domains of (a), (b), and (d) are CLκ, and the charge pair is a kappa 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 third CH1 domain of (a), (b), and (d).

[0058] In some embodiments, the CH1 domains of (a) and / or (b) of the trivalent T cell mobilization molecule, or the CH1 domains of (a), (b), and / or (d) of the tetravalent T cell mobilization molecule, can form disulfide links with the light chain constant domains of (a) and / or (b) of the trivalent T cell mobilization molecule, or with the light chains of (a), (b), and / or (d) of the tetravalent T cell mobilization molecule, via manipulated disulfide linkages.

[0059] In some embodiments, the CH1 domain of (b) of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule can form a disulfide linkage with the light chain constant domain via an engineered disulfide linkage, while the CH1 domain of (a) is linked to the light chain constant domain via a native disulfide linkage.

[0060] In some embodiments, the CH1 domains of (b) and (d) of the tetravalent T cell mobilization molecule can form disulfide links with the light chain constant domains of (b) and (d) via manipulated disulfide linkages, and the CH1 domain of (a) is linked to the light chain constant domain of (a) via a native disulfide linkage.

[0061] In some embodiments, the CH1 domain of the first T cell-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule is (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of a natural non-cysteine ​​amino acid with cysteine, The light chain constant domain of the second T cell binding arm is (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of a natural non-cysteine ​​amino acid with cysteine, Substituting cysteine ​​in the CH1 domain of the first T cell binding arm and substituting cysteine ​​in the light chain constant domain of the first T cell binding arm can form disulfide bonds.

[0062] In some embodiments, the CH1 domain of the first T cell-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule is (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 220, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, The light chain constant domain is (i) Substitution of the native cysteine ​​with a non-cysteine ​​amino acid at position 212, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 122, 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, and the numbering follows the EU index.

[0063] In some embodiments, the light chains of the first and second antigen-binding arms of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule contain native cysteine, the CH1 domains of the first and second antigen-binding arms contain native cysteine, the native cysteine ​​of the CH1 domain of the first antigen-binding arm and the native cysteine ​​of the constant domain of the light chain of the first antigen-binding arm can form a disulfide bond, and the native cysteine ​​of the CH1 domain of the second antigen-binding arm and the native cysteine ​​of the constant domain of the light chain of the second antigen-binding arm can form a disulfide bond.

[0064] In some embodiments, the VH of the first antigen-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization 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 to an amino acid sequence selected from SEQ ID NOs: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0065] In some embodiments, CH1 of the first antigen-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization 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 sequences described in SEQ ID NOs. 67 and 93.

[0066] In some embodiments, the VL of the first antigen-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization 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 sequences described in SEQ ID NOs: 8, 16, 24, 107, 115, 134, 142, and 150.

[0067] In some embodiments, the light chain constant domain of the first T cell-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization 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 described in SEQ ID NO: 63.

[0068] In some embodiments, the VL of the first T cell-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule includes VL-CDR1 as described in SEQ ID NO: 27 or 31, VL-CDR2 as described in SEQ ID NO: 28 or 32, and VL-CDR3 as described in SEQ ID NO: 29 or 33, and the VH of the first T cell-binding arm includes VH-CDR1 as described in SEQ ID NO: 36, 40, or 44, VH-CDR2 as described in SEQ ID NO: 37, 41, or 45, and VH-CDR3 as described in SEQ ID NO: 38, 42, or 46.

[0069] In some embodiments, the VL of the first T cell binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization 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 described in SEQ ID NO: 30 or 34.

[0070] In some embodiments, the VH of the first T cell-binding arm of a trivalent T cell mobilization molecule or a tetravalent T cell mobilization 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 sequences described in SEQ ID NOs. 39, 43, and 47.

[0071] In some embodiments, the VH of the second T cell-binding arm of the tetravalent T cell mobilization molecule includes VH-CDR1 as described in SEQ ID NO: 84, VH-CDR2 as described in SEQ ID NO: 85, and VH-CDR3 as described in SEQ ID NO: 86.

[0072] In some embodiments, the VH of the second T cell-binding arm of the tetravalent T cell mobilization 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 described in SEQ ID NO: 56 or 83.

[0073] In some embodiments, the tetravalent T cell mobilization molecule comprising a trivalent T cell mobilization molecule further comprises (d) a second T cell binding arm. In some embodiments, the second T cell binding arm comprises VL, which includes VL-CDR1 as described in SEQ ID NO: 51, VL-CDR2 as described in SEQ ID NO: 52, and VL-CDR3 as described in SEQ ID NO: 53, and VH, which includes VH-CDR1 as described in SEQ ID NO: 48, VH-CDR2 as described in SEQ ID NO: 49, and VH-CDR3 as described in SEQ ID NO: 50. In some embodiments, 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 described in SEQ ID NO: 54, and 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 described in SEQ ID NO: 56. In some embodiments, a trivalent T cell recruiting molecule or a tetravalent T cell recruiting molecule binds to an epitope on the extracellular loop of STEAP2.

[0074] In some embodiments, the trivalent T cell mobilization molecule or the tetravalent T cell mobilization molecule comprises a heavy chain including the heavy chain constant region of the second antigen-binding arm, and the first 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 described in SEQ ID NO: 81. In some embodiments, the heavy chain including the heavy chain constant regions of the first antigen-binding arm and 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 an amino acid sequence selected from SEQ ID NO: 87. In some embodiments, the trivalent T cell mobilization molecule comprises SEQ ID NOs: 25, 35, 81, 25, and 93. In some embodiments, the tetravalent T cell mobilization molecule includes SEQ ID NOs: 25, 35, 81, 25, and 87. In some embodiments, the tetravalent T cell mobilization molecule includes SEQ ID NOs: 151, 152, 153, 154, and 152. In some embodiments, the tetravalent T cell mobilization molecule includes SEQ ID NOs: 151, two copies of SEQ ID NOs: 152, SEQ ID NOs: 153, and SEQ ID NOs: 154.

[0075] In some embodiments, the T cell mobilization molecule is encoded by one or more nucleic acids. In some embodiments, the vector comprises nucleic acids. In some embodiments, the isolated host cell comprises nucleic acids or the vector. In other embodiments, the T cell mobilization molecule is formulated with a pharmaceutically acceptable carrier. This disclosure also relates to the treatment of a disease. In some embodiments, this disclosure relates to a method for treating a disease in a patient who requires treatment of the disease, the method comprising the step of administering an effective amount of the T cell mobilization molecule to the patient. In some embodiments, the disease is cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the T cell mobilization molecule is formulated for use as a pharmaceutical. In some embodiments, the T cell mobilization molecule is for use in the treatment of cancer. In some embodiments, the T cell mobilization molecule is used in the manufacture of a pharmaceutical for the treatment of cancer. [Brief explanation of the drawing]

[0076] [Figure 1A] A schematic diagram of T cell engaging molecules (TEDs) is shown. [Figure 1B] A schematic diagram of T cell engaging molecules (TED2) is shown. [Figure 1C] A schematic diagram of the trivalent T cell engaging molecule (TED3) is shown. [Figure 1D] A schematic diagram of the tetravalent T cell engaging molecule (TED4) is shown. [Figure 2] The diagram shows the kappa of the binding arm of a T cell engager and the ribbon of the CH1 chain interaction surface. [Figure 3] This diagram shows the ribbon-like surface of the lambda and CH1 chain interaction of the binding arms of T cell engagers. [Figure 4A] This shows the binding of the trivalent STEAP2 T cell engager molecules TED3 30D12 G96P / CD3 K29-SN75_3 and TED3 30D12 G96P / CD3 K29-SN75_V12 to C4-2 cells compared to the TED3 Nip228_V12 control. [Figure 4B] This shows the CD8 binding of the STEAP2 trivalent T cell recruitment molecules TED3 30D12 G96P / CD3 K29-SN75_3 and TED3 30D12 G96P / CD3 K29-SN75_V12. [Figure 4C] This shows the binding of the tetravalent STEAP2 T cell engager molecule 40A3LO12-TED4-K17 / E75 to C4-2 cells. [Figure 4D] This shows the CD8 binding of the STEAP2 tetravalent T cell mobilization molecule 40A3LO12-TED4-K17 / E7. [Figure 4E]This study demonstrates the cytotoxicity of the STEAP2 trivalent T cell engager molecules 30D12-TED3 K29 / SN75 and 30D12-TED3 K29 / SN75 V12 compared to 30D12-TED3-control-CD3, 30D12-TED3-control-CD3 V12, and TED3 NIP228 / control-CD3 / CD8 V12 control. [Figure 4F] This study demonstrates CD8+ T cell activation by the trivalent STEAP2 T cell mobilization molecules 30D12-TED3 K29 / SN75 and 30D12-TED3 K29 / SN75 V12, compared to 30D12-TED3-control-CD3, 30D12-TED3-control-CD3 V12, and TED3 NIP228 / control-CD3 / CD8 V12 controls. [Figure 4G] This study demonstrates CD4+ T cell activation by the trivalent STEAP2 T cell mobilization molecules 30D12-TED3 K29 / SN75 and 30D12-TED3 K29 / SN75 V12, compared to 30D12-TED3 control-CD3, 30D12-TED3 control-CD3 V12, and TED3 NIP228 / control-CD3 / CD8 V12 control. [Figure 4H] This study demonstrates that the STEAP2 T cell recruitment molecule 40A3LO12-TED2-K29 / SN75 exhibits high cytotoxicity against high-STEAP2-expressing cells (LNCap, C4-2, VCaP, and 22Rv1) and low cytotoxicity against low-STEAP2-expressing cells (DU145). [Figure 4I] This study demonstrates that the trivalent STEAP2 T cell recruitment molecule 30D12-TED3-K29 / SN75 exhibits high cytotoxicity against high-STEAP2-expressing cells (LNCap, C4-2, VCaP, and 22Rv1) and low cytotoxicity against low-STEAP2-expressing cells (DU145). [Figure 4J] This shows the EC50 of STEAP2 T cell mobilization molecules and trivalent STEAP2 T cell mobilization molecules in cell lines with different levels of STEAP2 expression. [Figure 4K] This shows the STEAP2 receptor density on different cell lines. [Figure 4L]The LNCaP, C4-2, and VCaP cell lines exhibit cytotoxicity and IL-6 and TNFα cytokine release induced by the STEAP2 T cell mobilization molecule 40A3LO12-TED2-K29 / SN75 and the trivalent STEAP2 T cell mobilization molecule 30D12-TED3-K29 / SN75. [Figure 5A] This demonstrates the activation of CD4 and CD8 T cells by the STEAP2 T cell recruitment molecule (TED) and the trivalent (TED3) STEAP2 CD8-inducible T cell recruitment molecule. [Figure 5B] This demonstrates T cell activation by the bivalent STEAP2 T cell recruitment molecule (TED2) and various tetravalent (TED4) STEAP2 CD8-inducible T cell recruitment molecules. [Figure 5C] This study demonstrates that 30D12-TED3-K29 / SN75 induced a reduction in IL-6 release compared to the 30D12-TED-K29 / SN75 molecule at a similar level of cytotoxicity. [Figure 5D-01] Compared to 40A3LO12-TED2-K29 / SN75, all variants of the TED4 T cell engager exhibited similar levels of cytotoxicity, but with lower levels of IL-6 release. [Figure 5D-02] Compared to 40A3LO12-TED2-K29 / SN75, all variants of the TED4 T cell engager exhibited similar levels of cytotoxicity, but with lower levels of IL-6 release. [Figure 5D-03] Compared to 40A3LO12-TED2-K29 / SN75, all variants of the TED4 T cell engager exhibited similar levels of cytotoxicity, but with lower levels of IL-6 release. [Figure 5D-04] Compared to 40A3LO12-TED2-K29 / SN75, all variants of the TED4 T cell engager exhibited similar levels of cytotoxicity, but with lower levels of IL-6 release. [Figure 6A]This shows the degree of inhibition of cell lysis by TED2 T cell recruiting molecules and TED3 trivalent STEAP2 T cell recruiting molecules at different Treg-to-T effector cell ratios. [Figure 6B] This shows Treg activation in untreated cells and in cells treated with T cell recruitment molecules (TED) and trivalent (TED3) STEAP2 CD8-inducible T cell recruitment molecules. [Figure 7A] This shows the fluorescence of mKate-labeled C4-2 spheroid tumors over time in the absence (control) and presence of gradually increasing concentrations of the trivalent STEAP2 T cell mobilization molecule (TED3). [Figure 7B] We demonstrated that tumor cell death of mKate 2-expressing C4-2 spheroids by PBMCs was observed in all variants of TED4 T cell engagers and was increased compared to 40A3LO12-TED2-K29 / SN75. [Figure 8A] The tumor volume is shown in human PBMC-transplanted mice either untreated or treated with either a non-targeted trivalent T cell recruitment molecule (NIP228-TED3-K29 / SN75 V12) or a trivalent (30D12-TED3-K29 / SN75 V12) STEAP2 CD8-inducible T cell engager molecule. [Figure 8B] This shows the tumor volume in human PBMC-transplanted mice either untreated or treated with either the TED2 T cell mobilization molecule or the trivalent STEAP2 CD8-inducible T cell mobilization molecule, with C4-2 tumor cells embedded. [Figure 8C] Bioluminescence images of mice transplanted with human PBMCs and intravenously injected with luciferase-expressing C4-2 tumor cells, either untreated (PBS) or treated with the trivalent STEAP2 CD8-inducible T cell recruiting molecule, are shown. [Figure 8D] Figure 8C shows a graph of bioluminescence in mice. [Figure 9]The following describes TNF-α levels in mice implanted with luciferase-expressing C4-2 tumor cells, transplanted with human PBMCs from two different donors, and treated either untreated (PBS) or with a STEAP2 T cell recruiting molecule with a CD3 control-binding arm (30D12-TED-CD3 control), a STEAP2 T cell recruiting molecule with a modified CD3-binding arm (30D12-TED-KN29 / SN75), or a trivalent STEAP2 CD8-inducible T cell recruiting molecule with a modified CD3-binding arm (30D12-TED3-K29 / SN75 V12). [Figure 10] This shows tumor volume in mice subcutaneously implanted with a mixture of human PBMCs and C4-2 tumor cells, either untreated (PBS) or treated with either the 40A3LO12-TED2-K29 / SN75 molecule or the 30D12-TED3-K29 / SN75 V12 molecule. [Figure 11] This shows tumor volume in human immune stem cell transplanted mice in which C4-2 tumor cells were subcutaneously implanted and either untreated or treated with the 30D12-TED3-K29 / SN75 V12 molecule. [Figure 12] This study shows a correlation between the EC50 of cytolysis of the tetravalent STEAP2 T cell mobilization molecule 40A3LO12-TED4-K17 / E75 and the level of STEAP2 cell surface expression (antigen binding capacity, ABC) across eight tumor cell lines. [Figure 13] This study demonstrates that the addition of regulatory T cells (Tregs) to a co-culture of effector T cells and tumor cells reduces granzyme B production in CD8 T cells in response to non-CD8-inducible T cell recruitment molecules, but Tregs do not show any effect in response to the CD8-inducible 40A3LO12-TED4-K17 / E75 molecule. [Figure 14] The study demonstrates persistent cytotoxicity of PBMCs after up to four rounds of co-culture with tumor cells in the presence of 40A3LO12-TED4-K17 / E75. [Figure 15]This study demonstrates that cytotoxicity of STEAP 2-negative cells occurs only when co-cultured with varying amounts of STEAP 2-positive cells. [Figure 16A] This study demonstrates the cytotoxicity of xenograft organoids derived from prostate cancer patients when co-cultured with T cells in the presence of 40A3LO12-TED4-K17 / E75. No cytotoxicity was observed in response to the untargeted NIP228-TED4-K17 / E75 molecule. [Figure 16B] This study demonstrates the cytotoxicity of xenograft organoids derived from prostate cancer patients when co-cultured with T cells in the presence of 40A3LO12-TED4-K17 / E75. No cytotoxicity was observed in response to the untargeted NIP228-TED4-K17 / E75 molecule. [Figure 16C] At the end of this assay, we observed an increase in the activation of CD8+ T cells compared to CD4+ T cells. [Figure 17] This shows increased IFNg release in primary prostate tumor slices treated with 40A3LO12-TED4-K17 / E75. [Figure 18A] This shows tumor volume in human PBMC-transplanted mice either untreated or treated with either a non-targeted T cell recruitment molecule (NIP228-TED4-K17 / E75) or a tetravalent (40A3LO12-TED4-K17 / E75) STEAP2 CD8-inducing T cell engager molecule. [Figure 18B] This shows that the levels of CD69 and CD25 on T cells in the blood of these animals did not change after receiving three doses of 40A3LO12-TED4-K17 / E75. [Figure 18C] In the tumors of these animals after receiving three doses of 40A3LO12-TED4-K17 / E75, we observed increased levels of CD69 and CD25 on CD8+ T cells, rather than on CD4+ T cells. [Figure 19]The tumor burden (measured by bioluminescence imaging) is observed in human PBMC-transplanted mice either untreated or treated with either a non-targeted T cell recruiting molecule (NIP228-TED4-K17 / E75) or a tetravalent (40A3LO12-TED4-K17 / E75) STEAP2 CD8-inducing T cell engager molecule, with luciferase-expressing C4-2 tumor cells implanted in the tibia. [Figure 20] This shows tumor volume in human PBMC-transplanted mice either untreated or treated with a tetravalent (40A3LO12-TED4-K17 / E75)STEAP2 CD8-inducing T-cell engager molecule, implanted with fragments from a xenograft model derived from prostate cancer patients. [Modes for carrying out the invention]

[0077] T cell mobilization molecules are provided that bind to antigens on target cells, e.g., human prostate 6-transmembrane epithelial antigen 2 (STEAP2) on cancer cells, and T cell antigens, e.g., differentiation antigen group 3 (CD3) and / or CD8 proteins. Methods for constructing and using T cell mobilization molecules are further provided. In some embodiments, the T cell mobilization molecule binds to one or more antigens, e.g., one or more epitopes on STEAP2 on cancer cells, and one or more T cell proteins, e.g., CD3 and / or CD8. While we do not wish to be bound by theory, it is hypothesized that the number and effectiveness of target protein-T cell interactions brought about by the T cell mobilization molecules described herein will enable increased target cell death while reducing the risk of inducing cytokine release syndrome.

