T cell engager molecules and uses thereof

Abstract

The present invention provides single chain T cell engager (TCE) molecules having a scFab that binds to a target antigen and a scFv that binds to CD3, and TCE molecules that bind to CCR8 and CD3. Methods of treating cancer are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 197,265, filed June 4, 2021, and U.S. Provisional Patent Application No. 63 / 236,547, filed August 24, 2021, each of which is incorporated by reference in its entirety herein.

[0002] FIELD OF THEINVENTION The present invention relates to the field of oncology, in particular to bispecific T cell engager (TCE) molecules and the treatment of cancer patients with said molecules. [Background technology]

[0003] 2. Background of the Invention Redirection of T cell activity against tumor cells by bispecific molecules independent of T cell receptor specificity is an evolving approach in immuno-oncology (Frankel SR, Baeuerle PA. Targeting T cells to tumor cells using bispecific antibodies. Curr Opin Chem Biol 2013; 17:385-92). Such novel protein-based drugs are typically capable of binding two different types of antigens simultaneously. Several structural formats are known and are currently being explored for applications in cancer immunotherapy and drug delivery (Fan, Gaowei; Wang, Zujian; Hao, Mingju; Li, Jinming (2015). “Bispecific antibodies and their applications”. Journal of Hematology & Oncology. 8:130).

[0004] Bispecific molecules useful in immuno-oncology can be antigen-binding polypeptides such as antibodies, for example IgG-like bispecific antibodies, i.e., full-length bispecific antibodies, or non-IgG-like bispecific antibodies that are not full-length antigen constructs. Full-length bispecific antibodies typically retain the traditional monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that the two Fab sites bind to different antigens. Non-full-length bispecific antibodies may completely lack the Fc region. These include chemically linked Fabs consisting of only the Fab region, as well as various types of bivalent and trivalent single-chain variable fragments (scFv). There are also fusion proteins that mimic the variable domains of two antibodies. An example of such a format is the Bispecific T Cell Engager (BiTE®) (Yang, Fa; Wen, Weihong; Qin, Weijun (2016). "Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies". International Journal of Molecular Sciences. 18(1):48).

[0005] BiTE molecules are recombinant protein constructs formed from two flexibly linked antibody-derived binding domains. One binding domain of BiTEs is specific for a selected tumor-associated surface antigen on target cells; the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. Due to their specific design, BiTE molecules are uniquely suited to transiently link T cells to target cells and, at the same time, potently activate the intrinsic cytolytic potential of T cells against target cells. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for bispecific molecules, specifically T cell engager ("TCE") molecules, that bind to a target antigen and CD3 and demonstrate enhanced lysis of target cells, and have desirable manufacturing properties, such as increased aggregation temperature and steeper HIC elution peak slope. The present invention provides single chain TCE molecules having a scFab that binds to a target antigen (e.g., a tumor antigen) and a scFv that binds to CD3. Some TCE molecules further have the scFc linked to the scFv by a linker to extend the half-life of the molecule. The TCE molecules of the present invention demonstrate enhanced lysis of target cells and improved manufacturing related properties.

[0007] The present invention also provides a CCR8 TCE molecule that binds to CCR8 and CD3. CC chemokine receptor type 8 (CCR8) is a member of the beta chemokine receptor family and is a seven transmembrane G protein-coupled receptor with a 35 amino acid extracellular N-terminus. The ligand for CCR8 is CCL1, and CCL1-induced CCR8 signaling occurs via G protein-coupled proteins. CCR8 is expressed on the surface of cancer-resident Tregs at a significantly higher prevalence and at higher levels compared to circulating or normal tissue Tregs and conventional T effector (Teff) cells. Treg cell infiltration in solid tumors is associated with poor clinical outcomes, and Tregs suppress anti-cancer immune responses through inhibition of Teff cytotoxicity.

[0008] The CCR8 TCE molecules of the present invention are believed to induce redirected T cell lysis of tumor-resident CCR8+ Tregs while excluding normal tissue Tregs that have little to no CCR8 expression. The CCR8 TCE molecules of the present invention are believed to have an improved safety profile compared to other Treg depletion therapeutic candidates that target other markers that do not specifically deplete cancer-resident Tregs.

[0009] The CCR8 TCE molecules of the present invention are single chain molecules, and have (i) an scFv that binds CCR8 and an scFv that binds CD3 (the two scFvs are linked by a linker); or (ii) an scFab that binds CCR8 and an scFv that binds CD3 (the scFab and scFv are linked by a linker). Some TCE molecules further have an scFc linked to an scFv that binds CD3 by a linker to extend the half-life of the molecule. The CCR8 TCE molecules of the present invention demonstrate pM range cytotoxicity and bind to both cynomolgus monkey and human CCR8. Interestingly, a CCR8 TCE molecule has been discovered that binds to a unique epitope on CCR8 and does not block ligand binding to CCR8. Binding to this unique epitope is believed to contribute to the high affinity and biological activity of the TCE molecules. Binding to this unique epitope may also contribute to an acceptable pharmacokinetic profile. [Means for solving the problem]

[0010] Summary of the Invention The present invention provides a T cell engager (TCE) molecule, which may be referred to as a scFab-containing TCE molecule, comprising: (i) an scFab that binds a tumor antigen, the scFab comprising a first heavy chain variable region (scFab VH), a CH1 domain, a first light chain variable region (scFab VL), and a Cκ or Cλ domain; and (ii) an scFv that binds CD3 and comprises a second VL and a second VH, the TCE molecule being single chain. In some embodiments, the scFab comprises a C-terminal portion linked to an N-terminal portion of the scFv by a linker. In some embodiments, the TCE molecule further comprises an scFc. In some embodiments, the scFc comprises an N-terminal portion linked to a C-terminal portion of the scFv by a linker. In certain embodiments, the scFv binds human CD3. In some embodiments, the tumor antigen is CCR8.

[0011] In some embodiments, the scFab of the TCE molecules of the invention has the following arrangement from N-terminus to C-terminus: VH, CH1, VL, and Cκ or Cλ. In other embodiments, the scFab has the following arrangement from N-terminus to C-terminus: VL, Cκ or Cλ, VH, and CH1. In some embodiments, the scFab includes a linker connecting the CH1 and VL, the linker being (G4S)6, (G4S)7, (G4S)8, (G4Q)6, (G4Q)7, or (G4Q)8. In some embodiments, the scFab includes a linker connecting the Cκ or Cλ and VH, the linker being (G4S)6, (G4S)7, (G4S)8, (G4Q)6, (G4Q)7, or (G4Q)8. In some embodiments, the scFab contains a natural cysteine ​​clamp between the heavy chain constant domain and the light chain constant domain. In some embodiments, the TCE molecule comprises an engineered cysteine ​​clamp in the scFab between residue 44 in the VH domain and residue 100 in the VL domain (Kabat numbering). In some embodiments, the scFab contains a native cysteine ​​clamp between the heavy and light chain constant domains and an engineered cysteine ​​clamp between residue 44 in the VH domain and residue 100 in the VL domain. In some embodiments, the TCE molecule CH1, Cκ, and / or Cλ domains are IgG, IgM, IgA, IgD, or IgE. In certain embodiments, the domains are IgG. In more particular embodiments, the domains are IgG1. In some embodiments, the domains are human domains. In certain embodiments, the domains are human IgG1.

[0012] The invention provides single chain TCE molecules having the following arrangement from N-terminus to C-terminus: scFab(VH, CH1, linker, VL, Cκ or Cλ), linker, scFv(VH, linker, VL). In one embodiment, the TCE molecule further comprises an scFc and has the following arrangement: scFab(VH, CH1, linker, VL, Cκ or Cλ), linker, scFv(VH, linker, VL), linker, Fc1(hinge, CH2, CH3), linker, Fc2(hinge, CH2, CH3).

[0013] The invention provides single chain TCE molecules having the following arrangement from N-terminus to C-terminus: scFab(VL, Cκ or Cλ, linker, VH, CH1), linker, scFv(VH, linker, VL). In one embodiment, the TCE molecule further comprises an scFc and has the following arrangement: scFab(VL, Cκ or Cλ, linker, VH, CH1), linker, scFv(VH, linker, VL), linker, Fc1(hinge, CH2, CH3), linker, Fc2(hinge, CH2, CH3).

[0014] The invention provides single chain TCE molecules with the following configuration: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL). In one embodiment, the TCE molecule further comprises an scFc and has the following configuration: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL)-linker-Fc1 (hinge, CH2, CH3), linker, Fc2 (hinge, CH2, CH3).

[0015] The present invention also provides a TCE molecule having the following configuration from N-terminus to C-terminus: scFv(VH, linker, VL) binding to CCR8-linker-scFv(VH, linker, VL) binding to CD3-linker-Fc1(CH2-CH3)-linker-Fc2(CH2-CH3). In one embodiment, the TCE molecule binds to CCR8 and CD3. The present invention provides a TCE molecule having the following configuration from N-terminus to C-terminus: scFv(VL-linker-VH) binding to CCR8-linker-scFv(VH-linker-VL) binding to CD3-linker-Fc1(CH2-CH3)-linker-Fc2(CH2-CH3). In one embodiment, the TCE molecule binds to CCR8 and CD3.

[0016] The present invention provides single chain TCE molecules having a scFab-scFv-scFv-scFc format. In some embodiments, the TCE molecule comprises the following configuration: VH-CH1-linker-VL-Cκ / Cλ-linker-VH-linker-VL-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises the following configuration: VL-Cκ / Cλ-linker-VH-CH1-linker-VH-linker-VL-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises the following configuration: VL-Cκ / Cλ-linker-VH-CH1-linker-VH-linker-VL-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises the following configuration: VL-Cκ / Cλ-linker-VH-CH1-linker-VL-linker-VH-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises the following configuration: VH-CH1-linker-VL-Cκ / Cλ-linker-VL-linker-VH-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises Cκ. In some embodiments, the TCE molecule comprises Cλ.

[0017] The invention also provides single chain TCE molecules having a scFab-scFab-scFv-scFc format. In some embodiments, the TCE molecule comprises the following configuration: VH-CH1-linker-VL-Cκ / Cλ-linker-VH-CH1-linker-VL-Cκ / Cλ-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises the following configuration: VL-Cκ / Cλ-linker-VH-CH1-linker-VH-CH1-linker-VL-Cκ / Cλ-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises the following configuration: VH-CH1-linker-VL-Cκ / Cλ-linker-VL-Cκ / Cλ-linker-VH-CH1-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises the following configuration: VL-Cκ / Cλ-linker-VH-CH1-linker-VL-Cκ / Cλ-linker-VH-CH1-linker-VH-linker-VL-linker-Fc1-linker-Fc2. In some embodiments, the TCE molecule comprises a Cκ. In some embodiments, the TCE molecule comprises a Cλ. In some embodiments, the TCE molecule comprises a Cκ and a Cλ.