[0078] i.Definition To facilitate understanding of this disclosure, certain terms are defined first. Further definitions are provided throughout the detailed description.

[0079] It should be noted that the terms "a" or "an" (representing a real entity) refer to one or more of those real entities; for example, "nucleotide sequence" is understood to represent one or more nucleotide sequences. Therefore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably in this specification.

[0080] Furthermore, when used herein, “and / or” should be interpreted as a specific disclosure of each of two designated features or components, with or without the other. Accordingly, the terms “and / or” as used in phrases such as “A and / or B” are intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the terms “and / or” as used in phrases such as “A, B, and / or C” are intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0081] Whenever an aspect is described herein using the word “comprising,” it is understood that other similar aspects described with respect to “consisting of” and / or “consisting essentially of” are also provided. As used herein, the terms “comprise” and “include” and their variations (e.g., “comprises,” “comprising,” “includes,” and “including”) are understood to include the described components, features, elements, or processes, or groups of components, features, elements, or processes, but not to exclude any other components, features, elements, or processes, or groups of components, features, elements, or processes. The terms “comprising,” “consisting essentially of,” and “consisting of” may be substituted for any of the other two terms, while retaining their usual meanings.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art relating to this disclosure. For example, see the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2 nd ed.,2002,CRC Press,The Dictionary of Cell and Molecular Biology,3 rd The ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide many general dictionaries of terms used in this disclosure for those skilled in the art.

[0083] Units, prefixes, and symbols are given in the format recognized by the International System of Units (SI). Numerical ranges include the number defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure and can be obtained by referring to this specification as a whole. Thus, the terms defined immediately below are more fully defined by referring to this specification as a whole.

[0084] The term "approximately" is used herein to mean roughly, roughly, approximately, or within that range. When the term "approximately" is used with a numerical range, it modifies that range by extending the boundary above and below the stated number. Generally, the term "approximately" can be used to modify a number above or below (higher or lower than) the stated value, for example, with a variation of 10 percent.

[0085] As used herein, the term “T cell recruiting molecule” refers to any molecule that can target an antigen on a target cell, e.g., STEAP2 on cancer cells, and can bind to a protein expressed on a T cell, e.g., CD3 and / or CD8 protein. A T cell recruiting 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. A T cell recruiting molecule may comprise a monoclonal antibody, a chimeric antibody, a humanized antibody, and a portion of a human antibody. For example, a T cell mobilization molecule may include an antigen-binding portion or arm of an antibody, such as an anti-STEAP 2 antibody and / or an anti-CD3 antibody and / or an anti-CD8 antibody, (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of VL, VH, LC and CH1 domains, (ii) an F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment containing two Fab fragments linked by disulfide crosslinking at a hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR); (vii) a combination of two or more isolated CDRs that can be joined by a synthetic linker of choice; or (viii) a single chain Fv. The antibody fragments include Fv and scFv. The antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for usefulness in the same manner as intact antibodies. The antigen-binding portion or arm can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Ribbon diagrams of the kappa and CH1 chain interaction surfaces of the binding arm of a T cell engager, and ribbon diagrams of the lambda and CH1 chain interaction surfaces of the binding arm of a T cell engager are shown in Figures 2 and 3, respectively.

[0086] As used herein, the terms “antigen-binding arm” and “antigen-binding moiety” refer to a portion of a molecule that binds to all or part of a target epitope and generally includes 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 a paratope of the antigen-binding moiety or arm, which is part of the antigen-binding moiety or arm that binds to the target epitope. The 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 to which the antigen-binding arm binds. A monoclonal monospecific IgG antibody molecule contains two antigen-binding arms, each of which can bind to the same epitope (i.e., is bivalent for a single epitope).

[0087] An example of a bispecific antibody form incorporating some of these modifications to improve the efficient production of these molecules is "DuetMab," described in Mazor 2015 and WO2013 / 096291. The DuetMab antibody molecule uses a knob-into-hole technique for the heterodimerization of two distinct heavy chains and increases the effectiveness of congeneral heavy-light chain pairing by replacing a native disulfide bond at one of the CH1-CL interfaces with an engineered disulfide bond.

[0088] As used herein, the term “valence” refers to the presence of a specific number of antigen-binding moieties or arms in a T-cell recruiting molecule that binds to an epitope. For example, as used herein, the term “trivalent” refers to a T-cell recruiting molecule having three moieties or arms that bind to an antigen. As used herein, the term “tetravalent” refers to a T-cell recruiting molecule having four moieties or arms that bind to an antigen. The antigen-binding moieties or arms of trivalent and / or tetravalent T-cell recruiting molecules can bind to the same antigen molecule, can bind to the same epitope on the antigen molecule, can bind to different epitopes on the same antigen molecule, and / or can bind to different epitopes on different antigen molecules. In some embodiments of the bivalent molecule 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 a bispecific T-cell engager DuetMab ("Bispecific T-cell Engager DuetMab, TED," see Figure 1A). In some embodiments of the trivalent molecules 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 engager DuetMab ("Bispecific T-cell Engager DuetMab, TED2," see Figure 1B). In some embodiments of the trivalent molecules 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, and this is referred to as the trispecific T-cell engager DuetMab ("Trispecific T-cell engager DuetMab, TED3," see Figure 1C). In some embodiments of the tetravalent molecules described herein, a single antigen-binding domain binds to CD3 in the tetravalent compound, another antigen-binding domain binds to CD8, and third and fourth antigen-binding domains each bind to a different target or the same target, and may be referred to as a tetraspecific T-cell engager DuetMab ("Tetraspecific T-cell engager DuetMab, TED4", Figure 1D).In some embodiments, the TED4 form includes two antigen-binding domains capable of binding to the same target.

[0089] The natural evolution of bispecific antibodies was the introduction of triplicate antibodies, but as used herein, the term “linker” refers to a peptide chain of at least two amino acids, e.g., glycine and / or serine, that connects two polypeptide chains, e.g., the heavy chain of the antigen-binding arm and the heavy chain of the T cell-binding arm. A linker may contain one or more glycine and / or serine amino acids. As used herein, the terms “linked,” “attached,” and “fused” refer to the association of two or more molecules. Linking may be covalent or non-covalent. Linking may also be genetic (i.e., recombinantly fused). Such linking may be achieved using a wide variety of techniques recognized in the field, such as chemical conjugation and recombinant protein production. For example, a linker may contain at least one “GGGGS.”

[0090] In some embodiments, the linkage between the binding domain and the Fc domain includes a standard hinge region. In some embodiments, 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.

[0091] As used herein, the phrase "capable of disulfide linking" refers to two polypeptide chains, each containing at least one cysteine, in a position that allows for the formation of a disulfide crosslink between the two polypeptide chains when the two polypeptide chains are present in a T cell mobilization molecule.

[0092] The term “antibody” in some embodiments refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as “VH”) and a heavy chain constant region (abbreviated herein as “CH”). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as “VL”) and a light chain constant domain. The light chain constant domain consists of one domain (abbreviated herein as “CL”). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are incorporated into more conserved regions called framework regions (FRs). The VH and VL regions contain a framework region (FR) on either side of each CDR, which provides a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region has the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus. Antibodies may be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species, including IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Antibodies, such as IgG1, exist in several allotypes, each differing in at most a few amino acids. Antibodies include, for example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies; human and non-human antibodies; and fully synthetic antibodies.

[0093] The variable regions of the heavy and light chains contain binding domains (also called paratopes) that interact with antigen molecules (or epitopes on antigen molecules, e.g., epitopes on CD3 molecules). The constant region of the antibody 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 of the classical complement system (C1q). The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, amino acids in the variable region are numbered using the Kabat numbering system, and amino acids in the constant region are numbered using the EU system (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 paratopes that bind to the antigen. The light chains in natural antibodies are either "lambda (λ)" or "kappa (κ)" light chains, which differ in their amino acid sequences. The light chains of the trivalent and / or tetravalent T cell engager molecules described herein may be chimeric light chains, for example, containing CLλ and VLκ.

[0094] Antibodies, and methods for their construction and use, are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23(9):1126-1136 (2005). Given the current technology for monoclonal antibody technology, antibodies can be prepared for most targets. It is possible to employ monoclonal and other antibody molecules and use recombinant DNA technology to produce other antibodies, chimeric molecules, and / or T-cell mobilization molecules. Such techniques may include introducing a CDR or variable region of one antibody into a different antibody molecule, or attaching a CDR or variable region to an antibody molecule.

[0095] As used herein, the term “affinity” refers to a measure of the strength of binding of an antigen or target (such as an epitope) to its homobinding domain (such as a paratope). As used herein, the term “avidity” refers to the overall stability of the complex between a population of epitopes and a paratope (i.e., the antigen and antigen-binding arm).

[0096] The term "epitope" refers to a site on an antigen (e.g., STEAP2, CD3, or CD8) to which trivalent and / or tetravalent T cell recruitment molecules can bind. Epitopes can be formed from both adjacent amino acids (usually linear epitopes) or non-adjacent amino acids juxtaposed by tertiary folding of the protein (usually conformational epitopes). Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, but not always, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a specific spatial conformation.

[0097] The term "binding to the same epitope" for two or more antigen-binding moieties means that the moieties bind to the same segment of amino acid residues. An antigen-binding moiety that "competes with another antibody for binding to the target" refers to an antigen-binding moiety that (partially or completely) inhibits the binding of another antibody to the target.

[0098] As used herein, the terms "specific binding", "selective binding", "selectively binds", and "specifically binds" refer to an antigen-binding portion or arm that binds to an epitope on a given antigen. Typically, the antigen-binding portion or arm binds with an equilibrium dissociation constant (K -7 ) of less than about 10 -8 M, e.g., less than about 10 -9 M, 10 -10 M, or less than 10 D M, or even lower, as determined by, for example, surface plasmon resonance (SPR) technology on a BIACORE® 2000 instrument using a given antigen, e.g., human STEAP2 or CD3 or CD8, as an analyte and a T cell mobilizing molecule as a ligand, or by Scatchard analysis of the binding of the T cell mobilizing molecule to antigen-positive cells, and (ii) binds to the given antigen with an affinity that is at least two-fold greater than the affinity for binding to non-specific antigens (e.g., BSA, casein) other than the given antigen or closely related antigens. Thus, a T cell mobilizing molecule that "specifically binds to human STEAP2" refers to an antigen-binding portion or arm that binds to human STEAP2 with a K -7 of 10 -8 M or less, e.g., less than about 10 -9 M, 10 -10 M, or less than 10 D M, or even lower.

[0099] As used herein, the term “polypeptide” is intended to encompass both singular and plural “polypeptides” and includes any chain of 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 a chain of two or more amino acids is included in the definition of “polypeptide,” even though each of these terms may have a more specific meaning. The term “polypeptide” can be used in place of or interchangeably with any of these terms. The term further includes polypeptides that have undergone post-translational or post-synthetic modifications, such as palmitoyl group conjugation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, disulfide bond formation, proteolytic cleavage, or modification with amino acids not naturally occurring. As used herein, the term “peptide” encompasses full-length peptides and their fragments, variants, or derivatives. As used herein, a “peptide” may be part of a fusion polypeptide that includes additional components, such as albumin or PEG moieties, to increase its half-life. Peptides as used herein can also be derivatized in many different ways. Peptides may include modifications such as the conjugation of a palmitoyl group.

[0100] As used herein, “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 are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, predicted non-essential amino acid residues in the antigen-binding arm may be substituted with other amino acid residues from the same side-chain family.

[0101] The percentage of identity between two arrays is a function of the number of identical positions shared by the arrays (i.e., homology % = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap. The comparison of arrays and the determination of the percentage of identity between two arrays can be achieved using mathematical algorithms, as described in the non-restrictive examples below.

[0102] The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) with the NWSgapdna.CMP matrix and gap 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 program (version 2.0) with the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. In addition, 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, using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, which is incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com).

[0103] The nucleic acid and protein sequences described herein can be further used, for example, as “query sequences” for performing searches against public databases to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described in Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed using the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, you can use the default parameters for each program (e.g., XBLAST and NBLAST).

[0104] As used herein, the term “nucleic acid molecule” is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded and may be cDNA.

[0105] As used herein, the terms “polynucleotide” and “nucleic acid molecule” are intended to include DNA molecules and RNA molecules. Polynucleotides or nucleic acid molecules may be single-stranded or double-stranded and may be cDNA.

[0106] As used herein, the term “promoter” refers to a DNA sequence recognized by a cellular mechanism or introduced synthetic mechanism that is required to initiate specific transcription of a gene. The term “promoter” also means encompassing sufficient nucleic acid elements for promoter-dependent gene expression that can be controlled with respect to cell-type, tissue-specific, or inducible expression by an external signal or drug, such elements may be located in the 5' or 3' region of the native gene. In some embodiments, a promoter may be a constitutively active promoter, a cell-type specific promoter, or an inducible promoter.

[0107] As used herein, the term "IRES" refers to elements that facilitate direct internal ribosome entry into start codons such as ATG in cistrons (protein-coding regions), thereby resulting in cap-independent translation of genes. See, for example, Jackson RJ et al., Trends Biochem Sci 15(12):477-83(199) and Jackson RJ and Kaminski, A. RNA 1(10):985-1000(1995). Under the translational control of IRES, translation proceeds cap-independently.

[0108] 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 required for endonuclease cleavage of the RNA transcript, followed by a polyadenylation consensus sequence AATAAA. The polyadenylation signal sequence provides a “poly-A site,” i.e., a site on the RNA transcript to which an adenine residue will be added by post-transcriptional polyadenylation.

[0109] As used herein, the terms “functionally linked,” “functionally inserted,” “functionally positioned,” “controlled,” or “transcriptionally regulated” 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 “operatably linked” means that the DNA sequence and regulatory sequence are linked in such a way that gene expression is possible when the appropriate molecule (e.g., a transcription activator protein) binds to the regulatory sequence. The term “operatably inserted” means that the DNA of interest introduced into a cell is located adjacent to a DNA sequence that directs the transcription and translation of the introduced DNA (i.e., promotes the production of the polypeptide encoded by the DNA of interest).

[0110] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors are capable of directing the expression of a gene to which they are operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. The terms “plasmid” and “vector” can be used interchangeably, as plasmids are the most commonly used form of vectors. However, other forms of expression vectors that perform equivalent functions are also included, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0111] 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 the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such a cell. Such offspring may not be identical to the parent cell in practice, as certain modifications may occur in subsequent generations due to mutation or environmental influences, but they are still included within the scope of the term “host cell” as used herein.

[0112] As used herein, the terms “subject,” “individual,” or “patient” refer to any organism to which the compositions disclosed herein, such as T-cell mobilization molecules, may be administered, for example, for experimental, diagnostic, prophylactic, 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. A subject may be a human or animal that is in need of, requires, is receiving, will receive, or is receiving care from a trained professional for a particular disease or condition.

[0113] As used herein, the terms “to treat,” “to cure,” or “to treat ~” mean to alleviate the pathology of a disease, reduce or eliminate the symptoms of a disease, promote increased survival rates, and / or reduce discomfort. For example, to treat can mean the ability of a treatment to alleviate the symptoms, signs, or causes of a disease when administered to a subject. To treat can also mean the reduction or decrease of at least one clinical symptom and / or the inhibition or delay of the progression of a condition and / or the prevention or delay of the onset of a disease or illness.

[0114] "Immune response," as understood in the art, generally refers to the biological response within vertebrates to foreign or abnormal substances, such as cancerous cells, which protect the organism from these drugs and the diseases they cause. The immune response is 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 either these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells, e.g., effector T cells, Th cells, CD4 + cells, CD8 + This includes activation or inhibition of T cells or Treg cells, or activation or inhibition of any other cells of the immune system, such as NK cells.

[0115] As used herein, the term “immunotherapy” refers to the treatment of a person who is suffering from a disease or at risk of suffering from or experiencing a relapse of a disease, by means of inducing, enhancing, suppressing or otherwise modifying the immune system or immune response.

[0116] As used herein, the term “cancer” refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distal parts of the body via the lymphatic system or bloodstream.

[0117] For example, the terms “effective dose,” “therapeutic effective dose,” and “sufficient dose” of T-cell mobilizing molecules or compositions described herein refer to an amount sufficient to produce a beneficial or desired outcome when administered to a subject, including humans, including relief of symptoms, reduction of the degree of a condition, disorder, or disease, achievement of a stabilized (i.e., non-exacerbated) state of a condition, disorder, or disease, delay or slowing of the onset of progression of a condition, disorder, or disease, improvement or remission (whether partial or complete), whether detectable or undetectable, improvement of at least one measurable physical parameter, not necessarily recognizable by the patient, or enhancement or improvement of a condition, disorder, or disease. In some embodiments, treatment includes inducing a clinically significant response without excessive levels of side effects. Thus, “therapeutic effective dose” or its synonyms depends on the context in which they are applied. In some embodiments, a therapeutic effective dose of a drug (e.g., a T-cell mobilizing molecule or composition described herein) is an amount that produces a beneficial or desired outcome in a subject compared to a control that does not receive the drug. The amount of a given drug (e.g., T cell mobilization molecule or composition) will vary depending on various factors such as the given drug, pharmaceutical formulation, route of administration, type of disease or disorder, the specificity of the subject (e.g., age, sex, and / or weight), or the host being treated.

[0118] As used herein, the term “prophylactic effective dose” refers to the amount of an agent (e.g., a T-cell mobilizing molecule or composition) that delays, prevents, or blocks the onset, development, or progression of a condition or disease over a period of time including weeks, months, or years. The prophylactic effective dose may vary depending on the characteristics of the agent, how the agent is administered, the degree of the disease risk, and the patient’s medical history, age, weight, family history, genetic makeup, any prior or concomitant treatments, as well as other individual characteristics of the patient being treated.

[0119] As used herein, the terms "ug" and "uM" are interchangeable with "μg" and "μM," respectively.

[0120] The various embodiments described herein are described in further detail in the following subsections.

[0121] II. Antigen-binding arm The antigen-binding arm of the T cell recruitment molecule described herein binds to STEAP2. In some embodiments, the T cell recruitment molecule includes one STEAP2-binding arm. In some embodiments, the T cell recruitment molecule binds to two STEAP2 antigens.

[0122] In some embodiments, the antigen-binding arm of the T cell recruitment molecule described herein may be of any form including Fab, Fab', F(ab')2, Fd, Fv, single-chain fragment variable (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some embodiments, the antigen-binding arm includes Fab.

[0123] In some embodiments, the antigen-binding arm comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein VH comprises VH complementarity-determining regions (CDR)1, VH-CDR2, and VH-CDR3, and VL comprises VL-CDR1, VL-CDR2, and VL-CDR3.

[0124] In some embodiments, the antigen-binding arm comprises VH-CDR1 containing an amino acid sequence selected from SEQ ID NOs: 1, 9, 17, 103, 111, 127, 135, and 143. In some embodiments, the antigen-binding arm comprises VH-CDR2 containing an amino acid sequence selected from SEQ ID NOs: 2, 10, 18, 104, 112, 128, 136, and 144. In some embodiments, the antigen-binding arm comprises VH-CDR3 containing an amino acid sequence selected from SEQ ID NOs: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137, and 145.

[0125] In some embodiments, the antigen-binding arm comprises VL-CDR1 containing an amino acid sequence selected from SEQ ID NOs: 4, 12, 20, 100, 108, 130, 138, and 146. In some embodiments, the antigen-binding arm comprises VL-CDR2 containing an amino acid sequence selected from SEQ ID NOs: 5, 13, 21, 101, 109, 131, 139, and 147. In some embodiments, the antigen-binding arm comprises VL-CDR3 containing an amino acid sequence selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148.

[0126] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 3, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 6.

[0127] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 9, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 10, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 11, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 12, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 13, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 14.

[0128] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 17, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 18, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 19, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 20, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 21, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 22.

[0129] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 94, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 6.

[0130] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 96, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 6.

[0131] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 98, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 6.

[0132] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 103, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 104, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 105, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 100, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 101, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 102.

[0133] In some embodiments, the antigen-binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 111, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 112, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 113, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 108, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 109, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 110.

[0134] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 7.

[0135] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 15.

[0136] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 23.

[0137] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 95.

[0138] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 97.

[0139] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 99.

[0140] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 106. In some embodiments, the antigen-binding arm comprises a VH having at least about 95% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 106.

[0141] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 114.

[0142] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 133.

[0143] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 141.

[0144] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, 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 described in SEQ ID NO: 149.

[0145] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 8. In some embodiments, the antigen-binding arm comprises a VL containing the amino acid sequence described in SEQ ID NO: 8.

[0146] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 16. In some embodiments, the antigen-binding arm comprises a VL containing the amino acid sequence described in SEQ ID NO: 16.

[0147] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 24.

[0148] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 107. In some embodiments, the antigen-binding arm comprises a VL containing the amino acid sequence described in SEQ ID NO: 107.

[0149] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 115.

[0150] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 134.

[0151] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 142.

[0152] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 150.

[0153] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 7, and 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 described in SEQ ID NO: 8. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 7, and a VL containing an amino acid sequence described in SEQ ID NO: 8.

[0154] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 15, and 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 described in SEQ ID NO: 16. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 15, and a VL containing an amino acid sequence described in SEQ ID NO: 16.

[0155] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 23, and 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 described in SEQ ID NO: 24. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 23, and a VL containing an amino acid sequence described in SEQ ID NO: 24.

[0156] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 95, and 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 described in SEQ ID NO: 8. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 95, and a VL containing an amino acid sequence described in SEQ ID NO: 8.

[0157] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 97, and 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 described in SEQ ID NO: 16. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 97, and a VL containing an amino acid sequence described in SEQ ID NO: 16.

[0158] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 99, and 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 described in SEQ ID NO: 24. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 99, and a VL containing an amino acid sequence described in SEQ ID NO: 24.

[0159] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 106, and 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 described in SEQ ID NO: 107. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 106, and a VL containing an amino acid sequence described in SEQ ID NO: 107.

[0160] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 114, and 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 described in SEQ ID NO: 115. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 114, and a VL containing an amino acid sequence described in SEQ ID NO: 115.

[0161] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 133, and 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 described in SEQ ID NO: 134. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 133, and a VL containing an amino acid sequence described in SEQ ID NO: 134.

[0162] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 141, and 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 described in SEQ ID NO: 142. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence described in SEQ ID NO: 141, and a VL containing an amino acid sequence described in SEQ ID NO: 142.

[0163] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 149, and 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 described in SEQ ID NO: 150.

[0164] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising: 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 to an amino acid sequence selected from SEQ ID NOs. 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149; and 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 to an amino acid sequence selected from SEQ ID NOs. 8, 16, 24, 107, 115, 134, 142, and 150. In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising a VH containing an amino acid sequence selected from SEQ ID NOs: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149, and a VL containing an amino acid sequence selected from SEQ ID NOs: 8, 16, 24, 107, 115, 134, 142, and 150.

[0165] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising 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 an amino acid sequence selected from SEQ ID NOs. 59, 60, and 61.

[0166] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm comprising 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 an amino acid sequence selected from SEQ ID NOs. 62, 63, and 64.

[0167] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 8, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 8, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0168] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 8, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 8, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0169] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 16, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 16, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0170] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 16, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 16, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0171] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 24, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 24, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0172] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 24, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 24, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0173] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 107, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 107, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0174] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 107, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 107, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0175] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 115, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 115, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0176] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 115, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 115, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0177] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 115, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 115, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0178] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 115, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 115, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0179] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 115, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 115, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0180] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 134, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 134, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0181] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 142, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 142, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0182] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 150, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the antigen-binding arm comprises a VL containing an amino acid sequence described in SEQ ID NO: 150, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0183] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 8, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0184] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 16, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0185] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 24, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0186] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 107, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0187] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 115, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0188] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 134, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0189] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 142, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0190] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 150, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0191] In some embodiments, the T cell mobilization 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 NOs.65-80.

[0192] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 7, and 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 NOs: 65-80.

[0193] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 15, and 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 NOs: 65-80.

[0194] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 23, and 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 NOs: 65-80.

[0195] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 95, and 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 NOs: 65-80.

[0196] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 97, and 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 to an amino acid sequence selected from SEQ ID NOs: 65-80.

[0197] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 99, and 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 to an amino acid sequence selected from SEQ ID NOs.

[0198] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 106, and 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 to an amino acid sequence selected from SEQ ID NOs.

[0199] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 114, and 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 to an amino acid sequence selected from SEQ ID NOs.

[0200] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 133, and 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 to an amino acid sequence selected from SEQ ID NOs.

[0201] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 141, and 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 to an amino acid sequence selected from SEQ ID NOs: 65-80.

[0202] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 149, and 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 to an amino acid sequence selected from SEQ ID NOs: 65-80.

[0203] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 7, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0204] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 15, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0205] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 23, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0206] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 95, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0207] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 97, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0208] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 99, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0209] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 106, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0210] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 114, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0211] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 133, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0212] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 141, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0213] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 149, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 88, and 91.

[0214] In some embodiments, the T cell mobilization molecule comprises a heavy chain containing a STEAP2 antigen-binding domain fused to a CD8-binding domain. In some embodiments, the antigen-binding arm further comprises a light chain containing a STEAP2-binding domain.

[0215] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 25.

[0216] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 116.

[0217] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 117.

[0218] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 118.

[0219] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 119.

[0220] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 26.

[0221] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 120.

[0222] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 121.

[0223] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 122.

[0224] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 123.

[0225] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 124.

[0226] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 125.

[0227] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 126.

[0228] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 26, and 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 described in SEQ ID NO: 25. In some embodiments, the T cell mobilization molecule includes a STEAP2 binding arm comprising a heavy chain containing an amino acid sequence described in SEQ ID NO: 26, and a light chain containing an amino acid sequence described in SEQ ID NO: 25.

[0229] III.T cell binding arm The T cell mobilizing molecules described herein include at least one T cell binding arm. In some embodiments, the T cell mobilizing molecules described herein include two T cell binding arms. In some embodiments, the T cell mobilizing molecule includes at least one T cell binding arm that binds to the CD3 antigen. In some embodiments, the T cell mobilizing molecule includes at least one T cell binding arm that binds to the CD8 antigen. In some embodiments, the T cell mobilizing molecule includes at least one T cell binding arm that binds to the CD3 antigen and at least one T cell binding arm that binds to the CD8 antigen. In some embodiments, the T cell binding arm includes a heavy chain variable domain and a light chain variable domain. In some embodiments, the T cell binding arm includes only the heavy chain variable domain.

[0230] In some embodiments, the T cell binding arms of the T cell mobilizing molecules described herein can be of any form including Fab, Fab’, F(ab’)2, Fd, Fv, single chain fragment variable (scFv), single chain antibody, VHH, vNAR, nanobody (single domain antibody), or any combination thereof. In some embodiments, the antigen binding arm includes Fab. In some embodiments, the antigen binding arm includes VHH.

[0231] In some embodiments, the T cell binding arm includes a variable heavy chain region (VH) and a variable light chain region (VL), where VH includes VH-CDR1, VH-CDR2, VH-CDR3, and VL includes VL-CDR1, VL-CDR2, and VL-CDR3.

[0232] In some embodiments, the T cell-binding arm of the T cell mobilizing molecule is capable of binding to CD3. CD3 (cluster of differentiation 3) is a protein complex composed of four subunits, the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3 associates with the T cell receptor and the ζ chain to generate activation signals in T lymphocytes. The T cell mobilizing molecule targeting CD3 and a target cell antigen (or antigens), e.g., STEAP2, can force a transient interaction between the target cell (or cells), e.g., STEAP2-expressing cancer cells, and T cells, causing crosslinking, T cell activation, and subsequent antigen-dependent T cell killing of the target cell. In some embodiments, the T cell mobilizing molecule binds monovalently to the CD3 protein, and the T cell receptor is crosslinked and activated only upon binding to the target cell.

[0233] In some embodiments, the T cell-binding arm comprises a VH-CDR1 comprising an amino acid sequence selected from SEQ ID NO: 36, 40, and 44. In some embodiments, the T cell-binding arm comprises a VH-CDR2 comprising an amino acid sequence selected from SEQ ID NO: 37, 41, and 45. In some embodiments, the T cell-binding arm comprises a VH-CDR3 comprising an amino acid sequence selected from SEQ ID NO: 38, 42, and 46.

[0234] In some embodiments, the T cell-binding arm comprises a VL-CDR1 comprising an amino acid sequence selected from SEQ ID NO: 27 and 31. In some embodiments, the T cell-binding arm comprises a VL-CDR2 comprising an amino acid sequence selected from SEQ ID NO: 28 and 32. In some embodiments, the T cell-binding arm comprises a VL-CDR3 comprising an amino acid sequence selected from SEQ ID NO: 29 and 33.

[0235] In some embodiments, the T cell binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 36, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 37, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 38, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 31, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 32, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 33.

[0236] In some embodiments, the T cell binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 36, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 37, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 38, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 27, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 28, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 29.

[0237] In some embodiments, the T cell binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 40, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 41, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 42, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 31, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 32, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 33.

[0238] In some embodiments, the T cell binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 40, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 41, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 42, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 27, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 28, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 29.

[0239] In some embodiments, the T cell binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 44, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 45, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 46, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 31, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 32, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 33.

[0240] In some embodiments, the T cell binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 44, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 45, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 46, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 27, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 28, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 29.

[0241] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 39. In some embodiments, the T cell binding arm comprises a VL containing the amino acid sequence described in SEQ ID NO: 39.

[0242] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 43. In some embodiments, the T cell binding arm comprises a VL containing the amino acid sequence described in SEQ ID NO: 43.

[0243] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 47. In some embodiments, the T cell binding arm comprises a VL containing the amino acid sequence described in SEQ ID NO: 47.

[0244] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 34.

[0245] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 30.

[0246] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 39, and 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 described in SEQ ID NO: 34.

[0247] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 39, and 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 described in SEQ ID NO: 30.

[0248] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 43, and 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 described in SEQ ID NO: 34.