[0018] In one embodiment, the scFab VH and CH1 of the scFab-containing TCE molecule of the invention comprise the amino acid sequences represented by SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, or SEQ ID NO:124. In one embodiment, the TCE molecule of the invention comprises a Cκ. In a specific embodiment, the scFab VL and Cκ of the scFab-containing TCE molecule of the invention comprise the amino acid sequences represented by SEQ ID NO:13, SEQ ID NO:29, SEQ ID NO:45, SEQ ID NO:61, SEQ ID NO:77, SEQ ID NO:93, SEQ ID NO:109, or SEQ ID NO:125. In another specific embodiment, the TCE molecule comprises the amino acid sequence represented by SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:46, SEQ ID NO:62, SEQ ID NO:78, SEQ ID NO:94, SEQ ID NO:110, or SEQ ID NO:126. In another specific embodiment, the TCE molecule comprises the amino acid sequence represented by SEQ ID NO:15, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:63, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:111, or SEQ ID NO:127. In another specific embodiment, the TCE molecule comprises the amino acid sequence represented by SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:48, SEQ ID NO:64, SEQ ID NO:80, SEQ ID NO:96, SEQ ID NO:112, or SEQ ID NO:128.

[0019] In another embodiment, the scFab VH and CH1, or scFab VL and Cκ, comprise a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequences of the scFab VH and CH1 or scFab VL and Cκ sequences described herein. In another embodiment, the TCE molecule comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequences of the TCE molecule sequences described herein.

[0020] The present invention also provides a TCE molecule comprising: (i) a first scFv that binds to CCR8, the first scFv comprising a first VH region (CCR8 scFv VH) and a first VL region (CCR8 scFv VL); and (ii) a second scFv that binds to CD3, the second scFv comprising a second VH region and a second VL region. A molecule having this structure and that binds to CCR8 and CD3 may be referred to as a CCR8 TCE molecule. In a preferred embodiment, the CCR8 TCE molecule is a single chain.

[0021] In one embodiment, the CCR8 TCE molecule scFv VH comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:39, SEQ ID NO:55, SEQ ID NO:71, SEQ ID NO:87, SEQ ID NO:103, or SEQ ID NO:119, and the CCR8 scFv VL comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:104, or SEQ ID NO:120. In another embodiment, the first scFv comprises the amino acid sequence set forth in SEQ ID NO:9, 25, 41, 57, 73, 89, 105, or 121. In another embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO:10, SEQ ID NO:26, SEQ ID NO:42, SEQ ID NO:58, SEQ ID NO:74, SEQ ID NO:90, SEQ ID NO:106, or SEQ ID NO:122. In another embodiment, the TCE molecule further comprises an scFc, wherein the TCE molecule comprises an amino acid sequence set forth by SEQ ID NO:11, SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:75, SEQ ID NO:91, SEQ ID NO:107, or SEQ ID NO:123. In another embodiment, the CCR8 scFv VH comprises a sequence of amino acids at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a CCR8 scFv VH sequence described herein. In another embodiment, the CCR8 scFv VL comprises a sequence of amino acids at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a CCR8 scFv VL sequence described herein.

[0022] In another embodiment, the first scFv (that binds CCR8) comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the first scFv sequence described herein.

[0023] In another embodiment, a CCR8 TCE molecule of the present invention comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a CCR8 TCE molecule sequence described herein.

[0024] In some embodiments, the first VH (scFab VH and / or CCR8 scFv VH) of the TCE molecule of the invention comprises HCDR1, HCDR2, HCDR3, and the first VL (scFab VL and / or CCR8 scFv VL) comprises LCDR1, LCDR2, and LCDR3: a) HCDR1 comprises the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:17, SEQ ID NO:33, SEQ ID NO:49, SEQ ID NO:65, SEQ ID NO:81, SEQ ID NO:97, or SEQ ID NO:113; b) HCDR2 comprises the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:18, SEQ ID NO:34, SEQ ID NO:50, SEQ ID NO:66, or SEQ ID NO:82; c) HCDR3 comprises an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:67, or SEQ ID NO:83; d) LCDR1 comprises the amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, or SEQ ID NO:84; e) LCDR2 comprises the amino acid sequence represented by SEQ ID NO:5, SEQ ID NO:21, SEQ ID NO:37, SEQ ID NO:53, SEQ ID NO:69, or SEQ ID NO:85; f) LCDR3 comprises the amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:22, SEQ ID NO:38, SEQ ID NO:54, SEQ ID NO:70, or SEQ ID NO:86.

[0025] In certain embodiments, HCDR1 comprises the amino acid sequence represented by SEQ ID NO:1, HCDR2 comprises the amino acid sequence represented by SEQ ID NO:2, HCDR3 comprises the amino acid sequence represented by SEQ ID NO:3, LCDR1 comprises the amino acid sequence represented by SEQ ID NO:4, LCDR2 comprises the amino acid sequence represented by SEQ ID NO:5, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO:6.

[0026] In another specific embodiment, HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 17, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 18, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 19, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 20, LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 21, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 22.

[0027] In another specific embodiment, HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 33, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 34, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 35, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 36, LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 37, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 38.

[0028] In yet another specific embodiment, HCDR1 comprises the amino acid sequence represented by SEQ ID NO:49, HCDR2 comprises the amino acid sequence represented by SEQ ID NO:50, HCDR3 comprises the amino acid sequence represented by SEQ ID NO:51, LCDR1 comprises the amino acid sequence represented by SEQ ID NO:52, LCDR2 comprises the amino acid sequence represented by SEQ ID NO:53, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO:54.

[0029] In another specific embodiment, HCDR1 comprises the amino acid sequence represented by SEQ ID NO:65, HCDR2 comprises the amino acid sequence represented by SEQ ID NO:66, HCDR3 comprises the amino acid sequence represented by SEQ ID NO:67, LCDR1 comprises the amino acid sequence represented by SEQ ID NO:68, LCDR2 comprises the amino acid sequence represented by SEQ ID NO:69, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO:70.

[0030] In another specific embodiment, HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 81, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 82, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 83, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 84, LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 85, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 86.

[0031] In yet another specific embodiment, HCDR1 comprises the amino acid sequence represented by SEQ ID NO:97, HCDR2 comprises the amino acid sequence represented by SEQ ID NO:98, HCDR3 comprises the amino acid sequence represented by SEQ ID NO:99, LCDR1 comprises the amino acid sequence represented by SEQ ID NO:100, LCDR2 comprises the amino acid sequence represented by SEQ ID NO:101, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO:102.

[0032] In another specific embodiment, HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 113, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 114, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 115, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 116 or SEQ ID NO: 336 (KSSQSVLYSSNNX1NYLA, X1 is K or R), LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 118.

[0033] The present invention provides a TCE molecule comprising the following arrangement from N-terminus to C-terminus: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL), wherein the scFv that binds CCR8 comprises CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid residues set forth in SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, and SEQ ID NO:222, respectively. In one embodiment, the scFv that binds CCR8 comprises VH and VL set forth in SEQ ID NO:223 and SEQ ID NO:224, respectively. In a particular embodiment, the scFv that binds CCR8 comprises the amino acid residues set forth in SEQ ID NO:225. In one embodiment, the TCE molecule comprises a G4S linker. In one embodiment, the TCE molecule comprises a G4Q linker. In one embodiment, the CD3 binding scFv is I2E. In another embodiment, the CD3 binding scFv is I2C. In another embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 226. In a further embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 227. In some such embodiments, the TCE molecule is TCE 1.1. In a preferred embodiment, the TCE molecule is a single chain. In some embodiments, the TCE molecule may have an arrangement such that the VL is N-terminal to the VH.

[0034] The present invention provides a TCE molecule comprising the following arrangement from N-terminus to C-terminus: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL)-linker-Fc1 (hinge, CH2, CH3), linker, Fc2 (hinge, CH2, CH3), wherein the scFv that binds CCR8 comprises CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising amino acid residues as set forth in SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, and SEQ ID NO:233, respectively. In one embodiment, the scFv that binds CCR8 comprises VH and VL as set forth in SEQ ID NO:234 ​​and SEQ ID NO:235, respectively. In a particular embodiment, the scFv that binds CCR8 comprises amino acid residues as set forth in SEQ ID NO:236. In one embodiment, the TCE molecule comprises a G4S linker. In one embodiment, the TCE molecule comprises a G4Q linker. In one embodiment, the CD3 binding scFv is I2E. In another embodiment, the CD3 binding scFv is I2C. In another embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 237. In a further embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 238. In some such embodiments, the TCE molecule is TCE 1.2. In a preferred embodiment, the TCE molecule is a single chain. In some embodiments, the TCE molecule may have an arrangement such that the VL is N-terminal to the VH.

[0035] The present invention provides a TCE molecule comprising the following arrangement from N-terminus to C-terminus: a CCR8-binding scFab (VH, CH1, linker, VL, Cκ or Cλ), a linker, a CD3-binding scFv (VH, linker, VL), wherein the CCR8-binding scFab comprises CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising amino acid residues set forth in SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, and SEQ ID NO:244, respectively. In one embodiment, the scFab comprises a VH and a VL set forth in SEQ ID NO:245 and SEQ ID NO:246, respectively. In a particular embodiment, the scFab comprises amino acid residues set forth in SEQ ID NO:247. In one embodiment, the TCE molecule comprises a G4S linker. In one embodiment, the TCE molecule comprises a G4Q linker. In one embodiment, the CD3-binding scFv is I2E. In another embodiment, the CD3-binding scFv is I2C. In another embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 248. In a further embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 249. In some such embodiments, the TCE molecule is TCE 1.3. In a preferred embodiment, the TCE molecule is a single chain. In some embodiments, the TCE molecule may have an arrangement such that the VL is N-terminal to the VH.

[0036] The present invention provides a TCE molecule comprising the following arrangement from N-terminus to C-terminus: scFab that binds CCR8 (VH, CH1, linker, VL, Cκ or Cλ), linker, scFv that binds CD3 (VH, linker, VL), linker, Fc1 (hinge, CH2, CH3), linker, Fc2 (hinge, CH2, CH3), wherein the scFab that binds CCR8 comprises CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising amino acid residues set forth in SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, and SEQ ID NO:255, respectively. In one embodiment, the scFab comprises a VH and a VL set forth in SEQ ID NO:256 and SEQ ID NO:257, respectively. In a particular embodiment, the scFab comprises amino acid residues set forth in SEQ ID NO:258. In one embodiment, the TCE molecule comprises a G4S linker. In one embodiment, the TCE molecule comprises a G4Q linker. In one embodiment, the CD3 binding scFv is I2E. In another embodiment, the CD3 binding scFv is I2C. In another embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 259. In a further embodiment, the TCE molecule comprises the amino acid sequence set forth in SEQ ID NO: 260. In some such embodiments, the TCE molecule is TCE 1.4. In a preferred embodiment, the TCE molecule is a single chain. In some embodiments, the TCE molecule may have an arrangement such that the VL is N-terminal to the VH.

[0037] The invention further provides a TCE molecule that binds to human CCR8, comprising an HCDR1 amino acid sequence of SEQ ID NO: 787; an HCDR2 amino acid sequence of SEQ ID NO: 788; an HCDR3 amino acid sequence of SEQ ID NO: 789; an LCDR1 amino acid sequence of SEQ ID NO: 790; an LCDR2 amino acid sequence of SEQ ID NO: 791; and an LCDR3 amino acid sequence of SEQ ID NO: 792. In some embodiments, the TCE molecule comprises a VH amino acid sequence of SEQ ID NO: 965 and a VL amino acid sequence of SEQ ID NO: 966.

[0038] The invention further provides a TCE molecule that binds to human CCR8, comprising an HCDR1 amino acid sequence of SEQ ID NO: 787; an HCDR2 amino acid sequence of SEQ ID NO: 788; an HCDR3 amino acid sequence of SEQ ID NO: 789; an LCDR1 amino acid sequence of SEQ ID NO: 336 (X1 is K or R); an LCDR2 amino acid sequence of SEQ ID NO: 791; and an LCDR3 amino acid sequence of SEQ ID NO: 792. In some embodiments, the TCE molecule comprises a VH amino acid sequence of SEQ ID NO: 965 and a VL amino acid sequence of SEQ ID NO: 342, wherein X1 is K or R, X2 is H or Q, and / or X3 is S or P.

[0039] The present invention further provides a TCE molecule that binds to human CCR8, comprising an HCDR1 amino acid sequence of SEQ ID NO: 805, an HCDR2 amino acid sequence of SEQ ID NO: 806, an HCDR3 amino acid sequence of SEQ ID NO: 807, an LCDR1 amino acid sequence of SEQ ID NO: 808, an LCDR2 amino acid sequence of SEQ ID NO: 809, and an LCDR3 amino acid sequence of SEQ ID NO: 810.

[0040] The present invention further provides a TCE molecule that binds to human CCR8, comprising: (a) an HCDR1 amino acid sequence of X1X2GX4H (SEQ ID NO: 1181), where (i) X1 is N, S, D, G, T, or R, (ii) X2 is C, N, Y, S, or F, and (iii) X4 is M or F; (b) any of the amino acids set forth in SEQ ID NOs: 596, 602, 608, 614, 620, 626, 632, 638, 644, 650, 656, 662, 668, 674, 680, 686, 692, 698, 704, 710, 716, 722, 728, 734, 740, 746, 752, 758, 764, 770, 776, 782, 788, 794, 800, 806, 815, 821, 827, 833, 839, 845, 851, 857, 863, 869, 875, 881, 887, or 893, or a variant thereof that contains one to four amino acid substitutions or is at least 90% identical to any one of the foregoing HCDR2 amino acid sequences; (c) SEQ ID NOs: 597, 603, 609, 615, 621, 627, 633, 639, 645, 651, 6 57, 663, 669, 675, 681, 687, 693, 699, 705, 711, 717, 723, 729, 735, 741, 747, 753, 759, 765, 771, 777, 783, 795, 801, 807, 816, 822, 828, 834, 840, 846, 852, 858, 864, 870, 876, 882, 888, or 894, or a variant thereof which contains one to four amino acid substitutions or is at least 90% identical to any one of the foregoing HCDR3 amino acid sequences; d) an LCDR1 amino acid sequence of SEQ ID NO: 598, 604, 610, 616, 622, 628, 634, 640, 646, 652, 658, 664, 670, 676, 682, 688, 694, 700, 706, 712, 718, 724, 730, 736, 742, 748, 754, 760, 766, 772, 778, 784, 796, 802, 808, 811, 817, 823, 829, 835, 841, 847, 853, 859, 865, 871, 877, 883, or 889, or one to four amino acid substitutions thereof;or a variant that is at least 90% identical to any one of the foregoing LCDR1 amino acid sequences; (e) an LCDR2 amino acid sequence of RX2X3X4RPS (SEQ ID NO: 1182), where (i) X2 is A, N, D, S, or Q, (ii) X3 is S, T, N, I, F, or A, and (iii) X4 is N or V; and (f) SEQ ID NOs: 600, 606, 612, 618, 624, 630, 636, 642, 648, 654, 660, 666, 672, 678, 684, 690, 696, 702, 708, 714, 720, 726, 732, 738, 744, 750, 756, 762, 768, 774, 780, 786, 798, 804, 810, 813, 819, 825, 831, 837, 843, 849, 855, 861, 867, 873, 879, 885, or 891, or a variant thereof which contains one to four amino acid substitutions or which is at least 90% identical to any one of the foregoing LCDR3 amino acid sequences. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 595, 601, 607, 613, 619, 625, 631, 637, 643, 649, 655, 661, 667, 673, 679, 685, 691, 697, 703, 709, 715, 721, 727, 733, 739, 745, 751, 757, 763, 769, 775, 781, 793, 799, 805, 814, 820, 826, 832, 838, 844, 850, 856, 862, 868, 874, 880, 886, or 892. In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO:599, 605, 611, 617, 623, 629, 635, 641, 647, 653, 659, 665, 671, 677, 683, 689, 695, 701, 707, 713, 719, 725, 731, 737, 743, 749, 755, 761, 767, 773, 779, 785, 797, 803, 809, 812, 818, 824, 830, 836, 842, 848, 854, 860, 866, 872, 878, 884, or 890. In some embodiments, the VH is selected from the group consisting of SEQ ID NOs: 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941,943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 967, 969, 971, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, or 1000 amino acid sequences. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 968, 970, 972, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, or 999.

[0041] In some embodiments, the TCE molecule comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 967 and a VL comprising the amino acid sequence of SEQ ID NO: 968; (b) a VH comprising the amino acid sequence of SEQ ID NO: 969 and a VL comprising the amino acid sequence of SEQ ID NO: 970; (c) a VH comprising the amino acid sequence of SEQ ID NO: 971 and a VL comprising the amino acid sequence of SEQ ID NO: 972; (d) a VH comprising the amino acid sequence of SEQ ID NO: 974 and a VL comprising the amino acid sequence of SEQ ID NO: 973; (e) a VH comprising the amino acid sequence of SEQ ID NO: 976 and a VL comprising the amino acid sequence of SEQ ID NO: 975; (f) a VH comprising the amino acid sequence of SEQ ID NO: 978 and a VL comprising the amino acid sequence of SEQ ID NO: 977; (g) a VH comprising the amino acid sequence of SEQ ID NO: 980 and a VL comprising the amino acid sequence of SEQ ID NO: 979; (h) a VH comprising the amino acid sequence of SEQ ID NO: 982 and a VL comprising the amino acid sequence of SEQ ID NO: 981; (i) a VH comprising the amino acid sequence of SEQ ID NO: 984 (j) a VH comprising an amino acid sequence of SEQ ID NO: 986 and a VL comprising an amino acid sequence of SEQ ID NO: 985; (k) a VH comprising an amino acid sequence of SEQ ID NO: 988 and a VL comprising an amino acid sequence of SEQ ID NO: 987; (l) a VH comprising an amino acid sequence of SEQ ID NO: 990 and a VL comprising an amino acid sequence of SEQ ID NO: 989; (m) a VH comprising an amino acid sequence of SEQ ID NO: 992 and a VL comprising an amino acid sequence of SEQ ID NO: 991; (n) a VH comprising an amino acid sequence of SEQ ID NO: 994 and a VL comprising an amino acid sequence of SEQ ID NO: 993; (o) a VH comprising an amino acid sequence of SEQ ID NO: 996 and a VL comprising an amino acid sequence of SEQ ID NO: 995; (p) a VH comprising an amino acid sequence of SEQ ID NO: 998 and a VL comprising an amino acid sequence of SEQ ID NO: 997; or (q) a VH comprising an amino acid sequence of SEQ ID NO: 1000 and a VL comprising an amino acid sequence of SEQ ID NO: 999.

[0042] In some embodiments, the TCE molecule comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 965, and a light chain variable region (VL) comprising the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNX1NYLAWYX2QKPGQX3PKLLISWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYYCQQYYSIPITFGGGTKVEIKR (SEQ ID NO: 342), where X1 is K or R, X2 is H or Q, and / or X3 is S or P. In some embodiments, the TCE molecule of the invention comprises a YTE motif in the Fc region, which corresponds to M252Y / S254T / T256E in the constant heavy chain region of IgG1 or IgG4. YTE extends the half-life of the molecule (see, e.g., Booth et al., MAbs 2018 Oct;10(7):1098-1110). In some embodiments, the TCE molecules of the present invention, including YTE, are TCE molecules that bind to CCR8 and CD3.

[0043] In some embodiments, the TCE molecule of the present invention comprises an I2E scFv. In some embodiments, the TCE molecule of the present invention comprises an I2C scFv. The amino acid sequence of I2E is represented by SEQ ID NOs: 199-206. The amino acid sequence of I2C is represented by SEQ ID NOs: 191-198.

[0044] The present invention provides additional TCE molecules, which are set forth in Table 25. The amino acid sequences of these TCE molecules are set forth in Table 25 by SEQ ID NOs: 261-589.

[0045] The present invention also provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a TCE molecule of the present invention. In one embodiment, the cancer is a solid tumor. In certain embodiments, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. In some embodiments, the method further comprises administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody. In some such embodiments, the PD-1 antagonist antibody or the PD-L1 antagonist antibody is administered prior to, simultaneously with, and / or after administration of the TCE molecule. In certain embodiments, the PD-1 antagonist antibody is pembrolizumab, nivolumab, cemiplimab, or antibody 20C1.009. In other specific embodiments, the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab. In some embodiments, the method further comprises administering to the patient a chemotherapeutic agent. In some such embodiments, the chemotherapeutic agent may be administered prior to, simultaneously with, or after administration of the TCE molecule. In some embodiments, the method comprises administering to the patient a TCE molecule of the invention and a chemotherapeutic agent. In some embodiments, the method comprises administering to the patient a TCE molecule of the invention, a PD-1 or PD-L1 antagonist antibody, and a chemotherapeutic agent.

[0046] The present invention provides a TCE molecule of the invention for use in therapy.

[0047] The present invention also provides a TCE molecule for use in treating cancer. In one embodiment, the cancer is a solid tumor. In a particular embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. In a more particular embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer. In some embodiments, the use further comprises administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody. In some such embodiments, the PD-1 antagonist antibody or the PD-L1 antagonist antibody is administered prior to, simultaneously with, and / or after administration of the TCE molecule. In a particular embodiment, the PD-1 antagonist antibody is pembrolizumab, nivolumab, cemiplimab, or antibody 20C1.009. In other specific embodiments, the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab. In some embodiments, the use further comprises administering to the patient a chemotherapeutic agent. In some such embodiments, the chemotherapeutic agent may be administered prior to, simultaneously with, or after administration of the TCE molecule. In some embodiments, the use comprises administering to the patient a TCE molecule of the invention and a chemotherapeutic agent. In some embodiments, the use comprises administering to the patient a TCE molecule of the invention, a PD-1 or PD-L1 antagonist antibody, and a chemotherapeutic agent.