[0249] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 43, and 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 described in SEQ ID NO: 30.

[0250] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 47, and 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 described in SEQ ID NO: 34.

[0251] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 47, and 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 described in SEQ ID NO: 30.

[0252] In some embodiments, the T cell binding arm comprises VH - CDR1, VH - CDR2, and VH - CDR3 present in a VH region having the amino acid sequence set forth in SEQ ID NO: 39, and VL - CDR1, VL - CDR2, and VL - CDR3 present in a VL region having the amino acid sequence set forth in SEQ ID NO: 34.

[0253] In some embodiments, the T cell binding arm comprises VH - CDR1, VH - CDR2, and VH - CDR3 present in a VH region having the amino acid sequence set forth in SEQ ID NO: 39, and VL - CDR1, VL - CDR2, and VL - CDR3 present in a VL region having the amino acid sequence set forth in SEQ ID NO: 30.

[0254] In some embodiments, the T cell binding arm comprises VH - CDR1, VH - CDR2, and VH - CDR3 present in a VH region having the amino acid sequence set forth in SEQ ID NO: 43, and VL - CDR1, VL - CDR2, and VL - CDR3 present in a VL region having the amino acid sequence set forth in SEQ ID NO: 34.

[0255] In some embodiments, the T cell binding arm comprises VH - CDR1, VH - CDR2, and VH - CDR3 present in a VH region having the amino acid sequence set forth in SEQ ID NO: 43, and VL - CDR1, VL - CDR2, and VL - CDR3 present in a VL region having the amino acid sequence set forth in SEQ ID NO: 30.

[0256] In some embodiments, the T cell binding arm comprises VH - CDR1, VH - CDR2, and VH - CDR3 present in a VH region having the amino acid sequence set forth in SEQ ID NO: 47, and VL - CDR1, VL - CDR2, and VL - CDR3 present in a VL region having the amino acid sequence set forth in SEQ ID NO: 34.

[0257] In some embodiments, the T cell binding arm includes VH-CDR1, VH-CDR2, and VH-CDR3 located in the VH region having the amino acid sequence described in SEQ ID NO: 47, and VL-CDR1, VL-CDR2, and VL-CDR3 located in the VL region having the amino acid sequence described in SEQ ID NO: 30.

[0258] In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising 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 an amino acid sequence selected from SEQ ID NOs. 59, 60, and 61.

[0259] In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising 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 an amino acid sequence selected from SEQ ID NOs. 62, 63, and 64.

[0260] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 34, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0261] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 34, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0262] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 30, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0263] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 30, and 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 to the amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0264] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 34, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0265] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 30, and 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 to an amino acid sequence selected from SEQ ID NOs: 60, 61, 63, and 64.

[0266] In some embodiments, the T cell mobilization 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 NOs.65-80.

[0267] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 39, and 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 NOs: 65-80.

[0268] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 43, and 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 NOs: 65-80.

[0269] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 47, and 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 NOs: 65-80.

[0270] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 39, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, and 80.

[0271] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 43, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, and 80.

[0272] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 47, and 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 to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 72, 73, 74, 75, 76, 77, 78, 79, and 80.

[0273] In some embodiments, the T cell mobilization molecule includes a T cell 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 respect to the amino acid sequence described in SEQ ID NO: 82. In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising a heavy chain containing an amino acid sequence described in SEQ ID NO: 81 or 82.

[0274] In some embodiments, one of the T cell binding arms can bind to CD8. CD8 (differentiation antigen group 8) is a dimer consisting of a pair of CD8 chains. The most common form of CD8 consists of a CD8-α chain and a CD8-β chain. CD8 acts as a co-receptor on MCHI-restricted T cells, enhancing the antigen sensitivity of CD8+ T cells by binding to a nearly immutable region of the MCHI at a site different from where the T cell receptor binds.

[0275] In some embodiments, the T cell recruitment molecule includes a T cell binding arm capable of binding to CD3 and a T cell binding arm capable of binding to CD8.

[0276] In some embodiments, the T cell binding arm comprises VH-CDR1 containing an amino acid sequence selected from SEQ ID NOs: 48 and 84. In some embodiments, the T cell binding domain comprises VH-CDR2 containing an amino acid sequence selected from SEQ ID NOs: 49 and 85. In some embodiments, the T cell binding domain comprises VH-CDR3 containing an amino acid sequence selected from SEQ ID NOs: 50 and 86.

[0277] In some embodiments, the T cell binding arm includes VL-CDR1 having the amino acid sequence described in SEQ ID NO: 51. In some embodiments, the T cell binding arm includes VL-CDR2 having the amino acid sequence described in SEQ ID NO: 52. In some embodiments, the T cell binding arm includes VL-CDR3 having the amino acid sequence described in SEQ ID NO: 53.

[0278] In some embodiments, the T cell binding arm includes VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 48, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 49, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 50, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 51, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 52, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 53.

[0279] In some embodiments, the T cell binding arm includes VH-CDR1 having the amino acid sequence described in SEQ ID NO: 84, VH-CDR2 having the amino acid sequence described in SEQ ID NO: 85, and VH-CDR3 having the amino acid sequence described in SEQ ID NO: 86. In some embodiments, the T cell binding arm includes VH-CDR1, VH-CDR2, and VH-CDR3 located in a VHH region having the amino acid sequence described in SEQ ID NO: 83.

[0280] In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 56.

[0281] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 54.

[0282] In some embodiments, the T cell mobilization 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 described in SEQ ID NO: 56, and 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 described in SEQ ID NO: 54.

[0283] In some embodiments, the T cell binding arm includes VH-CDR1, VH-CDR2, and VH-CDR3 located in the VH region having the amino acid sequence described in SEQ ID NO: 56, and VL-CDR1, VL-CDR2, and VL-CDR3 located in the VL region having the amino acid sequence described in SEQ ID NO: 54.

[0284] In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising 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 an amino acid sequence selected from SEQ ID NOs. 62, 63, and 64.

[0285] In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising 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 an amino acid sequence selected from SEQ ID NOs. 59, 60, and 61.

[0286] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 54, and 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 to an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64. In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising a VL containing an amino acid sequence described in SEQ ID NO: 54, and a light chain constant domain containing an amino acid sequence selected from SEQ ID NOs: 62, 63, and 64.

[0287] In some embodiments, the T cell mobilization 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 to the amino acid sequence described in SEQ ID NO: 54, and 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 to the amino acid sequence selected from SEQ ID NOs: 59, 60, and 61.

[0288] In some embodiments, the T cell mobilization 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 NOs. 57, 65, 66, and 67.

[0289] In some embodiments, the T cell mobilization molecule includes a T cell binding arm comprising a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 83.

[0290] IV. Charge pairs The terms “charge pair” and “charge variant” are used interchangeably herein and refer to inversely charged amino acids, e.g., 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 located in the heavy chain region (e.g., constant heavy chain region 1 (CH1)), in a position intended to promote the association of the light chain and the heavy chain. “Lambda charge pair” means a charge pair in which a positively or negatively charged amino acid residue is located in the lambda light chain (e.g., CLλ). “Kappa charge pair” means a charge pair in which a positively or negatively charged amino acid residue in the light chain is located in the kappa light chain (e.g., CLκ).

[0291] While we do not wish to be bound by theory, it is thought that the oppositely charged amino acid residues in charge pairs increase the attractive force 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.

[0292] At least one of the amino acid residues in a charge pair can be manipulated into an immunoglobulin and / or a 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 embodiments, both amino acid residues in a charge pair are manipulated into an immunoglobulin and / or a T cell binding arm (i.e., neither amino acid residue in the pair is a wild-type amino acid residue).

[0293] 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.

[0294] Charged amino acid residues are typically found in nature. Examples of naturally occurring positively charged amino acid residues according to this disclosure include arginine, lysine, and histidine. Examples of 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 as "uncharged" in the art, they have isoelectric points less than 6 and are therefore partially negatively charged at neutral pH. For the purposes of the charged pairs disclosed herein, serine and threonine are examples of negatively charged amino acid residues (along with glutamic acid and aspartic acid).

[0295] In some embodiments, a charge pair comprises a positively charged amino acid residue selected from arginine, lysine, or histidine located at one of the positions in the charge pair, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located 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.

[0296] Lambda charge pair By introducing lambda charge pairs as exemplified herein at several locations, the correct pairing of light and heavy chains in the antigen and / or T cell binding arm can be improved.

[0297] In some embodiments, the lambda charge pair includes a positively or negatively charged amino acid residue at positions 117, 119, 134, 136, or 178 of the steady-state light chain lambda region (CLλ). In some embodiments, the lambda charge pair includes a positively or negatively charged amino acid residue at positions 141, 185, 128, 145, 183, 185, 173, or 187 of the CH1 domain. As mentioned elsewhere, the numbering follows EU numbering.

[0298] In some embodiments, the lambda charge pair is located in one or more of the following 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, (ix) Position 117 in CLλ and position 187 in CH1.

[0299] In some embodiments, the lambda charge pair is located at position 117 in CLλ and position 141 in CH1. For example, the lambda charge pair 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, Lysine at position 117 of CLλ and serine at position 141 of CH1, and Lysine at position 117 of CLλ and threonine at position 141 of CH1.

[0300] In some embodiments, the lambda charge pair is selected from any one of a. to f. in the above list. In some embodiments, the lambda charge pair is selected from any one of a. to e. in the above list. In some embodiments, the lambda charge pair is selected from any one of a., b., and e. in the above list. In some embodiments, the lambda charge pair is a.

[0301] In some embodiments, the lambda charge pair is located at position 117 in CLλ and position 185 in CH1. For example, the lambda charge pair 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, Lysine at position 117 of CLλ and serine at position 185 of CH1, Lysine at position 117 of CLλ and threonine at position 185 of CH1.

[0302] In some embodiments, the lambda charge pair is located at position 119 in CLλ and position 128 in the CH1 domain. For example, the lambda charge pair 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, Lysine at position 119 of CLλ and serine at position 128 of CH1, Lysine at position 119 of CLλ and threonine at position 128 of CH1.

[0303] In some embodiments, the lambda charge pair is located at position 134 in CLλ and position 128 in the CH1 domain. For example, the lambda charge pair 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, Lysine at position 134 of CLλ and serine at position 128 of CH1, Lysine at position 134 of CLλ and threonine at position 128 of CH1.

[0304] In some embodiments, the lambda charge pair is located at position 134 in CLλ and position 145 in the CH1 domain. For example, the lambda charge pair 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, Lysine at position 134 of CLλ and serine at position 145 of CH1, Lysine at position 134 of CLλ and threonine at position 145 of CH1.

[0305] In some embodiments, the lambda charge pair is located at position 134 in CLλ and position 183 in the CH1 domain. For example, the lambda charge pair 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, Lysine at position 134 of CLλ and serine at position 183 of CH1, Lysine at position 134 of CLλ and threonine at position 183 of CH1.

[0306] In some embodiments, the lambda charge pair is lysine at position 134 of CLλ and aspartic acid or serine at position 183 of the CH1 domain.

[0307] In some embodiments, the lambda charge pair is located at position 136 in CLλ and position 185 in the CH1 domain. For example, the lambda charge pair 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, Lysine at position 136 of CLλ and serine at position 185 of CH1, Lysine at position 136 of CLλ and threonine at position 185 of CH1.

[0308] In some embodiments, the lambda charge pair is located at position 178 in CLλ and position 173 in the CH1 domain. For example, the lambda charge pair 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; Lysine at position 178 of CLλ and serine at position 173 of CH1, Lysine at position 178 of CLλ and threonine at position 173 of CH1.

[0309] In some embodiments, the antigen and / or T cell binding arm containing lambda charge pairs contains two or more lambda charge pairs. For example, the first antigen and / or T cell binding arm may contain 2, 3, 4, 5, 6, 7, 8, or 9 lambda charge pairs at positions (i) to (ix) above.

[0310] In some embodiments, the first antigen and / or T cell binding arm comprises a kappa charge pair, and both the second antigen-binding arm and / or the second T cell binding arm comprise a lambda charge pair, and the charged amino acid residue located on the second antigen-binding CH1 domain and CLλ has the opposite charge to that located on the second T cell-binding CH1 domain and CLλ.

[0311] In some exemplary embodiments, the positively charged amino acid residue in the lambda charge pair is located on the light chain, and the negatively charged amino acid residue in the lambda charge pair is located on the heavy chain. In some embodiments, the negatively charged amino acid residue is located on the light chain, and the positively charged amino acid residue in the lambda charge pair is located on the heavy chain.

[0312] Kappa charge pair In the T cell mobilization molecules described herein, at least one antigen and / or T cell binding arm comprises a kappa charge pair. As described above, the kappa charge pair refers to a positively charged amino acid residue and a negatively charged amino acid residue, one of which is located on the kappa light chain (e.g., CLκ) of the antigen and / or T cell binding arm, and the other is located on the heavy chain (e.g., CH1) at a position intended to facilitate the association of the light chain of a second antigen and / or T cell binding arm with CH1.

[0313] In some embodiments, the antigen and / or T cell binding arm containing CLκ includes a kappa charge pair located at position 133 in CLκ and position 183 in the second CH1. In some embodiments, the negatively charged amino acid residue in the kappa charge pair is located at position 133 in CLκ, and the positively charged amino acid residue in the kappa charge pair is located at position 183 in the second CH1. In some embodiments, the positively charged amino acid residue in the kappa charge pair is located at position 133 in CLκ, and the negatively charged amino acid residue in the kappa charge pair is located at position 183 in the second CH1. In some embodiments, 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 stated above, this numbering follows EU numbering.

[0314] In some embodiments, both the second antigen-binding arm and the second T-cell-binding arm include a kappa charge pair. In some embodiments, the first antigen-binding arm includes a kappa charge pair, and the second antigen-binding arm and the second T-cell-binding arm include a lambda charge pair.

[0315] In some embodiments, the positively charged amino acid residues in the kappa charge pairs in the second antigen-binding arm are located on the second CH1 and the negatively charged amino acid residues are located on the second light chain; the positively charged amino acid residues in the kappa charge pairs in the second T cell-binding arm are located on the third light chain and the negatively charged amino acid residues are located on the third CH1. In some embodiments, the positively charged amino acid residues in the kappa charge pairs in the second antigen-binding arm are located on the second light chain and the negatively charged amino acid residues are located on the second CH1; the positively charged amino acid residues in the kappa charge pairs in the second antigen-binding arm are located on the third CH1 and the negatively charged amino acid residues are located on the light chain.

[0316] In some embodiments, the first antigen-binding arm includes a lambda charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the first CH1 and CLλ; the second antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and CLκ of the second light chain; the second T cell-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the third CH1 and CLκ of the third light chain, wherein the charged amino acid residue located on the third CH1 and CLκ of the third light chain has the opposite charge to that of the charged amino acid residue located on the second CH1 and CLκ of the second light chain.

[0317] In other embodiments, the first antigen-binding arm contains a kappa charge pair, and the second antigen-binding arm and the second T-cell-binding arm contain a reversed lambda charge pair. In some embodiments, the first antigen-binding arm contains a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the first CH1 and CLκ, the second antigen-binding arm contains a lambda charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and the CLλ of the second light chain, and the second T-cell-binding arm contains a lambda charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the third CH1 and the CLλ of the third light chain, wherein the charged amino acid residue located on the third CH1 and the CLλ of the third light chain has the opposite charge to that of the charged amino acid residue located on the second CH1 and the CLλ of the second light chain.

[0318] For example, the charge pair in the second antigen-binding arm may be formed from a positively charged amino acid residue in the second CH1 and a negatively charged amino acid residue in the second light chain, and the kappa charge pair in the second T cell-binding arm may be formed from a negatively charged amino acid residue in the third CH1 and a positively charged amino acid residue in the third light chain.

[0319] Alternatively, the charge pair in the second antigen-binding arm may be formed from a negatively charged amino acid residue in the second CH1 and a positively charged amino acid residue in the second light chain, and the kappa charge pair in the second T cell-binding arm may be formed from a positively charged amino acid residue in the third CH1 and a negatively charged amino acid residue in the third light chain.

[0320] As described in the examples, several methods are known that can be used to determine the correct light chain pairing. These include mass spectrometry-based approaches that can be used to establish the correct heavy / light chain association. In some embodiments, if the T cell recruitment molecule contains a mixture of kappa and lambda light chains, the ratio of kappa to lambda light chains in the assembled T cell recruitment molecule can be determined using microfluidic electrophoresis as a readout for the correct light chain ratio.

[0321] Therefore, in some embodiments, T cell recruitment molecules containing lambda charge pairs exhibit improved correct light chain pairing compared to equivalent T cell recruitment molecules lacking lambda charge pairs. In some embodiments, trivalent antibodies containing lambda charge pairs optionally exhibit correct light chain ratios of 90%, 95%, 96%, 97%, 98%, or greater than 99% (determined, for example, using microfluidic electrophoresis) after the T cell recruitment molecules have been purified using light chain affinity purification.

[0322] As described herein, T cell recruitment molecules can be selectively purified based on their light chains using techniques such as light chain affinity chromatography utilizing affinity resins specific to either CLκ or CLλ. Examples of such affinity resins include LambdaFabSelect and KappaSelect resins available from GE Healthcare. Using such methods, T cell recruitment molecules containing both CLκ and CLλ can be selectively purified and therefore can be used to improve the production of T cell recruitment molecules.

[0323] V. Manipulated disulfide In some embodiments, T cell recruitment molecules contain engineered disulfides in addition to charge pairs. "Engineered disulfide" means that a native interchain disulfide bond at the CH1-CL interface of at least one of the first antigen-binding arm, the second antigen-binding arm, the first T cell-binding arm, or the second T cell-binding arm (e.g., at 220 of CH1 and 212 of LC) is replaced by an engineered (non-native) interchain disulfide, while other binding arms or multiple arms contain native interchain disulfide bonds at the CH1-CL interface. Engineered disulfides are typically formed by manipulating cysteine ​​in the CL of the light chain and the corresponding CH1 domain of the heavy chain, replacing cysteine ​​that would normally form interchain disulfides. Disclosures relating to the introduction of engineered disulfides into antibodies for the purpose of promoting heterodimerization can be found, for example, in U.S. Patent No. 9,527,927 and Mazor, 2015, which are incorporated herein by reference in their entirety.

[0324] The formation of disulfide bonds between cysteine ​​residues occurs during the folding of many proteins entering the secretory pathway. When a polypeptide chain breaks down, adjacent cysteines can form a covalent bond during a process catalyzed by members of the protein disulfide isomerase family. As used herein, the terms “disulfide-linked” or “disulfide-coupled” refer to a single covalent bond formed from the coupling of thiol groups, particularly cysteine ​​residues. In some embodiments, the covalent bond between two cysteines is located between the two sulfur atoms of each residue. However, depending on the environment, for example, in the event of disulfide reduction, not all protein species can always have the disulfide present. Therefore, the terms “disulfide-linked” or “disulfide-coupled” (whether natural or engineered) also refer, in some embodiments, to the presence of two cysteine ​​residues capable of forming a disulfide linkage, regardless of whether they are actually bonded at individual points in time.

[0325] In some embodiments, a disulfide linkage between the light chain of at least one of the antigen and / or T cell binding arms and CH1 can be formed between the light chain of that antigen and / or T cell binding arm and the CH1-operated cysteine ​​pair. In some embodiments, a disulfide linkage between the light chain of two of the 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) and CH1 can be formed between the light chain of those two binding arms and the CH1-operated cysteine ​​pair.

[0326] In some embodiments, a disulfide linkage between the first light chain and CH1 can be formed between the first light chain and the operand cysteine ​​pair at the first CH1. In some embodiments, a disulfide linkage between the third light chain and the third CH1 can be formed between the third light chain and the operand cysteine ​​pair at the third CH1. As described above, the light chain may contain CLλ or CLκ. In some embodiments, the operand cysteine ​​pair of CLλ and CH1 is located at position 122 of CLλ and position 126 of CH1, where 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 embodiments, the non-cysteine ​​residue is valine.

[0327] In some embodiments, the pair of kappa regions (CLκ) and manipulated cysteine ​​in CH1 are located at position 121 of CLκ and position 126 of CH1, the same CLκ containing a non-cysteine ​​residue at position 214, and the same CH1 containing a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0328] In some embodiments, the disulfide linkage between the first light chain and the first CH1 can be formed between the manipulated cysteine ​​pair in the first light chain and the first CH1, the disulfide linkage between the second light chain and the second CH1 can be formed between the native cysteine ​​pair, and the disulfide linkage between the third light chain and the third CH1 can be formed either between the native cysteine ​​pair or between the cysteine ​​pair inserted into the third light chain polypeptide and the third heavy chain polypeptide, wherein the cysteine ​​pair inserted into the third light chain polypeptide and the third heavy chain polypeptide is located at a different amino acid residue position than the cysteine ​​pair inserted into the first light chain polypeptide and the first heavy chain polypeptide.

[0329] In some embodiments, a disulfide linkage between a first light chain and a first CH1 can be formed between a pair of manipulated cysteines at 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; a disulfide linkage formed between a second light chain and a second CH1 can be formed between a pair of native cysteines; and a disulfide linkage that can be formed between a third light chain and a third CH1 can be formed between a pair of manipulated cysteines at 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.

[0330] In some embodiments, the T cell mobilization molecule comprises a first antigen-binding arm having the lambda charge pair and the engineered disulfide, a second antigen-binding arm having the kappa charge pair and the native disulfide, and a first T cell-binding arm having the kappa charge pair and the engineered disulfide.

[0331] In some embodiments, the T cell mobilization molecule comprises other combinations of charge pairs and engineered disulfides. In some embodiments, the first antigen-binding arm comprises a lambda charge pair and a native disulfide, and the first and second T cell-binding arms comprise a kappa charge pair and an engineered disulfide. In some embodiments, the first T cell-binding arm comprises a kappa charge pair and an engineered disulfide, and the first and second antigen-binding arms comprise a lambda charge pair and a native disulfide. In some embodiments, the second antigen-binding arm comprises a kappa charge pair and a native disulfide, and the first and second T cell-binding arms comprise a lambda charge pair and an engineered disulfide.

[0332] Modification of the VI.Fc region In some embodiments, the first antigen-binding arm and the second antigen-binding arm further include a first Fc region and a second Fc region (i.e., further include CH2 and CH3 regions of the heavy chain).

[0333] In some embodiments, the T cell recruitment molecule includes one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote the formation of a multivalent T cell recruitment molecule by promoting the pairing of a first Fc region and a second Fc region. In some embodiments, the T cell recruitment molecule includes knob-into-hole (KiH) Fc modifications based on single amino acid substitutions in the CH3 domain that promote heavy chain heterodimerization, as described in Ridgway, 1996. The knob variant heavy chain CH3 has a smaller amino acid substituted with a larger amino acid, thereby creating a bump (knob) on the surface of the CH3 domain, while the hole variant has a larger amino acid substituted with a smaller amino acid, thereby creating a cavity (hole) on the surface of the CH3 domain. Further modifications may also be introduced to stabilize the association between heavy chains.

[0334] In some embodiments, CH3 modifications to enhance heterodimerization include, for example, a “hole” mutation Y407V / T366S / L368A on one Fc region and a “knob” mutation T366W on the other Fc region. In some embodiments, the T cell recruitment molecule further includes a stabilizing cystine mutation Y349C (e.g., on an Fc region with a “hole” mutation) and a stabilizing S354C mutation on the other Fc region (e.g., on an Fc region with a “knob” mutation).

[0335] In some embodiments, the substitution for generating a knob is the substitution of tryptophan at position 366, and the substitution for generating a hole is one or more of the following: iv) Substitution with valine at position 407, ii) Substitution with serine at position 366, and iii) Substitution with alanine at position 368.

[0336] In some embodiments, the T cell mobilization molecule includes a “knob” located on a first antigen-binding arm containing a lambda charge pair, and a “hole” on a second antigen-binding arm containing one of the kappa charge pairs. However, the reverse configuration is also specifically intended, namely, the “hole” located on the CH3 of the first antigen and / or T cell-binding arm, and the “knob” located on the CH3 of the second antigen and / or T cell-binding arm.

[0337] Other examples of CH3 modifications for enhancing heterodimerization are, for example, Table 1 of Brinkmann and Kontermann, 2017 MABS 9(2), 182-212, which are specifically incorporated herein by reference.

[0338] For example, one Fc region may include modifications to enable fractional elution by protein A chromatography, as described in Tustian, 2016. Briefly, one of the Fc regions may include modifications to remove binding to protein A (called Fc*), enabling selective purification of the heterodimer FcFc* polyvalent product. Examples of suitable modifications for generating the Fc* region include substitution of H435 with arginine and substitution of Y436 with phenylalanine.

[0339] In addition to those used to enhance heterodimerization, other Fc modifications that can be used include those that reduce or eliminate the binding of one or more Fcγ receptors and / or complement T-cell recruitment molecules, such as FcγRI, FcγRIIa, FcγRIIb, and FcγRIII. Such mutations reduce or suppress Fc effector function. Mutations for reducing or eliminating the binding of one or more Fcγ receptors and complement antibody molecules are known, including, for example, the L234F / L235E / P331S “triple mutation” or “TM” described in Organesyan, 2008.

[0340] In some embodiments, the first antigen-binding arm comprises a lambda charge pair and a first Fc region, and the disulfide linkage between the first light chain and the first CH1 can be formed between the first light chain and a pair of manipulated cysteine ​​in the first CH1, the first Fc region comprising a "knob" mutation; the second antigen-binding arm comprises a kappa charge pair and a second Fc region, and the disulfide linkage formed between the second light chain and the second CH1 is formed between a pair of native cysteine ​​in the second Fc region comprising a "hole" mutation; the T cell-binding arm comprises a kappa charge pair and a third CH1 and a third light chain The charged amino acid residues located on CLκ have the opposite charge to those located on CLκ of the second CH1 and the second light chain, and the disulfide linkage formed between the third light chain and the third CH1 can be formed between pairs of native cysteine ​​or between pairs of cysteine ​​inserted into the third light chain polypeptide and the third heavy chain polypeptide, and the pairs of cysteine ​​inserted into the third light chain polypeptide and the third heavy chain polypeptide are at different amino acid residue positions than the pairs of cysteine ​​inserted into the first light chain polypeptide and the first heavy chain polypeptide.

[0341] Non-limiting examples of multivalent T cell recruitment molecules, including lambda charge pairs, kappa charge pairs, manipulated disulfides, and modifications to promote heterodimerization of the first and second Fc regions, are provided in the examples.

[0342] In some embodiments, the T cell mobilization molecule includes amino acid modifications. In some embodiments, the modification is the substitution of an amino acid residue with any other naturally occurring or non-naturally occurring amino acid residue.

[0343] Naturally occurring residues share common side-chain properties: 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 and partially negatively charged: serine (S), threonine (T), 4) Acidic (negatively charged): Aspartic acid (D), Glutamic acid AC I (E), 5) Basic (positively charged): Histidine (H), Lysine (K), Arginine I, 6) Aromatics: They can be classified into classes based on tryptophan (W), tyrosine (Y), and phenylalanine (F).

[0344] As described above, serine (S) and threonine (T) have isoelectric points less than 6 and are partially negatively charged at neutral pH; therefore, they are classified as "polar and partially negatively charged" in this specification.

[0345] Amino acid substitutions may be conservative amino acid substitutions. Conservative amino acid substitutions may involve the exchange of one member of these classes with another member of the same class. For example, a conservative amino acid substitution may involve using the acidic amino acid glutamic acid (E) instead of the acidic amino acid aspartic acid (D).

[0346] In one embodiment, the antibody or its antigen-binding fragment does not have one or more effector functions. For example, in one embodiment, the antibody or its antigen-binding fragment does not have antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In one embodiment, the antibody or its antigen-binding fragment does not bind to Fc receptors and / or complement factors. In one embodiment, the antibody or its antigen-binding fragment does not have effector functions. In one embodiment, the antibody or its antigen-binding fragment contains an Fc region with a triple mutation (TM) that reduces antibody-dependent cytotoxicity (ADCC) compared to an antibody with a wild-type Fc region. In some embodiments, the antibody or its antigen-binding fragment has an Fc region with the L234F / L235E / P331S triple mutation (TM). In one embodiment, the antibody or its antigen-binding fragment does not have effector functions or not. In one embodiment, the antibody or its antigen-binding fragment has an Fc region containing E233P / L234V / L235A / G236del / S267K. In one embodiment, E233P / L234V / L235A / G236del / S267K is referred to as "null" or "Fc null".

[0347] VII.T cell recruitment molecules This disclosure provides T cell mobilization molecules. The T cell mobilization molecules described herein are capable of binding to the same antigen, or in some embodiments, to any two different epitopes on different antigens. In some embodiments, the T cell mobilization molecule comprises at least one antigen-binding arm and at least one T cell-binding arm. According to this disclosure, the “antigen-binding arm” comprises a heavy chain comprising a VH domain and a CH1 domain, and a light chain comprising a VL domain and a light chain constant domain, wherein the light chain constant domain is disulfide-linked to the CH1 domain, and the antigen-binding arm binds to the antigen of interest. In some embodiments, the “T cell-binding arm” comprises a heavy chain comprising a VH domain and a CH1 domain, and a light chain comprising a VL domain and a light chain constant domain, wherein the light chain constant domain is disulfide-linked to the CH1 domain, and the T cell-binding arm binds to a T cell antigen.

[0348] In some embodiments, the antigen-binding arm and / or T-cell-binding arm in the T-cell recruitment molecule further comprises an Fc region. In some embodiments, the antigen-binding arm and / or T-cell-binding arm in the T-cell recruitment molecule comprises a complete heavy chain (i.e., a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain).

[0349] In some embodiments, the T cell recruitment molecule includes a T cell recruitment arm comprising (i) a first antigen-binding arm comprising a heavy chain comprising a heavy chain constant region comprising VH and CH1, CH2, and CH3 domains, and a light chain comprising VL and a light chain constant domain, and (ii) a T cell-binding arm comprising a heavy chain comprising a heavy chain comprising a heavy chain constant region comprising VH and CH1, CH2, and CH3 domains, and a light chain comprising VL and a light chain constant domain, 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 domain. An exemplary T cell recruitment molecule is shown in Figure 1A.

[0350] In some embodiments, the T cell recruitment molecule comprises: (i) a first antigen-binding arm comprising a heavy chain comprising a heavy chain constant region comprising VH and CH1, CH2, and CH3 domains, and a light chain comprising VL and a light chain constant domain; (ii) a T cell binding arm comprising a heavy chain comprising a heavy chain comprising a heavy chain constant region comprising VH and CH1, CH2, and CH3 domains, and a light chain comprising VL and a light chain constant domain; and (iii) a second antigen-binding arm comprising a heavy chain comprising a heavy chain comprising VH and CH1 domains, and a light chain comprising VL and a light chain constant domain, wherein the heavy chain of the second antigen-binding arm is attached to the heavy chain of the T cell binding arm via a peptide linker. Exemplary T cell recruitment molecules are shown in Figures 1A to 1D.

[0351] In some embodiments, the light chain of the antigen-binding arm is disulfide-linked to the heavy chain of the antigen-binding arm in the T cell recruitment molecule via a naturally occurring interchain disulfide bond (e.g., a disulfide bond present in an IgG antibody). In some embodiments, the light chain of the antigen-binding arm is disulfide-linked to the heavy chain of the antigen-binding arm in the T cell recruitment molecule via an engineered disulfide bond. In some embodiments, the light chain of the T cell-binding arm is disulfide-linked to the heavy chain of the T cell-binding arm in the T cell recruitment molecule via a naturally occurring interchain disulfide bond. In some embodiments, the light chain of the T cell-binding arm is disulfide-linked to the heavy chain of the T cell-binding arm in the T cell recruitment molecule via an engineered disulfide bond.

[0352] In some embodiments, 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 embodiments, the heavy chain of an antigen-binding arm is attached to a T cell-binding arm via a peptide linker. In some embodiments, 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 embodiments, the peptide linker is formed mainly from glycine and serine amino acid residues, and in some embodiments, it contains the amino acid sequence GGGGS or SGGGGS. In some embodiments, the peptide linker contains or consists of SEQ ID NO: 89. In some embodiments, the linker contains 1 to 10 copies of SEQ ID NO: 89. In some embodiments, the linker contains 2 copies of SEQ ID NO: 89.