[0048] The present invention provides the use of the TCE molecule of the present invention for the manufacture of a medicament for the treatment of cancer.In one embodiment, the cancer is a solid tumor.In a particular embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer.In a more particular embodiment, the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer.

[0049] The invention also provides pharmaceutical compositions comprising a TCE molecule of the invention and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0050] The present invention also provides polynucleotides that encode the amino acid sequences of the TCE molecules of the present invention. The term "encoding" refers to a polynucleotide sequence that encodes one or more amino acids. The term does not require a start or stop codon. The present invention encompasses nucleic acid molecules that encode anti-CCR8 TCE polypeptide sequences.

[0051] In one embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence set forth in SEQ ID NO: 590. In a specific embodiment, the TCE molecule encoded by the polynucleotide sequence set forth in SEQ ID NO: 590 comprises the amino acid sequence set forth in SEQ ID NO: 227.

[0052] In one embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence set forth in SEQ ID NO: 592. In a particular embodiment, the TCE molecule encoded by the polynucleotide sequence set forth in SEQ ID NO: 592 comprises the amino acid sequence set forth in SEQ ID NO: 249.

[0053] In one embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence set forth in SEQ ID NO: 593. In a particular embodiment, the TCE molecule encoded by the polynucleotide sequence set forth in SEQ ID NO: 593 comprises the amino acid sequence set forth in SEQ ID NO: 260.

[0054] In one embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence set forth in SEQ ID NO: 591. In a specific embodiment, the TCE molecule encoded by the polynucleotide sequence set forth in SEQ ID NO: 591 comprises the amino acid sequence set forth in SEQ ID NO: 238.

[0055] The present invention also provides a DNA molecule comprising a polynucleotide encoding the amino acid sequence of the TCE molecule of the present invention. In one embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence shown by SEQ ID NO:590. In a particular embodiment, the TCE molecule encoded by the polynucleotide sequence shown by SEQ ID NO:590 comprises the amino acid sequence shown by SEQ ID NO:227. In another embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence shown by SEQ ID NO:592. In a particular embodiment, the TCE molecule encoded by the polynucleotide sequence shown by SEQ ID NO:592 comprises the amino acid sequence shown by SEQ ID NO:249. In another embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence shown by SEQ ID NO:593. In a particular embodiment, the TCE molecule encoded by the polynucleotide sequence shown by SEQ ID NO:593 comprises the amino acid sequence shown by SEQ ID NO:260. In another embodiment, the TCE molecule of the present invention is encoded by a polynucleotide sequence shown by SEQ ID NO:591. In a particular embodiment, the TCE molecule encoded by the polynucleotide sequence shown by SEQ ID NO:591 comprises the amino acid sequence shown by SEQ ID NO:238.

[0056] The present invention further provides a mammalian cell transformed with a DNA molecule of the present invention, the transformed mammalian cell being capable of expressing a TCE molecule of the present invention.

[0057] The invention also provides a process for producing a TCE molecule of the invention, the process comprising culturing a mammalian cell under conditions such that the TCE molecule is expressed, and recovering the expressed TCE molecule. The invention also provides a mammalian cell transformed with a DNA molecule of the invention, the transformed mammalian cell being capable of expressing a TCE molecule of the invention. The invention also provides a TCE molecule obtainable by the process.

[0058] In another embodiment, the invention provides a CCR8 TCE molecule that binds to human CCR8 at an epitope, the epitope comprising at least one residue of SEQ ID NO: 134. In one embodiment, the epitope comprises at least two residues of SEQ ID NO: 134. In one embodiment, the epitope comprises at least three residues of SEQ ID NO: 134. In one embodiment, the epitope comprises at least four residues of SEQ ID NO: 134. In one embodiment, the epitope comprises at least five residues of SEQ ID NO: 134. In one embodiment, the epitope comprises six or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises seven or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises eight or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises nine or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises ten or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises eleven or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises the 12 residues of SEQ ID NO: 134. In a particular embodiment, the epitope comprises the threonine residue at position 4 of SEQ ID NO: 134. As used herein, the term "epitope" refers to a site of an antigen that contacts (e.g. binds) a molecule. An epitope may be determined by methods known to those of skill in the art, including flow cytometry, hydrogen-deuterium exchange, alanine scanning, and / or X-ray crystallography of TCE molecules bound to a peptide. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by TCE molecules that bind to a CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening TCE molecules that bind to CCR8 by phage display. In one embodiment, the epitope is determined by determining binding to a CCR8 peptide expressed in a human cell, the peptide comprising the amino acid sequence set forth by SEQ ID NO: 134, or amino acid residues 1-12 of SEQ ID NO: 133. In some embodiments, the epitope is determined by an anti-CCR8 TCE molecule that binds to a T4R mutation in cynomolgus CCR8.In one embodiment, binding to T4R mutation is determined by cell-based affinity assay, and TCE molecule binding to cells expressing cynomolgus monkey CCR8 containing T4R mutation is compared to TCE molecule binding to cells expressing wild-type cynomolgus monkey CCR8 (containing threonine at position 4). In some embodiments, anti-CCR8 TCE molecule is bound to threonine at position 4 if it shows reduced binding to CCR8 containing T4R mutation. In certain embodiments, anti-CCR8 TCE molecule is bound to threonine at position 4 if it shows no detectable binding to CCR8 containing T4R mutation. In some embodiments, wild-type cynomolgus monkey CCR8 comprises the amino acid sequence shown by SEQ ID NO: 129. In some embodiments, cynomolgus monkey CCR8 containing T4R mutation comprises the amino acid sequence shown by SEQ ID NO: 130.

[0059] The present invention provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a CCR8 TCE molecule that binds to human CCR8 at an epitope, the epitope comprising at least one residue of SEQ ID NO: 134. In one embodiment, the epitope comprises at least two residues of SEQ ID NO: 134. In one embodiment, the epitope comprises at least three residues of SEQ ID NO: 134. In one embodiment, the epitope comprises at least four residues of SEQ ID NO: 134. In one embodiment, the epitope comprises at least five residues of SEQ ID NO: 134. In one embodiment, the epitope comprises six or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises seven or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises eight or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises nine or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises ten or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises 11 or more residues of SEQ ID NO: 134. In one embodiment, the epitope comprises 12 residues of SEQ ID NO: 134. In one embodiment, the epitope comprises the threonine residue at position 4 of SEQ ID NO: 134. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by TCE molecules that bind to CCR8 peptide-nanobody complexes. In one embodiment, the epitope is determined by screening TCE molecules that bind to CCR8 by phage display. In one embodiment, the epitope is determined by determining binding to a CCR8 peptide expressed in a human cell, the peptide comprising the amino acid sequence set forth by SEQ ID NO: 134 or amino acid residues 1-12 of SEQ ID NO: 133. In some embodiments, the epitope is determined by anti-CCR8 TCE molecules that bind to a T4R mutation in cynomolgus CCR8.In one embodiment, binding to T4R mutation is determined by cell-based affinity assay, and TCE molecule binding to cells expressing cynomolgus monkey CCR8 containing T4R mutation is compared to TCE molecule binding to cells expressing wild-type cynomolgus monkey CCR8 (containing threonine at position 4). In some embodiments, anti-CCR8 TCE molecule is bound to threonine at position 4 if it shows reduced binding to CCR8 containing T4R mutation. In certain embodiments, anti-CCR8 TCE molecule is bound to threonine at position 4 if it shows no detectable binding to CCR8 containing T4R mutation. In some embodiments, wild-type cynomolgus monkey CCR8 comprises the amino acid sequence shown by SEQ ID NO: 129. In some embodiments, cynomolgus monkey CCR8 containing T4R mutation comprises the amino acid sequence shown by SEQ ID NO: 130.

[0060] In another embodiment, the invention provides a CCR8 TCE molecule that binds to human CCR8 at an epitope, the epitope consisting of one residue of SEQ ID NO: 134. In one embodiment, the epitope consists of two residues of SEQ ID NO: 134. In one embodiment, the epitope consists of three residues of SEQ ID NO: 134. In one embodiment, the epitope consists of four residues of SEQ ID NO: 134. In one embodiment, the epitope consists of five residues of SEQ ID NO: 134. In one embodiment, the epitope consists of six residues of SEQ ID NO: 134. In one embodiment, the epitope consists of seven residues of SEQ ID NO: 134. In one embodiment, the epitope consists of eight residues of SEQ ID NO: 134. In one embodiment, the epitope consists of nine residues of SEQ ID NO: 134. In one embodiment, the epitope consists of ten residues of SEQ ID NO: 134. In one embodiment, the epitope consists of eleven residues of SEQ ID NO: 134. In one embodiment, the epitope consists of 12 residues of SEQ ID NO: 134. In one embodiment, the epitope consists of the threonine residue at position 4 of SEQ ID NO:134.

[0061] The present invention provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a CCR8 TCE molecule that binds to human CCR8 at an epitope, wherein the epitope consists of one residue of SEQ ID NO: 134. In one embodiment, the epitope consists of two residues of SEQ ID NO: 134. In one embodiment, the epitope consists of three residues of SEQ ID NO: 134. In one embodiment, the epitope consists of four residues of SEQ ID NO: 134. In one embodiment, the epitope consists of five residues of SEQ ID NO: 134. In one embodiment, the epitope consists of six residues of SEQ ID NO: 134. In one embodiment, the epitope consists of seven residues of SEQ ID NO: 134. In one embodiment, the epitope consists of eight residues of SEQ ID NO: 134. In one embodiment, the epitope consists of nine residues of SEQ ID NO: 134. In one embodiment, the epitope consists of ten residues of SEQ ID NO: 134. In one embodiment, the epitope consists of eleven residues of SEQ ID NO: 134. In one embodiment, the epitope consists of 12 residues of SEQ ID NO: 134. In one embodiment, the epitope consists of the threonine residue at position 4 of SEQ ID NO:134.

[0062] In another embodiment, the invention provides a CCR8 TCE molecule that binds to human CCR8 at an epitope, the epitope comprising at least one residue of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least two residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least three residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least four residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least five residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises six or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises seven or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises eight or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 9 or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 10 or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 11 or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 12 residues of amino acid residues 1-12 of SEQ ID NO: 133. In a particular embodiment, the epitope comprises the threonine residue at position 4 of amino acid residues 1-12 of SEQ ID NO: 133. The term "epitope" as used herein refers to a site of an antigen that contacts (e.g., binds) a molecule. Epitopes may be determined by methods known to those of skill in the art, including flow cytometry, hydrogen-deuterium exchange, alanine scanning, and / or x-ray crystallography of TCE molecules bound to a peptide. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by TCE molecules that bind to the CCR8 peptide-nanobody complex. In one embodiment, the epitope is determined by screening TCE molecules that bind to CCR8 by phage display.In one embodiment, the epitope is determined by determining binding to a CCR8 peptide expressed in human cells, the peptide comprising the amino acid sequence set forth by SEQ ID NO: 134, or amino acid residues 1-12 of SEQ ID NO: 133. In some embodiments, the epitope is determined by an anti-CCR8 TCE molecule binding to a T4R mutation in cynomolgus CCR8. In one embodiment, binding to a T4R mutation is determined by a cell-based affinity assay, in which TCE molecule binding to cells expressing cynomolgus CCR8 containing a T4R mutation is compared to TCE molecule binding to cells expressing wild-type cynomolgus CCR8 (containing a threonine at position 4). In some embodiments, the anti-CCR8 TCE molecule is bound to the threonine at position 4 if it shows reduced binding to CCR8 containing a T4R mutation. In certain embodiments, the anti-CCR8 TCE molecule is bound to the threonine at position 4 if it shows no detectable binding to CCR8 containing a T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the cynomolgus CCR8 comprising a T4R mutation comprises the amino acid sequence set forth in SEQ ID NO: 130.

[0063] The present invention provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a CCR8 TCE molecule that binds to human CCR8 at an epitope, the epitope comprising at least one residue of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least two residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least three residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least four residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises at least five residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises six or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises seven or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises eight or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 9 or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 10 or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 11 or more residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises 12 residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope comprises the threonine residue at position 4 of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by TCE molecules that bind to CCR8 peptide-nanobody complexes. In one embodiment, the epitope is determined by screening TCE molecules that bind to CCR8 by phage display. In one embodiment, the epitope is determined by determining binding to a CCR8 peptide expressed in a human cell, wherein the peptide comprises the amino acid sequence set forth by SEQ ID NO:134, or amino acid residues 1-12 of SEQ ID NO:133.In some embodiments, the epitope is determined by anti-CCR8 TCE molecule binding to T4R mutation in cynomolgus CCR8. In one embodiment, binding to T4R mutation is determined by cell-based affinity assay, and TCE molecule binding to cells expressing cynomolgus CCR8 containing T4R mutation is compared to TCE molecule binding to cells expressing wild-type cynomolgus CCR8 (containing threonine at position 4). In some embodiments, the anti-CCR8 TCE molecule is bound to threonine at position 4 if it shows reduced binding to CCR8 containing T4R mutation. In certain embodiments, the anti-CCR8 TCE molecule is bound to threonine at position 4 if it shows no detectable binding to CCR8 containing T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence shown by SEQ ID NO: 129. In some embodiments, the cynomolgus CCR8 containing T4R mutation comprises the amino acid sequence shown by SEQ ID NO: 130.

[0064] In another embodiment, the present invention provides a CCR8 TCE molecule that binds to human CCR8 at an epitope, the epitope consisting of at least one residue of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of two residues of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of three residues of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of four residues of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of five residues of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of six residues of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of seven residues of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of eight residues of amino acid residues 1 to 12 of SEQ ID NO: 133. In one embodiment, the epitope consists of the nine residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the ten residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the eleven residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the twelve residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the threonine residue at position 4, amino acid residues 1 to 12, of SEQ ID NO: 133.

[0065] The present invention provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a CCR8 TCE molecule that binds to human CCR8 at an epitope, the epitope consisting of one residue of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of two residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of three residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of four residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of five residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of six residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of seven residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of eight residues of amino acid residues 1-12 of SEQ ID NO: 133. In one embodiment, the epitope consists of the nine residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the ten residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the eleven residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the twelve residues, amino acid residues 1 to 12, of SEQ ID NO: 133. In one embodiment, the epitope consists of the threonine residue at position 4, amino acid residues 1 to 12, of SEQ ID NO: 133.

[0066] In one embodiment, the epitope is determined by epitope binning. In one embodiment, the epitope is determined by TCE molecules binding to CCR8 peptide-nanobody complexes. In one embodiment, the epitope is determined by screening TCE molecules binding to CCR8 by phage display. In one embodiment, the epitope is determined by determining binding to a CCR8 peptide expressed in human cells, the peptide comprising the amino acid sequence set forth by SEQ ID NO: 134, or amino acid residues 1-12 of SEQ ID NO: 133. In some embodiments, the epitope is determined by anti-CCR8 TCE molecules binding to a T4R mutation in cynomolgus CCR8. In one embodiment, binding to a T4R mutation is determined by a cell-based affinity assay, where TCE molecule binding to cells expressing cynomolgus CCR8 containing a T4R mutation is compared to TCE molecule binding to cells expressing wild-type cynomolgus CCR8 (containing a threonine at position 4). In some embodiments, the anti-CCR8 TCE molecule is bound to the threonine at position 4 if it shows reduced binding to CCR8 with T4R mutation. In certain embodiments, the anti-CCR8 TCE molecule is bound to the threonine at position 4 if it does not show detectable binding to CCR8 with T4R mutation. In some embodiments, the wild-type cynomolgus CCR8 comprises the amino acid sequence shown by SEQ ID NO: 129. In some embodiments, the cynomolgus CCR8 with T4R mutation comprises the amino acid sequence shown by SEQ ID NO: 130.

[0067] In some embodiments, the present invention provides a molecule that competes with the CCR8 TCE molecule of the present invention for binding to CCR8. Such molecules that compete for binding can be, for example, TCE molecules, antibodies, antibody fragments, or polypeptides. In some embodiments, the present invention provides a molecule that binds to the same epitope as the CCR8 TCE molecule of the present invention.

[0068] In some embodiments, the TCE molecules of the present invention may be administered simultaneously with, prior to, or after a variety of drugs and treatments commonly used in cancer treatment, such as chemotherapeutic agents, non-chemotherapeutic agents (e.g., anti-PD-1 or anti-PD-L1 inhibitors, such as antagonist antibodies), antineoplastic agents, and / or radiation. For example, administration may occur prior to, during, and / or after any treatment described herein. Examples of chemotherapeutic agents are described herein and include, but are not limited to, cisplatin, taxol, etoposide, mitoxantrone (Novantrone®), actinomycin D, cycloheximide, camptothecin (or water-soluble derivatives thereof), methotrexate, mitomycin (e.g., mitomycin C), dacarbazine (DTIC), antineoplastic antibiotics such as adriamycin (doxorubicin) and daunomycin, and all chemotherapeutic agents described herein.

[0069] In some embodiments, the TCE molecules of the invention may be administered simultaneously with, prior to, or after a PD-1 antagonist antibody or a PD-L1 antagonist antibody. The term "PD-1 antagonist antibody" refers to an antibody that specifically binds to PD-1 and reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-1 and one or more of its ligands, such as PD-L1 and PD-L2. In some embodiments, a PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1 and / or PD-L2. The term "PD-L1 antagonist antibody" refers to an antibody that specifically binds to PD-L1 and reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L1 with the PD-1 receptor. In some embodiments, a PD-L1 antagonist antibody inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 antagonist is any one of antibody 20C1.006 (SEQ ID NOs: 179-188), antibody 20C1.009 (SEQ ID NOs: 139-148 or 139-147 and 212), antibody 20A2.3 (SEQ ID NOs: 149-158), antibody 20D4.6 (SEQ ID NOs: 159-168), or antibody 20D4.17 (SEQ ID NOs: 169-178). In one embodiment, the PD-1 antagonist antibody is pembrolizumab. In another embodiment, the PD-1 antagonist antibody is nivolumab. In yet another embodiment, the PD-1 antagonist antibody is cemiplimab. In certain embodiments, the PD-1 antagonist antibody is antibody 20C1.009, the CDRs, variable region, and complete light and heavy chain amino acid sequences of which are set forth in SEQ ID NOs: 139-148 and 212. 20C1.009 is also known as AMG 404, and is also known as zeluvalimab. In an exemplary embodiment, an anti-PD-1 antibody such as 20C1.009 comprises a HC that includes a C-terminal lysine as in SEQ ID NO: 148. In an alternative embodiment, the antibody comprises a HC without a C-terminal lysine as in SEQ ID NO:212.

[0070] In some embodiments, 1185 to 1200 [Brief description of the drawings]

[0071] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] 1 shows the domains and domain order of the TCE molecules of the invention. An exemplary TCE molecule comprises the following domain order from N-terminus to C-terminus: VH-linker-VL-linker-VH-linker-VL-linker-Fc1-linker-Fc2 (left; "CCR8-CD3 TCE"). Another exemplary TCE molecule of the invention comprises the following domain order from N-terminus to C-terminus: VH-CH1-linker-VL-Ck / Clambda-linker-VH-linker-VL-linker-Fc1-linker-Fc2 (right; "scFab TCE"). Abbreviations: scFab = single chain Fab (VH-CH1-linker-VL-Ck / Clambda); scFv = single chain Fv (VH-linker-VL); scFc = single chain Fc. The formats shown may contain scFab or scFv in any configuration, from N-terminus to C-terminus: VH-VL, VL-VH, VH-CH1-VL-Ck / Clambda, or VL-Ck / Clambda-VH-CH1 (including linker). For simplicity, the VH-VL and VH-CH1-VL-Ck / Clambda configurations are shown. The formats shown may contain a G4S linker or a G4Q linker. For simplicity, the G4S linker is shown. [Diagram 2]1 shows the domains and domain order of the multi-target BiTE HLE formats of the invention. Multi-target BiTE HLE molecules in scFv-scFv-scFv-scFc format contain the following domain order from N-terminus to C-terminus: VH-linker-VL-linker-VH-linker-VL-linker-Fc1-linker-Fc2, while the scFab-scFv-scFv-scFc format contains VH-CH1-linker-VL-Cκ / Cλ-linker-VH-linker-VL-linker-VH-linker-VL-linker-Fc1-linker-Fc2. The scFab-scFab-scFv-scFc format contains VH-CH1-linker-VL-Cκ / Cλ-linker-VH-CH1-linker-VL-Cκ / Cλ-linker-VH-linker-VL-linker-Fc1-linker-Fc2. The BiTE formats shown can contain scFab or scFv in either configuration, HL or LH; for simplicity, only the HL configuration is shown. Cκ / Cλ=Cκ or Cλ. The BiTE formats shown can contain G4S or G4Q linkers. For simplicity, the G4S linker is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Detailed Description of the Invention The present disclosure provides a single chain TCE molecule comprising an scFab that binds to a target antigen and an scFv that binds to CD3. The present disclosure also provides a TCE molecule comprising an scFv that binds to CCR8 and an scFv that binds to CD3. Also provided are methods of treating cancer and methods of producing the TCE molecule.

[0073] A "single-chain variable fragment" ("scFv") is a fusion protein in which a VL region and a VH region are linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, the linker being long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)). In the context of other additional moieties (e.g., Fc regions), scFvs can be configured, for example, as VH-linker-VL (anti-CD3 scFv) or VL-linker-VH. An anti-targeting scFv is an scFv that binds to an antigen, such as a tumor antigen. An anti-targeting scFv can bind to CCR8. An anti-CD3 scFv binds to CD3. Examples of anti-CD3 scFv include I2E and I2C (represented by amino acid sequences 199-206 and 191-198, respectively).