[0353] In some embodiments, the T cell mobilization molecule comprises: (a) at least one antigen-binding arm that binds to STEAP2 and comprises a heavy chain comprising a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), and a light chain comprising a light chain variable domain (VL) and a light chain constant domain (CL); (b) a T cell-binding arm comprising a heavy chain comprising a VH domain and a CH1 domain, and a light chain comprising a VL domain and a light chain constant domain; and (c) an Fc domain comprising a first Fc region and a second Fc region, wherein each Fc region comprises a CH2 domain and a CH3 domain, and the Fc domain further comprises at least one modification to promote heterodimerization.

[0354] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm that binds to STEAP2 and comprises a VH domain containing 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, VH-CDR3 selected from SEQ ID NOs: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137, and 145, as well as a heavy chain containing a CH1 domain. In some embodiments, the antigen-binding arm of the T cell recruitment molecule further comprises a VL domain including 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, VL-CDR3 selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148, and a light chain including a light chain constant domain.

[0355] In some embodiments, the T cell mobilization molecule further comprises a T cell binding arm comprising a heavy chain comprising a VH domain that binds to CD3 and includes 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, as well as a CH1 domain. In some embodiments, the T cell binding arm further comprises a VL comprising 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, as well as a light chain comprising a light chain constant domain. In some embodiments, the T cell mobilization molecule further comprises 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.

[0356] In some embodiments, the T cell mobilization molecule further comprises a second antigen-binding arm that binds to STEAP2 and includes a VH domain comprising 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, VH-CDR3 selected from SEQ ID NOs: 3, 11, 19, 94, 96, 98, 105, 113, 129, 137, and 145, as well as a heavy chain comprising a CH1 domain. In some embodiments, the second antigen-binding arm of the T cell recruitment molecule further comprises a VL including 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, VL-CDR3 selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148, and a light chain including a light chain constant domain.

[0357] In some embodiments, one CH3 domain of the Fc region of the T cell mobilization molecule contains a knob mutation, and the other CH3 domain contains a hole mutation.

[0358] In some embodiments, one or more CH1 domains and light chain constant domains of the antigen-binding arm and T-cell-binding arm of the T-cell mobilization molecule further include charge-pair substitutions comprising a first charged amino acid substitution in CH1 and a second charged amino acid substitution in the light chain constant domain, wherein the first and second charged amino acid substitutions have opposite charges.

[0359] In some embodiments, one or more light chain constant domains of the antigen-binding arm and the T cell-binding arm are lambda light chain constant domains (CLλ), and the charge pair is a lambda 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. In some embodiments, the charged amino acids of the lambda charge pair are located at the following positions: (i) Position 117 in CLλ and position 141 in the CH1 domain, (ii) Position 117 in CLλ and position 185 in the CH1 domain, (iii) Position 119 in CLλ and position 128 in the CH1 domain, (iv) Position 134 in CLλ and position 128 in the CH1 domain, (v) Position 134 in CLλ and position 145 in the CH1 domain, (vi) Position 134 in CLλ and position 183 in the CH1 domain, (vii) Position 136 in CLλ and position 185 in the CH1 domain, (viii) Position 178 in CLλ and position 173 in the CH1 domain, and / or (ix) Located at one or more of the following positions: position 117 in CLλ and position 187 in the CH1 domain, with numbering following the EU index.

[0360] In some embodiments, one or more light chain constant domains in each of the antigen-binding arm and the T cell-binding arm are lambda light chain constant domains (CLλ), and the charge pair is a lambda charge pair. (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.

[0361] In some embodiments, one or more light chain constant domains of the antigen-binding arm and the T-cell-binding arm of the T-mobilization molecule are kappa light chain constant domains (CLκ), and the charge pair is a kappa 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, and the kappa charge pair is located at position 133 in CLκ and position 183 in CH1.

[0362] In some embodiments, one or more light chain constant domains of the antigen-binding arm and the T-cell-binding arm of the T-mobilization molecule are kappa light chain constant domains (CLκ), the charge pair is a kappa charge pair, the charged amino acid at position 133 is glutamic acid 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 glutamic acid.

[0363] In some embodiments, the light chain constant domain of the T cell binding arm is CLλ, the charge pair is a lambda charge pair as described herein, the light chain constant domain of the first antigen-binding domain and / or the second antigen-binding domain is a lambda or kappa charge pair, and the first antigen-binding arm contains a charged amino acid at CH1 which has the same charge as the charged amino acid at the CH1 domain of the second antigen-binding arm.

[0364] In some embodiments, the light chain constant domain of the T cell binding arm is CLλ, the charge pair is a lambda charge pair as described herein, the light chain constant domains of the first and second antigen-binding arms are kappa charge pairs, and the charged amino acid in the CH1 domain of the first antigen-binding arm has the same charge as the charged amino acid in the CH1 domain of the second antigen-binding arm.

[0365] In some embodiments, the CH1 domain of the antigen-binding arm or T cell-binding arm can be linked to the light chain constant domain via an engineered disulfide linkage.

[0366] In some embodiments, the CH1 domain, which can be linked to the light chain constant domain via an manipulated disulfide linkage, comprises (i) substitution of a natural cysteine ​​with a non-cysteine ​​amino acid, and (ii) substitution of a natural non-cysteine ​​amino acid with cysteine, and the light chain constant domain comprises (i) substitution of a natural cysteine ​​with a non-cysteine ​​amino acid, and (ii) substitution of a natural non-cysteine ​​amino acid with cysteine, and the substituted cysteine ​​of the light chain constant domain and the substituted cysteine ​​of the CH1 domain can form a disulfide bond.

[0367] In some embodiments, the CH1 domain, which can be linked to the light chain constant domain via an manipulated disulfide linkage, comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 220, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, and the light chain constant domain comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 212, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 122, and 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, and the numbering follows the EU index.

[0368] In some embodiments, the CH1 domain, which can be linked to the light chain constant domain via an manipulated disulfide linkage, comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 220, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, and the light chain constant domain comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 214, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 121, and 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, and the numbering follows the EU index.

[0369] In some embodiments, the antigen-binding arm of the T cell recruitment molecule includes a VH 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 to an amino acid sequence selected from SEQ ID NOs. 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0370] In some embodiments, the antigen-binding arm of the T cell recruitment molecule includes a heavy 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 respect to the amino acid sequence described in SEQ ID NO: 90.

[0371] In some embodiments, the antigen-binding arm of the T cell mobilization molecule comprises a heavy chain comprising 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 to an amino acid sequence selected from SEQ ID NOs. 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149, and a CH1 domain containing an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 96%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence described in SEQ ID NO. 90.

[0372] In some embodiments, the antigen-binding arm of the T cell recruitment 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 to an amino acid sequence selected from SEQ ID NOs: 8, 16, 24, 107, 115, 134, 142, and 150.

[0373] In some embodiments, the antigen-binding arm of the T cell recruitment molecule includes a 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 respect to the amino acid sequence described in SEQ ID NO: 63.

[0374] In some embodiments, the antigen-binding arm of the T cell mobilization molecule comprises a light chain comprising 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 to an amino acid sequence selected from SEQ ID NOs. 8, 16, 24, 107, 115, 134, 142, and 150, and 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 to the amino acid sequence described in SEQ ID NO. 63.

[0375] In some embodiments, the antigen-binding arm of the T cell recruitment molecule includes a VH 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 to an amino acid sequence selected from SEQ ID NOs. 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0376] In some embodiments, the antigen-binding arm of the T cell recruitment molecule includes a heavy 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 respect to the amino acid sequence described in SEQ ID NO: 88.

[0377] In some embodiments, the antigen-binding arm of the T cell mobilization molecule comprises a heavy chain comprising 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 to an amino acid sequence selected from SEQ ID NOs. 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149, and a CH1 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 to the amino acid sequence described in SEQ ID NO. 88.

[0378] In some embodiments, the T cell-binding arm of the T cell mobilization molecule includes 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. 39, 43, and 47.

[0379] In some embodiments, the first T cell binding arm of the T cell mobilization molecule includes a heavy 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 respect to the amino acid sequence described in SEQ ID NO: 79.

[0380] In some embodiments, the T cell binding arm of the T cell mobilization molecule includes a heavy chain comprising 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 to an amino acid sequence selected from SEQ ID NOs. 39, 43, and 47, and 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 to the amino acid sequence described in SEQ ID NO. 79.

[0381] In some embodiments, the T cell-binding arm of the T cell mobilization molecule includes a VL 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. 30 and 34.

[0382] In some embodiments, the T cell-binding arm of the T cell mobilization molecule includes a 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 respect to the amino acid sequence described in SEQ ID NO: 61.

[0383] In some embodiments, the T cell binding arm of the T cell mobilization molecule includes a light chain comprising 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 NOs. 30 and 34, and 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 described in SEQ ID NO. 61.

[0384] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 25. In some embodiments, the T cell mobilization molecule comprises a light chain comprising the amino acid sequence described in SEQ ID NO: 25.

[0385] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 116.

[0386] In some embodiments, the T cell mobilization molecule includes 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 respect to the amino acid sequence described in SEQ ID NO: 117.

[0387] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 118.

[0388] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 119.

[0389] In some embodiments, the T cell mobilization molecule includes 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 described in SEQ ID NO: 35. In some embodiments, the T cell mobilization molecule includes 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 described in SEQ ID NO: 81. In some embodiments, the T cell mobilization molecule includes 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 described in SEQ ID NO: 88.

[0390] In some embodiments, the T cell mobilization molecule includes the amino acid sequence described in SEQ ID NO: 25. In some embodiments, the T cell mobilization molecule includes the amino acid sequence described in SEQ ID NO: 35. In some embodiments, the T cell mobilization molecule includes the amino acid sequence described in SEQ ID NO: 81. In some embodiments, the T cell mobilization molecule includes the amino acid sequence described in SEQ ID NO: 88.

[0391] In some embodiments, the T cell recruitment molecule comprises the amino acid sequences described in SEQ ID NOs. 25, 26, 35, 81, and 92.

[0392] VIII. Trivalent trispecific T cell recruitment molecule This disclosure provides a trivalent, triplicate T cell recruiting molecule. The trivalent T cell recruiting molecules described herein are capable of binding to any three different epitopes on the same antigen, or, in some embodiments, on different antigens. In some embodiments, the trivalent T cell binding molecule comprises at least one antigen-binding arm and at least two different T cell binding arms. In some embodiments, the antigen-binding arm is referred herein as the “first antigen-binding arm,” and the T cell binding arms are referred herein as the “first T cell binding arm” and the “second T cell binding arm.” According to this disclosure, the “antigen-binding arm” comprises a heavy chain comprising a VH domain and a CH1 domain, and a light chain comprising a VL domain and a light chain constant domain, wherein the light chain constant domain is disulfide-linked to the CH1 domain. In some embodiments, the “T cell binding arm” comprises a heavy chain comprising a VH domain and a CH1 domain, and a light chain comprising a VL domain and a light chain constant domain, wherein the light chain constant domain is disulfide-linked to the CH1 domain. In some embodiments, the T cell-binding arm contains only the VH domain.

[0393] In some embodiments, the antigen-binding arm and / or at least one T cell-binding arm in the trivalent T cell recruitment molecule further comprises an Fc region. In some embodiments, the antigen-binding arm and / or T cell-binding arm in the trivalent T cell recruitment molecule comprises a complete heavy chain (i.e., a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain).

[0394] In some embodiments, the trivalent T cell recruitment molecule comprises: (i) an antigen-binding arm comprising a heavy chain comprising a heavy chain constant region comprising VH and CH1, CH2, and CH3 domains, and a light chain comprising VL and a light chain constant domain; (ii) a first T cell binding arm comprising a heavy chain comprising a heavy chain constant region comprising VH and CH1, CH2, and CH3 domains, and a light chain comprising VL and a light chain constant domain; and (iii) a second T cell binding arm comprising a heavy chain comprising VH and CH1 domains, and a light chain comprising VL and a light chain constant domain, 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 recruitment molecule is shown in Figure 1C.

[0395] In some embodiments, the light chain of the antigen-binding arm is disulfide-linked to the heavy chain of the antigen-binding arm in the trivalent T cell mobilization molecule via a naturally occurring interchain disulfide bond (e.g., a disulfide bond present in an IgG antibody). In some embodiments, the light chain of the antigen-binding arm is disulfide-linked to the heavy chain of the antigen-binding arm in the trivalent T cell mobilization molecule via an engineered disulfide bond. In some embodiments, the light chain of the first T cell-binding arm and / or the second T cell-binding arm is disulfide-linked to the heavy chain of the first T cell-binding arm and / or the second T cell-binding arm in the trivalent T cell mobilization molecule via a naturally occurring interchain disulfide. In some embodiments, the light chain of the first T cell-binding arm and / or the second T cell-binding arm is disulfide-linked to the heavy chain of the first T cell-binding arm and / or the second T cell-binding arm in the T cell mobilization molecule via an engineered disulfide bond.

[0396] In some embodiments, 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 embodiments, the heavy chain of an antigen-binding arm is attached to a T cell-binding arm via a peptide linker. In some embodiments, 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 embodiments, the peptide linker is formed mainly from glycine and serine amino acid residues, and in some embodiments, it contains the amino acid sequence GGGGS or SGGGGS. In some embodiments, the peptide linker contains or consists of SEQ ID NO: 89. In some embodiments, the linker contains 1 to 10 copies of SEQ ID NO: 89. In some embodiments, the linker contains 2 copies of SEQ ID NO: 89.

[0397] In some embodiments, the trivalent T cell mobilization molecule comprises: (a) at least one antigen-binding arm that binds to STEAP2 and comprises a heavy chain comprising a VH domain and a CH1 domain, and a light chain comprising a VL domain and a light chain constant domain; (b) at least one T cell-binding arm comprising a heavy chain comprising a VH domain and a CH1 domain, and a light chain comprising 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 further comprising at least one modification to promote heterodimerization.

[0398] In some embodiments, the trivalent T cell mobilization molecule includes (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.

[0399] In some embodiments, one CH3 domain of the Fc region of the trivalent T cell mobilization molecule contains a knob mutation, and the other CH3 domain contains a whole mutation.

[0400] In some embodiments, one or more CH1 domains and light chain constant domains of the antigen-binding arm and T-cell-binding arm of the trivalent T-cell mobilization molecule further include charge-pair substitutions comprising a first charged amino acid substitution in CH1 and a second charged amino acid substitution in the light chain constant domain, wherein the first and second charged amino acid substitutions have opposite charges.

[0401] In some embodiments, one or more light chain constant domains of the antigen-binding arm and T-cell-binding arm of the trivalent T-mobilization molecule are lambda light chain constant domains (CLλ), and the charge pair is a lambda 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. In some embodiments, the charged amino acids of the lambda charge pair are located at the following positions: (i) Position 117 in CLλ and position 141 in the CH1 domain, (ii) Position 117 in CLλ and position 185 in the CH1 domain, (iii) Position 119 in CLλ and position 128 in the CH1 domain, (iv) Position 134 in CLλ and position 128 in the CH1 domain, (v) Position 134 in CLλ and position 145 in the CH1 domain, (vi) Position 134 in CLλ and position 183 in the CH1 domain, (vii) Position 136 in CLλ and position 185 in the CH1 domain, (viii) Position 178 in CLλ and position 173 in the CH1 domain, and / or (ix) Located at one or more of the following positions: position 117 in CLλ and position 187 in the CH1 domain, with numbering following the EU index.

[0402] In some embodiments, one or more light chain constant domains of the antigen-binding arm and T cell-binding arm of the trivalent T-cell mobilization molecule are lambda light chain constant domains (CLλ), and the charge pair is a lambda charge pair. (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.

[0403] In some embodiments, one or more light chain constant domains of the antigen-binding arm and the T cell-binding arm of the trivalent T-mobilization molecule are kappa light chain constant domains (CLκ), and the charge pair is a kappa 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, and the kappa charge pair is located at position 133 in CLκ and position 183 in CH1.