[0074] A "single-chain antigen-binding fragment" ("scFab") is a fusion protein in which a VH and a CH1 are linked to a VL and a Cκ or Cλ via a linker to form a continuous protein chain, the linker being long enough to allow the protein chain to fold back on itself to form a configuration-independent monovalent antigen-binding site. The linker can be, for example, a (G4S)6, (G4S)7, or (G4S)8 linker. A G4S linker is a linker made up of the amino acids GGGGS (SEQ ID NO: 189) from the N-terminus to the C-terminus, which may be repeated multiple times. A (G4S)4 linker refers, for example, to a linker comprising the following amino acids from the N-terminus to the C-terminus: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 190). Alternatively, the linker can be, for example, a (G4Q)6, (G4Q)7, or (G4Q)8 linker. The G4Q linker is a linker made of amino acids GGGGQ (SEQ ID NO: 207) from the N-terminus to the C-terminus, and may be repeated multiple times. The (G4Q)4 linker means, for example, a linker comprising the following amino acids from the N-terminus to the C-terminus: GGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 208). The CCR8 TCE of the present invention comprises a G4Q linker.

[0075] The scFab, scFv, and / or scFc may also have a cysteine ​​clamp. A "cysteine ​​clamp" refers to the introduction of a cysteine ​​at a specific position into a polypeptide domain, typically by replacing an existing amino acid at that position, such that when brought into close proximity with another polypeptide domain also having a cysteine ​​introduced at that position, a disulfide bond ("cysteine ​​clamp") can form between the two domains. In certain embodiments, the scFC comprises at least one cysteine ​​clamp that creates a disulfide bond across both CH2 domains. In certain further embodiments, the scFC comprises at least two cysteine ​​clamps that create a disulfide bond across both CH2 domains. In other embodiments, the VH and VL domains of the binding construct may comprise cysteine ​​clamps that form disulfide bonds between the VH and VL domains. These cysteine ​​clamps stabilize the VH and VL domains in the antigen-binding configuration.

[0076] The cysteine ​​clamp may be naturally occurring or may be the result of a molecule engineered to contain a cysteine. For example, an scFab may have a natural cysteine ​​clamp between the heavy and light chain constant domains. An scFab may also have a natural cysteine ​​clamp between the heavy and light chain constant domains and an engineered cysteine ​​clamp between cysteine ​​residue 44 of the heavy chain variable region and cysteine ​​residue 100 of the light chain variable region. In addition, an anti-target scFv may also contain a cysteine ​​clamp between cysteine ​​residue 44 of the heavy chain variable region and cysteine ​​residue 100 of the light chain variable region, while an anti-CD3 scFv does not contain an engineered cysteine ​​clamp. An scFc may contain a hinge cysteine ​​clamp, a natural CH2 / CH3 cysteine ​​clamp, and / or an engineered CH2 cysteine ​​clamp (intrachain).

[0077] VH and VL contain CDRs, which are interspersed with more conserved regions called framework regions ("FRs"). Each variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of VL are referred to as "LCDR1, LCDR2, and LCDR3" and the three CDRs of VH are referred to as "HCDR1, HCDR2, and HCDR3". The CDRs contain most of the residues that form specific interactions with the antigen; i.e., the CDRs contain most of the residues that contact with antigen residues. The assignment of amino acids to the CDR domains within the VL and HL regions of the TCE molecules of the present invention is based on the well-known Kabat numbering convention (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 (1991)). It is also understood that other numbering conventions may be used, such as those of Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) and / or North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

[0078] In the most general sense, the T cell engager ("TCE") molecules described herein comprise a single chain polypeptide capable of binding two different antigens. "TCE molecule" may be used interchangeably with "BiTE molecule." BiTE molecules may comprise scFv or scFab, so long as they are bispecific (meaning they bind two targets simultaneously (target antigen and CD3)). TCE molecules are antigen-binding molecules. TCE molecules of the invention may comprise an scFab that binds to a target (e.g., a tumor or target antigen) and an scFv that binds to CD3. Such molecules may have the following configuration from N-terminus to C-terminus: scFab (VH, CH1, linker, VL, Cκ or Cλ), linker, scFv (VH, linker, VL). Such molecules may also have the following configuration from N-terminus to C-terminus: scFab (VL, Cκ or Cλ, linker, VH, CH1), linker, scFv (VH, linker, VL). In some embodiments, the scFab binds CCR8. In certain embodiments, the TCE molecule comprises Cκ.

[0079] Alternatively, the TCE molecule of the present invention may be composed of an scFv that binds CCR8 and an scFv that binds CD3. Such a TCE molecule may have the following configuration from N-terminus to C-terminus: scFv that binds CCR8 (VH, linker, VL), linker, scFv that binds CD3 (VH, linker, VL).

[0080] The TCE molecules of the present invention may also have a half-life extension (HLE) moiety. The HLE moiety may extend the in vivo half-life of the TCE molecules of the present invention. Non-limiting examples of half-life extension moieties include Fc polypeptides, single chain Fc polypeptides (scFc), albumin, albumin fragments, moieties that bind to albumin or neonatal Fc receptor (FcRn), derivatives of fibronectin engineered to bind to albumin or a fragment thereof, peptides, single domain protein fragments, or other polypeptides that may extend serum half-life. In other embodiments, the half-life extension moiety may be a non-polypeptide molecule, such as, for example, polyethylene glycol (PEG). In some embodiments, the HLE is a single chain Fc ("scFc").

[0081] An scFc is a fusion protein in which a CH2 and CH3 (Fc1) are linked to another CH2 and CH3 (Fc2) via a linker to form a continuous protein chain, the linker being long enough to allow the protein chain to fold back on itself. In some embodiments, an scFc includes a cysteine ​​clamp. An scFc may also include an Ig-Fc hinge region, or a portion of an Ig-Fc hinge region. The hinge is the amino terminal group towards the CH2 domain, and an scFc may have the following configuration: (Fc1: hinge, CH2, CH3), linker, (Fc2: hinge, CH2, CH3). It is envisioned that the hinge region promotes dimerization. Such Fc polypeptide molecules can be obtained, for example, but not limited to, by papain digestion of the immunoglobulin region, which results in a dimer of two Fc polypeptides. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the following Fc polypeptide sequences: IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc region, IgD Fc region, and IgE Fc region (see, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)).

[0082] The TCE molecules of the invention having an HLE portion (e.g., scFc) may have the following configuration: scFab (VH, CH1, linker, VL, Ck), linker, scFv (VH, linker, VL), linker, scFc (hinge, CH2, CH3, linker, hinge, CH2, CH3). The TCE molecules of the invention having an HLE portion may also have the following configuration: scFab (VL, Cκ or Cλ, linker, VH, CH1), linker, scFv (VH, linker, VL). The TCE molecules of the invention having an HLE portion may also have the following configuration: scFv binding to CCR8 (VH, linker, VL), linker, scFv binding to CD3 (VH, linker, VL), scFc (hinge, CH2, CH3, linker, hinge, CH2, CH3). The scFc may also be referred to herein as Fc1 (hinge, CH2, CH3), linker, Fc2 (hinge, CH2, CH3).

[0083] FIG. 1 shows an example of the structure of a TCE molecule of the present invention.

[0084] It will be appreciated that the TCE molecules of the present invention may have at least one amino acid substitution, provided that the TCE molecule retains the same or better desired binding specificity (e.g., binding to CCR8 and / or CD3). Thus, modifications to the TCE molecule structure are encompassed within the scope of the present invention. Such modifications may include amino acid substitutions that may be conservative or non-conservative without impairing the desired binding ability of the binding construct. Conservative amino acid substitutions may include non-natural amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in a living system. These include peptidomimetics and other forms in which amino acid moieties are reversed or inverted. Conservative amino acid substitutions may also include the replacement of natural amino acid residues with normative residues, which have little effect on the polarity or charge of the amino acid residue at that position.

[0085] The TCE molecules of the present invention may include fragments of the amino acid sequences described herein.

[0086] The TCE molecules of the present invention can bind to a target antigen (e.g., an antigen expressed on a tumor cell) and to CD3 expressed on a T cell. The target antigen can be a human protein, or a protein from another species, e.g., mouse, rat, rabbit, and / or cynomolgus monkey. The target antigen can be any protein expressed on a tumor cell when treating cancer. Non-limiting examples of target antigens include CCR8, claudin-6, and MAGE-B2.

[0087] In another embodiment, the invention provides a vector comprising a nucleic acid encoding a polypeptide of the invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0088] A recombinant expression vector of the invention may contain a nucleic acid of the invention in a form suitable for expressing the nucleic acid in a host cell. The recombinant expression vector contains one or more regulatory sequences selected based on the host cell to be used for expression, which are operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., the SV40 early gene enhancer, the Rous sarcoma virus promoter, and the cytomegalovirus promoter), those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences; see Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 11:287, which are incorporated herein by reference in their entireties). 236:1237), and those that induce inducible expression of a nucleotide sequence in response to a particular treatment or condition (e.g., the metallothionin promoter in mammalian cells, and tet- and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems. One of ordinary skill in the art will appreciate that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein.

[0089] In another embodiment, the present invention provides a host cell into which a recombinant expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells or their derivatives, such as Veggie CHO and related cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or the CHO line DXB-11, which is deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20). Additional CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861), and UV20 (ATCC#CRL-1862). Additional host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), AM-1 / D cells (described in U.S. Pat. No. 6,210,924), HeLa cells, the BHK (ATCC CRL 10) cell line, the CV1 / EBNA cell line (ATCC CCL 70) derived from the African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10:2821), 293, 293 EBNA, or MSR. Examples of such cells include human embryonic kidney cells such as 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary explants, HL-60 cells, U937 cells, HaK cells, or Jurkat cells.Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0090] Typically, expression vectors used in any of the host cells contain sequences for maintaining the plasmid, as well as sequences for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences", typically include one or more of the following nucleotide sequences in certain embodiments: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. The leader sequence may include the amino acid sequence represented by SEQ ID NO: 213 (MDMRVPAQLL GLLLLWLRGA RC), which is encoded by SEQ ID NO: 214 (atggacatga gagtgcctgc acagctgctg ggcctgctgc tgctgtggct gagaggcgcc agatgc). The leader sequence may comprise the amino acid sequence represented by SEQ ID NO: 215 (MAWALLLLTL LTQGTGSWA), which is encoded by SEQ ID NO: 216 (atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcc). The leader polynucleotide sequence may comprise the polynucleotide sequence represented by SEQ ID NO: 594 (ATGGACATGAGAGTGCCTGCACAGCTGCTGGGCCTGCTGCTGCTGTGGCTGAGAGGCGCCAGATG).

[0091] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or gene transfer techniques. For stable transfection of mammalian cells, it is known that only a small percentage of cells can integrate the foreign DNA into their genome, depending on the expression vector and gene transfer technique used. To identify and select such integrants, a gene encoding a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Additional selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells that have been stably transfected with the introduced nucleic acid can be identified by drug selection, among other methods (e.g., cells that have integrated the selectable marker gene survive, while other cells die).

[0092] The polynucleotides encoding the amino acid sequences of the TCE molecules of the present invention can be of any length appropriate for the desired use or function, can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. Those skilled in the art will recognize that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein is encoded by a multitude of other nucleic acid sequences. Also, mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues can be performed.