[0404] In some embodiments, one or more light chain constant domains of the antigen-binding arm and the T cell-binding arm of the trivalent T-mobilization molecule are kappa light chain constant domains (CLκ), the charge pair is a kappa charge pair, the charged amino acid at position 133 is glutamic acid 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 glutamic acid.

[0405] In some embodiments, the light chain constant domain of the T cell binding arm of the trivalent T-mobilization molecule is CLλ, the charge pair is a lambda charge pair as described herein, the light chain constant domains of the antigen-binding arm and the second T cell binding arm of the trivalent T-mobilization molecule are lambda or kappa charge pairs, and the antigen-binding arm contains a charged amino acid in the CH1 domain which has the same charge as the charged amino acid in the CH1 domain of the second T cell binding arm.

[0406] In some embodiments, the light chain constant domain of the T cell binding arm is CLλ, the charge pair is a lambda charge pair as described herein, the antigen binding domain and the light chain constant domain of the second T cell binding arm are kappa charge pairs, 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.

[0407] In some embodiments, the CH1 domain of the antigen-binding arm or T-cell-binding arm of a trivalent T-cell mobilization molecule can be linked to the light chain constant domain via an engineered disulfide linkage.

[0408] In some embodiments, the CH1 domain, which can be linked to the light chain constant domain via an manipulated disulfide linkage, comprises (i) substitution of a natural cysteine ​​with a non-cysteine ​​amino acid, and (ii) substitution of a natural non-cysteine ​​amino acid with cysteine, and the light chain constant domain comprises (i) substitution of a natural cysteine ​​with a non-cysteine ​​amino acid, and (ii) substitution of a natural non-cysteine ​​amino acid with cysteine, and the substituted cysteine ​​of the light chain constant domain and the substituted cysteine ​​of the CH1 domain can form a disulfide bond.

[0409] In some embodiments, the CH1 domain, which can be linked to the light chain constant domain via an manipulated disulfide linkage, comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 220, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, and the light chain constant domain comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 212, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 122, and 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, and the numbering follows the EU index.

[0410] In some embodiments, the CH1 domain, which can be linked to the light chain constant domain via an manipulated disulfide linkage, comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 220, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, and the light chain constant domain comprises (i) substitution of a native cysteine ​​with a non-cysteine ​​amino acid at position 214, and (ii) substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 121, and 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, and the numbering follows the EU index.

[0411] In some embodiments, the trivalent T cell mobilization molecule includes an antibody-binding arm that binds to STEAP2 and comprises a VH domain including 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, VH-CDR3 selected from SEQ ID NOs: 3, 11, 19, 94, 96, 98, 105, and 113, and a heavy chain including a CH1 domain. In some embodiments, the antigen-binding arm of the trivalent T cell mobilization molecule further comprises a VL including 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, VL-CDR3 selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148, and a light chain including a light chain constant domain.

[0412] In some embodiments, the trivalent T cell mobilization molecule includes an antigen-binding arm comprising a heavy chain containing a VH domain that binds to STEAP2 and has sequence identity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with respect to an amino acid sequence selected from SEQ ID NOs: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

[0413] In some embodiments, the T cell mobilization molecule includes an antigen-binding arm that binds to STEAP2 and comprises 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 respect to the amino acid sequence described in SEQ ID NO: 91. In some embodiments, the trivalent T cell mobilization molecule includes an antigen-binding arm that binds to STEAP2 and comprises a heavy chain having at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the amino acid sequence described in SEQ ID NO: 91.

[0414] In some embodiments, the trivalent T cell mobilization molecule includes an antigen-binding arm comprising: a heavy chain containing a VH domain that binds to STEAP2 and has at least about 80%, at least about 85%, at least about 90%, 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; and a heavy chain containing an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, 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 described in SEQ ID NO. 91. In some embodiments, the trivalent T cell mobilization molecule includes an antigen-binding arm comprising a heavy chain containing a VH domain that binds to STEAP2 and contains an amino acid sequence selected from SEQ ID NOs: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149, and a heavy chain containing the amino acid sequence described in SEQ ID NO: 91.

[0415] In some embodiments, the trivalent T cell mobilization molecule further comprises at least one T cell binding arm comprising a heavy chain comprising a VH domain that binds to CD3 and includes 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, as well as a CH1 domain. In some embodiments, the T cell binding arm further comprises a light chain comprising a VL including 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, as well as a light chain comprising a constant light chain domain. In some embodiments, the trivalent T cell mobilization molecule further comprises 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.

[0416] In some embodiments, at least one T cell binding arm that binds to CD3 includes 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: 39, 43, and 47.

[0417] In some embodiments, at least one T cell binding arm that binds to CD3 includes a 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 respect to the amino acid sequence described in SEQ ID NO: 79.

[0418] In some embodiments, the trivalent T cell mobilization molecule includes a T cell binding arm comprising a VH domain that binds to CD3 and 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 to an amino acid sequence selected from SEQ ID NOs. 39, 43, and 47, and a heavy chain that 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 to the amino acid sequence described in SEQ ID NOs. In some embodiments, the trivalent T cell mobilization molecule includes a T cell binding arm comprising a VH domain that binds to CD3 and contains an amino acid sequence selected from SEQ ID NOs. 39, 43, and 47, and a heavy chain that contains the amino acid sequence described in SEQ ID NOs. 79.

[0419] In some embodiments, the trivalent T cell mobilization molecule further comprises at least one T cell binding arm comprising a heavy chain comprising a VH domain that binds to CD8 and includes VH-CDR1 selected from SEQ ID NOs. 48 and 84, VH-CDR2 selected from SEQ ID NOs. 49 and 85, and VH-CDR3 selected from SEQ ID NOs. 50 and 86, as well as a CH1 domain. In some embodiments, the T cell binding arm further comprises a VL comprising VL-CDR1 as described in SEQ ID NOs. 51, VL-CDR2 as described in SEQ ID NOs. 52, and VL-CDR3 as described in SEQ ID NOs. 53, as well as a light chain comprising a light chain constant domain. In some embodiments, the trivalent T cell mobilization molecule further comprises 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.

[0420] In some embodiments, at least one T cell binding arm that binds to CD8 includes 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 respect to the amino acid sequence described in SEQ ID NO: 56.

[0421] In some embodiments, at least one T cell binding arm that binds to CD8 includes a CH1 domain having 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 respect to the amino acid sequence described in SEQ ID NO: 57.

[0422] In some embodiments, the trivalent T cell mobilization molecule includes a T cell binding arm comprising a VH domain that binds to CD8 and 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 to the amino acid sequence described in SEQ ID NO: 56, and a CH1 domain that 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 to the amino acid sequence described in SEQ ID NO: 57. In some embodiments, the trivalent T cell mobilization molecule includes a T cell binding arm comprising a VH domain that binds to CD8 and contains the amino acids described in SEQ ID NO: 56, and a CH1 domain that contains the amino acid sequence described in SEQ ID NO: 57.

[0423] In some embodiments, at least one T cell binding arm that binds to CD8 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 respect to the amino acid sequence described in SEQ ID NO: 54.

[0424] In some embodiments, at least one T cell binding arm that binds to CD8 includes a 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 respect to the amino acid sequence described in SEQ ID NO: 64.

[0425] In some embodiments, the trivalent T cell mobilization molecule includes a T cell binding arm comprising a VL domain that binds to CD8 and 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 to the amino acid sequence described in SEQ ID NO: 54, and a light chain constant domain that 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 to the amino acid sequence described in SEQ ID NO: 64. In some embodiments, the trivalent T cell mobilization molecule includes a T cell binding arm comprising a VL domain that binds to CD8 and contains the amino acids described in SEQ ID NO: 54, and a light chain constant domain that contains the amino acid sequence described in SEQ ID NO: 64.

[0426] In some embodiments, the trivalent T cell mobilization 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 described in SEQ ID NO: 25.

[0427] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 116.

[0428] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 117.

[0429] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 118.

[0430] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 119.

[0431] In some embodiments, the trivalent T cell mobilization 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 described in SEQ ID NO: 26.

[0432] In some embodiments, the T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO...

Claims

1. (a) A variable heavy chain domain (VH) that binds to an epitope on six-transmembrane epithelial antigen 2 (STEAP2) of human prostate and includes a variable heavy chain complementarity determining region 1 (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. An antigen-binding arm comprising a heavy chain containing a heavy chain CH1 domain, a light chain containing a variable light chain complementarity determining region 1 (VL-CDR1) selected from SEQ ID NOs: 4, 12, 20, 100, 108, 130, 138, and 146, a VL-CDR2 selected from SEQ ID NOs: 5, 13, 21, 101, 109, 131, 139, and 147, a VL-CDR3 selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148, and a light chain containing a light chain constant domain, (b) A first T cell binding arm that binds to differentiation antigen group 3 (CD3), comprising a heavy chain comprising a heavy chain variable domain (VH) including VH-CDR1 selected from SEQ ID NOs: 36, 40, and 44, VH-CDR2 selected from SEQ ID NOs: 37, 41, and 45, VH-CDR3 selected from SEQ ID NOs: 38, 42, and 46, and a heavy chain comprising a heavy chain CH1 domain, and a light chain comprising a light chain variable domain (VL) including VL-CDR1 selected from SEQ ID NOs: 27 and 31, VL-CDR2 selected from SEQ ID NOs: 28 and 32, VL-CDR3 selected from SEQ ID NOs: 29 and 33, and a light chain comprising a light chain constant domain, (c) A T cell mobilization molecule comprising an Fc domain including a first Fc region and a second Fc region, wherein each Fc region includes a CH2 domain and a CH3 domain, and the Fc domain further includes at least one modification for promoting heterodimerization.

2. (d) A trivalent T cell mobilization molecule comprising the T cell mobilization molecule according to claim 1, further comprising a second T cell binding arm comprising a heavy chain comprising a heavy chain variable domain (VH) containing VH-CDR1 described in SEQ ID NO: 48, VH-CDR2 described in SEQ ID NO: 49, and VH-CDR3 described in SEQ ID NO: 50, and a heavy chain comprising a heavy chain CH1 domain, and a light chain comprising a light chain variable domain (VL) containing VL-CDR1 described in SEQ ID NO: 51, VL-CDR2 described in SEQ ID NO: 52, and VL-CDR3 described in SEQ ID NO: 53, and a light chain comprising a light chain constant domain.

3. The trivalent T cell mobilization molecule according to claim 2, 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 linker.

4. The trivalent T cell mobilization molecule according to claim 2, wherein the heavy chain of the second T cell binding arm is attached to the heavy chain of the antigen binding arm via a linker.

5. The trivalent T cell mobilization molecule according to claim 3 or 4, wherein the linker comprises the amino acid sequence of SEQ ID NO:

89.

6. The trivalent T cell mobilization molecule according to any one of claims 3 to 5, wherein the linker comprises one to about ten copies of Sequence ID No.

89.

7. The trivalent T cell mobilization molecule according to any one of claims 3 to 6, wherein the linker comprises two copies of Sequence ID No.

89.

8. A T cell mobilization molecule or a trivalent T cell mobilization molecule according to any one of claims 1 to 7, wherein one of the two CH3 domains contains a knob mutation, and the other of the two CH3 domains contains a hole mutation.

9. A T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 8, wherein one or more of the CH1 domains and light chain constant domains of each of (a) the antigen-binding arm, (b) the first T cell-binding arm, and (d) the second T cell-binding arm further comprises a charge pair substitution including 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.

10. Each of (a), (b), and (d) is a lambda light chain constant domain (CLλ), wherein the charge pair is a lambda 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, the numbering follows the EU index, and the charged amino acid of the lambda charge pair is in the following positions: (i) Position 117 in CLλ and position 141 in CH1 domain, (ii) Position 117 in CLλ and position 185 in the CH1 domain, (iii) Position 119 in CLλ and position 128 in the CH1 domain, (iv) Position 134 in CLλ and position 128 in the CH1 domain, (v) Position 134 in CLλ and position 145 in the CH1 domain, (vi) Position 134 in CLλ and position 183 in the CH1 domain, (vii) Position 136 in CLλ and position 185 in the CH1 domain, (viiii) Position 178 in CLλ and position 173 in the CH1 domain, and / or (ix) A T cell mobilization molecule or trivalent T cell mobilization molecule according to claim 9, located at one or more of the positions 117 in the CLλ and 187 in the CH1 domain.

11. (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, (viiii) 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, the charged amino acid at position 187 is aspartic acid, the charged amino acid at position 117 is arginine, the charged amino acid at position 187 is glutamic acid, the charged amino acid at position 117 is arginine, the charged amino acid at position 187 is serine, the charged amino acid at position 117 is arginine, the charged amino acid at position 187 is threonine, the charged The T cell mobilization molecule or trivalent T cell mobilization molecule according to claim 10, wherein the amino acid 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.

12. The T cell mobilization molecule or trivalent T cell mobilization molecule according to claim 9, wherein one or more of the light chain constant domains in (a), (b), and (d) are kappa light chain constant domains (CLκ), the charge pair is a kappa 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, the numbering follows the EU index, and the kappa charge pair is located at position 133 in the CLκ and at position 183 in the CH1 domain.

13. The T cell mobilization molecule or trivalent T cell mobilization molecule according to claim 12, wherein the charged amino acid at position 133 is glutamic acid 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 glutamic acid.

14. A T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 13, wherein one of the light chain constant domains of (a), (b), and (d) is CLλ and the charge pair is a lambda charge pair, the second and third light chain constant domains of (a), (b), and (d) are CLλ or CLκ and the charge pair is a lambda or kappa charge pair, and the second of (a), (b), and (d) comprises a charged amino acid in the CH1 domain having the same charge as the charged amino acid in the third CH1 domain of (a), (b), and (d).

15. A T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 14, wherein one of the light chain constant domains of (a), (b), and (d) is CLλ, the charge pair is a lambda charge pair, the second and third light chain constant domains of (a), (b), and (d) are kappa charge pairs, and the second of (a), (b), and (d) contains a charged amino acid in the CH1 domain having the same charge as the charged amino acid in the third CH1 domain of (a), (b), and (d).

16. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 15, wherein the CH1 domain of (a), (b), and / or (d) can be linked to the light chain constant domain via an manipulated disulfide linkage.

17. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 16, wherein the CH1 domains of (b) and (d) can be linked to the light chain constant domain via manipulated disulfide linkages, and the CH1 domain of (a) is linked to the light chain constant domain via innate disulfide linkages.

18. The CH1 domain, which is capable of binding to the light chain constant domain via an manipulated disulfide linkage, (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of natural non-cysteine ​​amino acids with cysteine, The light chain constant domain, (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of natural non-cysteine ​​amino acids with cysteine, The T cell mobilization molecule or trivalent T cell mobilization molecule according to claim 16 or 17, wherein the substituted cysteine ​​of the light chain constant domain and the substituted cysteine ​​of the CH1 domain can form a disulfide bond.

19. The CH1 domain, which is capable of binding to the light chain constant domain via an manipulated disulfide linkage, (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 220, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, The light chain constant domain, (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 212, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 122, The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 16 to 18, 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, and the numbering follows the EU index.

20. The CH1 domain, which is capable of binding to the light chain constant domain via an manipulated disulfide linkage, (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 220, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, The light chain constant domain, (i) Substitution of the native cysteine ​​with a non-cysteine ​​amino acid at position 214, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 121, The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 16 to 18, 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, and the numbering follows the EU index.

21. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 20, wherein the antigen-binding arm comprises a VH having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to an amino acid sequence selected from SEQ ID NOs: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

22. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 21, wherein the antigen-binding arm is attached to the Fc domain and comprises 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. 66, 67, 69, 72, 73, 76, 80, 82, 88, and 91.

23. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 22, wherein the antigen-binding arm comprises 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 NOs: 8, 16, 24, 107, 115, 134, 142, and 150.

24. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 23, wherein the antigen-binding arm comprises 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 respect to the amino acid sequence described in SEQ ID NO:

63.

25. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 24, wherein the VL of the T cell binding arm of the T cell mobilization molecule or the VL of the first T cell binding arm or second T cell binding arm of the trivalent T cell mobilization molecule 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, and the VH 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.

26. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 25, wherein the VL of the T cell binding arm of the T cell mobilization molecule or the VL of the first T cell binding arm or second T cell binding arm of the trivalent T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 30 or 34.

27. A T cell mobilization molecule or a trivalent T cell mobilization molecule according to any one of claims 1 to 26, wherein the light chain constant domain of the T cell mobilization molecule or the light chain constant domain of the first T cell binding arm or the second T cell binding arm of the trivalent T cell mobilization 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 described in SEQ ID NO: 60 or 61.

28. A T cell mobilization molecule or a trivalent T cell mobilization molecule according to any one of claims 1 to 27, wherein the VH of the T cell binding arm of the T cell mobilization molecule, or the VH of the first T cell binding arm or the second T cell binding arm of the trivalent T cell mobilization 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 respect to the amino acid sequence described in SEQ ID NO: 39, 43, or 47.

29. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 28, wherein the heavy chain of the T cell binding arm of the T cell mobilization molecule or the first T cell binding arm or second T cell binding arm of the trivalent T cell mobilization molecule is attached to the Fc domain and includes a heavy chain constant region 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. 66, 67, 69, 74, 75, 77, 78, and 79.

30. The trivalent T cell mobilization molecule according to any one of claims 1 to 29, wherein the VH of the first T cell binding arm or the second T cell binding arm includes VH-CDR1 described in SEQ ID NO: 48, VH-CDR2 described in SEQ ID NO: 49, and VH-CDR3 described in SEQ ID NO: 50, and the VL includes VL-CDR1 described in SEQ ID NO: 51, VL-CDR2 described in SEQ ID NO: 52, and VL-CDR3 described in SEQ ID NO:

53.

31. The trivalent T cell mobilization molecule according to any one of claims 1 to 30, wherein the VL of the first T cell binding arm or 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 respect to the amino acid sequence described in SEQ ID NO:

54.

32. The trivalent T cell mobilization molecule according to any one of claims 1 to 31, wherein the constant light chain domain of the first T cell binding arm or 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 respect to the amino acid sequence described in SEQ ID NO:

64.

33. The trivalent T cell mobilization molecule according to any one of claims 1 to 32, wherein the VH of the first T cell binding arm or 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 respect to the amino acid sequence described in SEQ ID NO:

56.

34. The trivalent T cell mobilization molecule according to any one of claims 1 to 33, wherein the constant heavy chain domain of the first T cell binding arm or 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 an amino acid sequence selected from SEQ ID NOs. 57, 66, 67, 69, 74, 75, 77, 78, and 79.

35. The T cell mobilization molecule or trivalent T cell mobilization molecule according to any one of claims 1 to 34, wherein the antigen-binding arm binds to an epitope on the extracellular loop of STEAP2.

36. The T cell mobilization molecule according to any one of claims 1 to 35, wherein the T cell mobilization molecule includes SEQ ID NOs: 25, 26, 35, 81, and 92.

37. The trivalent T cell mobilization molecule according to any one of claims 1 to 35, wherein the trivalent T cell mobilization molecule comprises SEQ ID NOs. 25, 26, 35, 55, and 58.

38. (a) Heavy chain variable domains (VH), heavy chain CH, each containing a variable heavy chain complementarity determining region 1 (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, each binding to an epitope on six-transmembrane epithelial antigen 2 (STEAP2) of the human prostate, A first antigen-binding arm and a second antigen-binding arm comprising a heavy chain containing one domain, a variable light chain complementarity determining region 1 (VL-CDR1) selected from SEQ ID NOs: 4, 12, 20, 100, 108, 130, 138, and 146, a VL-CDR2 selected from SEQ ID NOs: 5, 13, 21, 101, 109, 131, 139, and 147, a VL-CDR3 selected from SEQ ID NOs: 6, 14, 22, 102, 110, 132, 140, and 148, and a light chain containing a constant light chain domain, (b) A first T cell binding arm comprising a VH containing VH-CDR1 selected from SEQ ID NOs: 36, 40, and 44, VH-CDR2 selected from SEQ ID NOs: 37, 41, and 45, VH-CDR3 selected from SEQ ID NOs: 38, 42, and 46, and a heavy chain containing a heavy chain CH1 domain; a VL containing VL-CDR1 selected from SEQ ID NOs: 27 and 31, VL-CDR2 selected from SEQ ID NOs: 28 and 32, VL-CDR3 selected from SEQ ID NOs: 29 and 33, and a light chain containing a light chain constant domain; (c) An Fc domain comprising a first Fc region and a second Fc region, wherein each Fc region comprises a CH2 domain and a CH3 domain, and the Fc domain further comprises at least one modification for promoting heterodimerization, A trivalent T cell mobilization molecule in which 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.

39. (d) A tetravalent T cell mobilization molecule comprising the trivalent T cell mobilization molecule according to claim 38, further comprising a second T cell binding arm comprising a heavy chain containing a variable heavy chain domain (VHH) that binds to differentiation antigen group 8 (CD8) and includes VH-CDR1 as described in SEQ ID NO: 84, VH-CDR2 as described in SEQ ID NO: 85, and VH-CDR3 as described in SEQ ID NO:

86.

40. The tetravalent T cell mobilization molecule according to claim 39, wherein the heavy chain of the second T cell binding arm is attached to the heavy chain of the first antigen binding arm.

41. The tetravalent T cell mobilization molecule according to claim 39 or 40, wherein the heavy chain of the second antigen-binding arm is attached to the heavy chain of the first T cell-binding arm via a linker.

42. The tetravalent T cell mobilization molecule according to claim 39 or 41, wherein the heavy chain of the second T cell binding arm is attached to the heavy chain of the first antigen binding arm via a linker.

43. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to claim 41 or 42, wherein the linker comprises the amino acid sequence of SEQ ID NO:

89.

44. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to any one of claims 41 to 43, wherein the linker comprises one to about ten copies of Sequence ID No.

89.

45. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to any one of claims 41 to 44, wherein the linker comprises two copies of Sequence ID No.

89.

46. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 45, wherein one of the two CH3 domains contains a knob mutation and the other of the two CH3 domains contains a hole mutation.

47. (a) one or more CH1 domains and light chain constant domains of the first antigen-binding arm or the second antigen-binding arm, and (b) the first T cell-binding arm, each comprising a charge pair substitution including 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, according to any one of claims 38 to 46.

48. (a) one or more of the light chain constant domains of the first antigen-binding arm or the second antigen-binding arm, and (b) the first T cell-binding arm, are lambda light chain constant domains (CLλ), wherein the charge pair is a lambda 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, the numbering follows the EU index, and the lambda charge pair is located at the following positions: (i) Position 117 in CLλ and position 141 in CH1 domain, (ii) Position 117 in CLλ and position 185 in the CH1 domain, (iii) Position 119 in CLλ and position 128 in the CH1 domain, (iv) Position 134 in CLλ and position 128 in the CH1 domain, (v) Position 134 in CLλ and position 145 in the CH1 domain, (vi) Position 134 in CLλ and position 183 in the CH1 domain, (vii) Position 136 in CLλ and position 185 in the CH1 domain, (viiii) Position 178 in CLλ and position 173 in the CH1 domain, and (ix) A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to claim 47, located at one or more of the positions 117 in the CLλ and 187 in the CH1 domain.

49. (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, (viiii) 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 The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to claim 48, wherein the amino acid 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.

50. (a) one or more of the light chain constant domains of the first antigen-binding arm or the second antigen-binding arm, and (b) the first T cell-binding arm, are kappa light chain constant domains (CLκ), the charge pair is a kappa 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, the numbering follows the EU index, and the kappa charge pair is located at position 133 in the CLκ and at position 183 in the CH1, according to any one of claims 47 to 49.

51. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to claim 50, wherein the charged amino acid at position 133 is glutamic acid 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 glutamic acid.

52. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 51, wherein one of the light chain constant domains of (a), (b), and (d) is CLλ and the charge pair is a lambda charge pair, the second and third light chain constant domains of (a), (b), and (d) are CLλ or CLκ and the charge pair is a lambda or kappa charge pair, and the second of (a), (b), and (d) contains a charged amino acid in the CH1 domain having the same charge as the charged amino acid in the third CH1 domain of (a), (b), and (d).

53. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 51, wherein one of the light chain constant domains of (a), (b), and (d) is CLλ and the charge pair is a lambda charge pair, the second and third light chain constant domains of (a), (b), and (d) are CLκ and the charge pair is a kappa charge pair, and the second of (a), (b), and (d) contains a charged amino acid in the CH1 domain having the same charge as the charged amino acid in the third CH1 domain of (a), (b), and (d).

54. The trivalent T cell mobilizing molecule or tetravalent T cell mobilizing molecule according to any one of claims 38 to 53, wherein the CH1 domain of (a) and / or (b) of the trivalent T cell mobilizing molecule, or the CH1 domain of (a), (b), and / or (d) of the tetravalent T cell mobilizing molecule, can form disulfide links with the light chain constant domain of (a) and / or (b) of the trivalent T cell mobilizing molecule, or the light chain of (a), (b), and / or (d) of the tetravalent T cell mobilizing molecule, via manipulated disulfide linkages.

55. The trivalent T cell mobilization molecule according to any one of claims 38 to 54, wherein the CH1 domain of (b) is capable of forming a disulfide linkage with the light chain constant domain via an manipulated disulfide linkage, and the CH1 domain of (a) is linked to the light chain constant domain via a natural disulfide linkage.

56. The tetravalent T cell mobilization molecule according to any one of claims 38 to 54, wherein the CH1 domains of (b) and (d) are capable of forming disulfide links with the light chain constant domains of (b) and (d) via manipulated disulfide linkages, and the CH1 domain of (a) is linked to the light chain constant domain of (a) via a natural disulfide linkage.

57. The CH1 domain of the first T cell binding arm is (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of natural non-cysteine ​​amino acids with cysteine, The light chain constant domain of the second T cell binding arm is (i) Substitution of natural cysteine ​​with non-cysteine ​​amino acids, (ii) Substitution of natural non-cysteine ​​amino acids with cysteine, The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to any one of claims 38 to 56, wherein the substituted cysteine ​​of the CH1 domain of the first T cell binding arm and the substituted cysteine ​​of the light chain constant domain of the first T cell binding arm can form a disulfide bond.

58. The CH1 domain, (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 220, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 126, The light chain constant domain, (i) Substitution of native cysteine ​​with a non-cysteine ​​amino acid at position 212, (ii) Substitution of a native non-cysteine ​​amino acid with cysteine ​​at position 122, The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to claim 57, 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, and the numbering follows the EU index.

59. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 58, wherein the light chain of the first antigen-binding arm and the second antigen-binding arm contains natural cysteine, the CH1 domain of the first antigen-binding arm and the natural cysteine ​​of the CH1 domain of the first antigen-binding arm and the natural cysteine ​​of the light chain constant domain of the first antigen-binding arm can form a disulfide bond, and the natural cysteine ​​of the CH1 of the second antigen-binding arm and the natural cysteine ​​of the light chain constant domain of the second antigen-binding arm can form a disulfide bond.

60. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 59, wherein the VH of the first antigen-binding arm and / or the second antigen-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 an amino acid sequence selected from SEQ ID NOs: 7, 15, 23, 95, 97, 99, 106, 114, 133, 141, and 149.

61. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 59, wherein the heavy chain of the first antigen-binding arm and / or the second antigen-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 an amino acid sequence selected from SEQ ID NOs. 67 and 93.

62. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 60, wherein the VL of the first antigen-binding arm and / or the second antigen-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 an amino acid sequence selected from SEQ ID NOs: 8, 16, 24, 107, 115, 134, 142, and 150.

63. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 61, wherein the light chain constant domain of the first antigen-binding arm and / or the second antigen-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 respect to the amino acid sequence described in SEQ ID NO:

63.

64. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to any one of claims 38 to 62, wherein the VL of the first T cell binding arm includes VL-CDR1 described in SEQ ID NO: 27 or 31, VL-CDR2 described in SEQ ID NO: 28 or 32, and VL-CDR3 described in SEQ ID NO: 29 or 33, and the VH of the first T cell binding arm includes VH-CDR1 described in SEQ ID NO: 36, 40, or 44, VH-CDR2 described in SEQ ID NO: 37, 41, or 45, and VH-CDR3 described in SEQ ID NO: 38, 42, or 46.

65. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to any one of claims 38 to 63, wherein the VL of the first 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 respect to the amino acid sequence described in SEQ ID NO: 30 or 34.

66. A trivalent T cell mobilization molecule or a tetravalent T cell mobilization molecule according to any one of claims 38 to 64, wherein the VH of the first 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 an amino acid sequence selected from SEQ ID NOs: 39, 43, and 47.

67. The tetravalent T cell mobilization molecule according to any one of claims 38 to 65, wherein the VH of the second T cell binding arm includes VH-CDR1 described in SEQ ID NO: 84, VH-CDR2 described in SEQ ID NO: 85, and VH-CDR3 described in SEQ ID NO:

86.

68. The tetravalent T cell mobilization molecule according to any one of claims 38 to 66, 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 respect to the amino acid sequence of SEQ ID NO: 56 or 83.

69. (d) A tetravalent T cell mobilization molecule comprising the trivalent T cell mobilization molecule according to claim 38, further comprising a second T cell binding arm.

70. The tetravalent T cell mobilization molecule according to claim 69, wherein the second T cell binding arm comprises VL, which includes VL-CDR1 as described in SEQ ID NO: 51, VL-CDR2 as described in SEQ ID NO: 52, and VL-CDR3 as described in SEQ ID NO: 53, and VH, which includes VH-CDR1 as described in SEQ ID NO: 48, VH-CDR2 as described in SEQ ID NO: 49, and VH-CDR3 as described in SEQ ID NO:

50.

71. The tetravalent T cell mobilization molecule according to claim 70, wherein 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 respect to the amino acid sequence described in SEQ ID NO: 54, and 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 respect to the amino acid sequence described in SEQ ID NO:

56.

72. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to any one of claims 38 to 70, wherein the first antigen-binding arm and the second antigen-binding arm bind to an epitope on the extracellular loop of STEAP2.

73. The trivalent T cell mobilization molecule or tetravalent T cell mobilization molecule according to any one of claims 38 to 71, wherein the heavy chain, including the heavy chain constant region of the second antigen-binding arm and the first 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 respect to the amino acid sequence described in SEQ ID NO:

81.

74. A tetravalent T cell mobilization molecule according to any one of claims 38 to 67, 71, and 72, wherein the heavy chain, including the heavy chain constant region of the first antigen-binding arm and 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 respect to an amino acid sequence selected from SEQ ID NO:

87.

75. The trivalent T cell mobilization molecule according to any one of claims 38 to 65, 71, and 72, wherein the trivalent T cell mobilization molecule comprises SEQ ID NOs: 25, 35, 81, 25, and 93.

76. The tetravalent T cell mobilization molecule includes SEQ ID NOs. 25, 35, 81, 25, and 87, or The tetravalent T cell mobilization molecule according to any one of claims 39 to 67, 71, and 73, wherein the tetravalent T cell mobilization molecule comprises SEQ ID NOs: 151, 152, 153, 154, and 152.

77. One or more nucleic acids encoding a T cell mobilization molecule according to any one of claims 1 to 76.

78. A vector comprising the nucleic acid described in claim 77.

79. An isolated host cell comprising the nucleic acid described in claim 77 or the vector described in claim 78.

80. A pharmaceutical composition comprising a T cell mobilization molecule according to any one of claims 1 to 76 and a pharmaceutically acceptable carrier.

81. A method for treating a disease in a patient requiring treatment for the disease, comprising administering to the patient an effective amount of a T cell mobilization molecule according to any one of claims 1 to 76, or a pharmaceutical composition according to claim 80.

82. The method according to claim 81, wherein the disease is cancer or prostate cancer.

83. A T-cell mobilization molecule according to any one of claims 1 to 76, or a pharmaceutical composition according to claim 80, for use as a pharmaceutical.

84. A T-cell mobilization molecule according to any one of claims 1 to 76 or a pharmaceutical composition according to claim 80, for use in the treatment of cancer.

85. Use of a T-cell mobilization molecule according to any one of claims 1 to 76 or a pharmaceutical composition according to claim 80 for the manufacture of a pharmaceutical for the treatment of cancer.

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