[0093] Transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification procedures. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian polypeptides that are substantially free of contaminating endogenous materials. Cells containing nucleic acids encoding the TCE molecules of the invention also include hybridomas.

[0094] In some embodiments, a vector is provided that comprises the nucleic acid molecule described herein.In some embodiments, the present invention comprises a host cell that comprises the nucleic acid molecule described herein.In some embodiments, a nucleic acid molecule is provided that encodes the TCE molecule described herein.In some embodiments, a pharmaceutical composition is provided that comprises at least one TCE molecule described herein.

[0095] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the invention.

[0096] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of either C-terminal carboxyl group.

[0097] Another type of covalent modification of TCE molecules that is included within the scope of the present invention involves altering the glycosylation pattern of a protein. Glycosylation patterns, as known in the art, can depend both on the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues, discussed below) or on the host cell or organism in which the protein is produced. Particular expression systems are discussed below.

[0098] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a single sugar, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0099] The TCR-CD3 complex is a heteromultimer that includes heterodimers containing TCRα and TCRβ or TCRγ and TCRδ, as well as various CD3 chains from among the CD3 zeta (CD3ζ), CD3 epsilon (CD3ζ3), CD3 gamma (CD3γ), and CD3 delta (CD3δ) chains.

[0100] The CD3 receptor complex is a protein complex and is composed of four chains. In mammals, the complex contains the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains bind to the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor CD3 complex and generate activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are very closely related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as the immunoreceptor tyrosine-based activation motif or ITAM for short, which is essential for the signaling ability of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene, which is present on chromosome 11 in humans. The most preferred epitope of CD3 epsilon is comprised within amino acid residues 1 to 27 of the extracellular domain of human CD3 epsilon. The TCE molecules according to the invention are typically and advantageously expected to exhibit less undesired non-specific T cell activation in specific immunotherapy, which translates into a lower risk of side effects.

[0101] In some embodiments, the effector cell protein can be human CD3 epsilon (CD3ζ) chain, which can be part of a multimeric protein. Alternatively, the effector cell protein can be human and / or cynomolgus TCR alpha, TCR beta, TCR delta, TCR gamma, CD3 beta (CD3β) chain, CD3 gamma (CD3γ) chain, CD3 delta (CD3δ) chain, or CD3 zeta (CD3ζ) chain.

[0102] Additionally, in some embodiments, the TCE molecule may bind to CD3 zeta chains from non-human species such as mice, rats, rabbits, New World monkeys, and / or Old World monkey species, including, but not limited to, the following mammalian species: Mus musculus, Rattus, Rattus norvegicus, Macaca fascicularis, Papio hamadryas, Papio, Papio anubis, Papio cynocephalus, Papio ursinus, Callithrix jacchus, Saguinus oedipus, and Saimiri sciureus. Therapeutic molecules with comparable activity in humans and species commonly used for preclinical testing, such as mice and monkeys, could simplify and expedite drug development and ultimately lead to improved outcomes. Such advantages can be critical in the lengthy and costly process of bringing a drug to market.

[0103] "Treatment" and / or "treating" and / or "treating", as used interchangeably herein, are intended to refer to any process in which there may be a slowing, interruption, arrest, control, halt, or reversing of the progression of the disorders described herein, but does not necessarily indicate a complete elimination of all disorder symptoms. Treatment includes administration of the TCE molecules of the present invention for the treatment of a disease or condition in a human that will benefit from the activity of the TCE molecules of the present invention, and includes: (a) inhibiting further progression of the disease; and (b) relieving the disease, i.e., causing remission of the disease or disorder, or alleviating the symptoms or complications thereof.

[0104] Suitable PD-L1 antagonist antibodies for use in combination with the TCE molecules of the invention include, but are not limited to, atezolizumab, avelumab, or durvalumab. Examples of PD-1 antagonist antibodies suitable for use in the methods of the invention include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, pidilizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, antibody 20C1.006 (SEQ ID NOs: 72-81), antibody 20C1.009 (SEQ ID NOs: 32-41 or SEQ ID NOs: 32-40 and SEQ ID NO: 212), antibody 20A2.003 (SEQ ID NOs: 42-51), antibody 20D4.006 (SEQ ID NOs: 52-61), or antibody 20D4.17 (SEQ ID NOs: 62-71), and any PD-1 antagonist antibody described in WO 2019 / 140196.

[0105] A therapeutically effective dose of the TCE molecule may be administered. The amount of the TCE molecule that constitutes a therapeutic dose may vary depending on the indication being treated, the patient's weight, and the patient's calculated skin surface area. The administration of the TCE molecule may be adjusted to achieve the desired effect. In many cases, repeated administration may be required. The dosage and frequency of administration may vary according to factors such as the route of administration, the specific TCE molecule used, the nature and severity of the disease to be treated, whether the condition is acute or chronic, and the size and general condition of the subject.

[0106] As used herein, "effective amount" refers to an amount of the TCE molecule of the present invention or a pharmaceutical composition containing the TCE molecule that will induce the desired therapeutic biological or medical response on a tissue, system, animal, mammal, or human as desired by a researcher, physician, or other clinician. The effective amount of the TCE molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the TCE molecule to induce the desired response in the individual. An effective amount is also an amount in which any toxic or adverse effects of the TCE molecule are outweighed by the therapeutically beneficial effects. Such benefits include ameliorating signs or symptoms of cancer. An effective amount can be readily determined by one of skill in the art using known techniques and by observing results obtained under analogous circumstances. An effective amount of the TCE molecule of the present invention may be administered in a single dose or in multiple doses. In determining the effective amount for a patient, attending medical practitioners will take into account several factors, including, but not limited to: the patient's size (e.g., weight or mass), body surface area, age, and health; the specific disease or disorder involved; the extent, involvement, or severity of the disease or disorder; the individual patient's response; the particular compound administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances known to the medical practitioner.

[0107] TCE molecules or pharmaceutical compositions containing such molecules can be administered by any feasible method. In the absence of some special formulation or circumstances, protein therapeutics are usually administered parenterally, for example, by injection, since oral administration causes protein hydrolysis in the acidic environment of the stomach. Possible administration routes are subcutaneous injection, intramuscular injection, intravenous injection, intraarterial injection, intralesional injection, or peritoneal bolus injection. TCE molecules can also be administered via injection, for example, intravenous injection or subcutaneous injection.

[0108] The TCE molecule may be administered in the form of a composition that includes one or more additional components, such as a physiologically acceptable carrier, excipient, or diluent. In some cases, the composition additionally includes one or more physiologically active agents. In various specific embodiments, the composition includes one, two, three, four, five, or six physiologically active agents in addition to the one or more TCE molecules. EXAMPLES

[0109] Working Example Example: TCE molecule affinity Claudin-6 TCE molecular affinity Claudin-6 T cell engager ("TCE") molecules are tested for affinity to human claudin-6. The TCE molecules are represented below in Table 1 by their unique identifiers. For example, the TCE molecule "CL6 3C1 HL CC x I2C x scFc" refers to a TCE molecule having, from N-terminus to C-terminus, an engineered cysteine ​​clamp ("CC"; clamp between VH44 and VL100 (Kabat numbering)) that binds claudin-6 ("CL6") and has a VH N-terminal to the VL, an I2C scFv that targets CD3 (VH N-terminal to the VL), and an scFc. The TCE molecule "CL6 3C1 HL scFab x I2C x scFc" refers to a TCE molecule having an scFab that binds claudin-6 with a VH N-terminus toward the VL, an I2C scFv that targets CD3 (VH N-terminus toward the VL), and an scFc. "x" represents a linker. The CDR sequences for both CL6 3C1 molecules are identical. Figure 1 shows the general structure of each molecule. The human claudin-6 sequence is represented by UniProt entry P56747, including its variants and isoforms.

[0110] The cell-based affinity of the TCE molecules is determined by nonlinear regression (monosite-specific binding) analysis. CHO cells transfected with human claudin-6 are incubated with decreasing concentrations of TCE molecules (up to 50 nM, step 1:1, 10 steps) for 16 hours at 4°C. Bound TCE molecules are detected by Alexa Fluor 488-conjugated AffiniPure Fab Fragment Goat Anti-Human IgG (H+L). Fixed cells are detected by FACS flow and signals are detected by fluorescence cytometry. The equilibrium dissociation constant (Kd) values ​​of each are calculated by the monosite-specific binding evaluation tool of GraphPad Prism software. The average Kd values ​​and standard deviations are calculated by Microsoft Excel. The average Kd values ​​are calculated from three independent experiments.

[0111] Essentially following the procedure described above, the following affinities were obtained:

[0112] [Table 1]

[0113] As shown in Table 1, these data demonstrate that TCE molecules with scFab targeted binders exhibit higher affinity for human claudin-6 compared to TCE molecules with scFv targeted binders.

[0114] Mage-B2 TCE molecule affinity Similarly, cell-based affinity of MAGE-B2 TCE molecules is determined by nonlinear regression (single-site specific binding) analysis. HLA-A*02:01 expressing T2 cells exogenously loaded with human MAGE-B2 peptides are incubated with decreasing concentrations of TCE molecules (up to 400 nM, 1:2 dilution, 11 steps) for 16 h at 4°C. Bound TCE molecules are detected by Alexa Fluor 488 conjugated AffiniPure Fab Fragment Goat Anti-Human IgG (H+L). Fixed cells are stained with DRAQ5, Far-Red Fluorescent Live-Cell Permeant DNA Dye and signals are detected by fluorescence cytometry. Respective equilibrium dissociation constant (Kd) values ​​are calculated by the single-site specific binding evaluation tool of GraphPad Prism software. Mean Kd values ​​and standard deviations are calculated by Microsoft Excel. Mean Kd values ​​are calculated from three independent experiments.

[0115] Essentially following the procedures described above, the following data was obtained.

[0116] [Table 2]

[0117] As shown in Table 2, these data demonstrate that TCE molecules with scFab targeted binders exhibit higher affinity for human MAGE-B2 compared to TCE molecules with scFv targeted binders.

[0118] Example: FACS-based cytotoxicity assay with unstimulated human PBMCs Human peripheral blood mononuclear cells (PBMCs) are prepared by Ficoll density gradient centrifugation from enriched lymphocyte preparations (buffy coats), a by-product of blood banks that collect blood for transfusion. Buffy coats are provided by local blood banks, and PBMCs are prepared the day after blood collection. After Ficoll density centrifugation and extensive washing with Dulbecco's PBS (Gibco), residual red blood cells are removed from PBMCs via incubation with red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 100 μM EDTA). Residual lymphocytes include mainly B and T lymphocytes, NK cells, and monocytes. PBMCs are maintained in culture at 37°C / 5% CO2 in RPMI medium (Gibco) with 10% FCS (Gibco).

[0119] Human T cells are isolated from PBMCs using a human pan T cell isolation kit (Miltenyi Biotec, # 130-096-535) according to the manufacturer's protocol. T cells are isolated using LS columns (Milteny Biotec, # 130-042-401). T cells are cultured in RPMI complete medium (RPMI1640; Biochrom AG, # FG1215) supplemented with 10% FBS (Bio West, # S1810), 1x non-essential amino acids (Biochrom AG, # K0293), 10 mM Hepes buffer (Biochrom AG, # L1613), 1 mM sodium pyruvate (Biochrom AG, # L0473), and 100 U / mL penicillin / streptomycin (Biochrom AG, # A2213) at 37°C in an incubator until required.

[0120] For analysis of cell lysis by flow cytometry assay, target antigen positive cells (claudin-6 stably transfected CHO cells or DAN-G stably transfected MAGE-B2 cells) are labeled as target cells and distinguished from effector cells using fluorescent membrane dye DiOC18 (DiO) (Thermo Fisher, #V22886). Briefly, cells were harvested, washed once with PBS, and resuspended in 2% (v / v) FBS and membrane dye DiO (5 μL / 10 e6 cells) in PBS containing 10 e After 3 min incubation at 37°C, cells were washed twice in complete RPMI medium and the cell count was adjusted to 1.25 x 10 e Adjust to 5 cells / mL. Determine cell viability using a Nucleocounter NC-250 (Chemometec) and Solution 18 Dye (Chemometec) containing acridine orange and DAPI.

[0121] To quantify the lysis of target antigen-positive cell lines in the presence of serial dilutions of TCE molecules, equal volumes of DiO-labeled target cells and effector cells (i.e., PBMCs devoid of CD14+ cells) are mixed to give a 10:1 E:T cell ratio. 160 μl of this suspension is transferred to each well of a 96-well plate. 40 μl of the corresponding serial dilutions of TCE molecules, negative control, or RPMI complete medium as an additional negative control are added. The TCE molecule-mediated cytotoxic reaction is allowed to proceed for 48 hours in a humidified incubator at 7% CO2. Cells are transferred to a new 96-well plate and loss of target cell membrane integrity is monitored by adding propidium iodide (PI) to a final concentration of 1 μg / mL. PI is a membrane-impermeable dye that is normally excluded from viable cells, but dead cells take it up and can be identified by fluorescence emission.

[0122] Samples are measured by flow cytometry on an iQue Plus (Intellicyt, now Sartorius) instrument and analyzed by Forecyt software (Intellicyt). Target cells are identified as DiO positive cells. PI negative target cells are classified as viable target cells. The percentage of cytotoxicity is calculated as dead target cells / target cells x 100. The percentage of cytotoxicity is plotted against the corresponding TCE molecule concentration using GraphPad Prism 7.04 software (Graph Pad Software, San Diego). Dose-response curves were analyzed by a four parametric logistic regression model for evaluation of sigmoidal dose-response curves with a fixed Hill slope to determine EC 50 Calculate the value.

[0123] Essentially following the procedures described above, the following data was obtained.

[0124] [Table 3]

[0125] These data demonstrate that claudin-6 TCE molecules with scFab targeted binders demonstrated improved potency of claudin-6 transfected CHO cells compared to TCE molecules with scFv targeted binders.

[0126] Data demonstrating MAGE-B2 TCE molecule cytotoxicity are shown in Table 4.

[0127] [Table 4]

[0128] These data demonstrate that MAGE-B2 TCE molecules with scFab targeted binders demonstrated enhanced lysis of DAN-G cells compared to TCE molecules with scFv targeted binders.

[0129] In similar experiments, scFab-containing TCE molecules with a disulfide bridge (cysteine ​​clamp ("CC"); cysteines at Kabat residues VH44 / VL100; CL6 3C1-02scFabCC x I2C x scFc) are tested for cytotoxic activity. These data (shown in Table 5) demonstrate that the scFab-containing TCE molecules with a disulfide bridge exhibited similar activity compared to the scFab-containing TCE molecule without this disulfide bridge. Both scFab-containing TCE molecules demonstrated increased cytotoxicity compared to the TCE molecules with the scFv target binder.

[0130] [Table 5]

[0131] Data from TCE molecules binding to target antigen and CD3 are shown below in Table 6. TCE molecules contained a (G4S)8 linker (top row), a (G4S)6 linker (second row), a disulfide bridge stabilized (Kabat VH44 / VL100) scFv target binding portion (third row), or a (G4S)8 linker (bottom row) in the scFab(VH-CH1-linker-VL-Ck)-linker-aCd3scFv(VH-linker-VL)-linker-scFc(Fc-linker-Fc) configuration.

[0132] [Table 6]

[0133] These data demonstrate that the scFab-containing TCE molecules with the scFab(G4S)8 linker exhibited the highest cytotoxic activity compared to the other molecules tested. The scFab-containing TCE molecules with the VL-Ck-linker-VH-CH1-aCD3-scFc configuration demonstrated the lowest activity compared to the scFab-containing TCE molecules with the VH-CH1-VL-Ck-aCD3-scFc configuration. This may be due to the configuration difference.

[0134] Example: Application of the scFab portion of the TCE format Multi-target TCE molecules are tested for cytotoxicity. As shown in the structure of FIG. 2, the TCE molecules tested have two anti-target scFvs (CD22 11-C3 CC scFv x CD20 29-F5 CC scFv x I2C x scFc and CD20 99-E5 CC scFv x CD22 28-B7N655 CC scFv x I2C x scFc), an anti-target scFab, and an anti-target scFv (CD20 99-E5 scFab x CD22 28-B7N655 CC scFv x I2C x scFc), or two anti-target scFabs (CD22 11-C3 scFab x CD20 29-F5 scFab x I2C x scFc). The TCE molecules tested have an anti-CD3 scFv ("I2C") and scFc. 11-C3, 29-F5, 99-E5, and 28-B7N655 refer to target binders such as 11-C3 scFab that have the same CDRs as 11-C3 scFv. Cytotoxicity of Raji cells (double positive for CD20 and CD22) was determined essentially as described above, giving the following data:

[0135] [Table 7]

[0136] As shown in Table 7, TCE molecules with scFab as both target binders (first row) have improved potency against single positive target cells (CD20 transfected CHO) and against double positive Raji cells compared to TCE molecules with scFv as both target binders (second row). In addition, TCE molecules with CD20 binding scFab (third row) demonstrate improved potency for CD20 transfected CHO cells and double positive Raji cells compared to TCE molecules with CD20 binding scFv (fourth row). .

[0137] Example: Evaluation of TCE molecule protein surface hydrophobicity To measure protein surface hydrophobicity, the isolated and prepared TCE molecule monomers adjusted to a specified protein concentration are transferred into an autosampler fitting sample vial and measured by FPLC system. A hydrophobic interaction chromatography (HIC) column is equilibrated with preparation buffer, and a predetermined volume of protein solution is applied at a constant preparation buffer flow rate. Detection is performed by light absorption at OD280nm.

[0138] Elution behavior is determined by peak shape and mathematically calculating the downward sloping slope of each signal peak, with a steeper slope / higher slope value indicating less hydrophobic interactions on the protein surface compared to constructs with flatter elution behavior and lower slope values.

[0139] Essentially following the procedures described above, the following data was obtained: "I2C" refers to an scFv that binds CD3. "CC" refers to an scFv that contains an engineered cysteine ​​clamp between the cysteine ​​at position 44 (VH) and the cysteine ​​at position 100 (VL) (Kabat).

[0140] [Table 8]

[0141] [Table 9]

[0142] [Table 10]

[0143] [Table 11]

[0144] These data demonstrate the degree of interaction of the applied constructs with the hydrophobic column matrix surface. In most cases, the peak slope for the scFab containing TCE molecule is steeper and has a higher mathematical value compared to the other TCE molecules tested. Constructs with higher surface hydrophobicity will elute with a larger elution volume and a smaller curve steepness since they interact more strongly with the matrix compared to constructs with lower surface hydrophobicity.

[0145] Example: Evaluation of TCE molecular aggregation temperature Elevated temperature can destabilize protein constructs, exposing structures that were originally buried by protein folding. The structures can be sticky and can come into contact with other constructs, causing aggregation and therefore a larger hydrodynamic radius. Molecules with higher aggregation temperatures are more stable compared to molecules with lower aggregation temperatures.

[0146] To determine the aggregation temperature of scFab-containing TCE molecules, isolated and prepared TCE molecule monomers adjusted to a defined protein concentration are pipetted in duplicate into a 96-well plate and covered with paraffin oil. The 96-well plate is transferred to a dynamic light scattering DLS reader that can heat the plate at a defined rate within a defined temperature range. Measurements are performed from 40°C to 70°C at a defined rate of temperature increase. Detection is performed by dynamic light scattering, which determines the hydrodynamic radius of the construct across a temperature gradient. The temperature at the onset of the increase in hydrodynamic radius is defined as the aggregation temperature. "I2C" refers to scFvs that bind to CD3. "CC" refers to scFvs that contain an engineered clamp cysteine ​​(cysteine ​​clamp between cysteine ​​at position 44 (VH) and cysteine ​​at position 100 (VL) (Kabat)) that binds to the target (i.e., MAGE-B2 in Table 11).

[0147] [Table 12]

[0148] [Table 13]

[0149] [Table 14]

[0150] These data demonstrate that the scFab TCE molecules are more stable than TCE molecules with scFv target binders, as they exhibit a higher average aggregation temperature compared to the other TCE molecules tested (with scFv target binders).

[0151] Example: Cell-based CCR8 binding competition assay The effect of biochemical competition with the CCR8 ligand CCL1 on the CCR8 binding properties of CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules is assessed by flow cytometry based on an engineered variant of the human T lymphocyte cell line HuT 78, which expresses native CCR8 but does not express the human CD3 epsilon chain. The cell line had a defined knockout in the CD3E gene.

[0152] Fifty thousand cells are incubated with 200 nM recombinant human CCL1 (Abcam, cat. no. ab9854) (diluted in PBS / 2% FCS) or with PBS / 2% FCS in 50 μl for 55 min at 4° C. Purified CCR8-binding TCE and scFab-containing CCR8-binding TCE molecules (diluted in PBS / 2% FCS) are added to a final concentration of 100 nM in a total volume of 100 μl and then incubated for 45 min at 4° C. For negative controls, PBS / 2% FCS is added instead of purified CCR8-binding TCE or scFab-containing CCR8-binding TCE molecules. After three washes, bound molecules are detected with the Fc gamma fragment-specific antibody R-phycoerythrin-conjugated anti-human IgG (Jackson, cat. no. 109-116-098) (diluted 1:50 in PBS / 2% FCS) for 45 min at 4° C. After 3 washes, samples are measured on a FACSCanto II instrument (Becton Dickinson). All conditions are run in triplicate.

[0153] Data was analyzed by FlowJo software (FlowJo / Becton Dickinson) to generate median values ​​for PE signals (median PE). From three replicates per condition, the mean median PE values ​​(with standard deviation) and the ratio of mean median PE in CCL1-treated conditions to mean median PE in CCL1-untreated conditions were calculated using Excel software (Microsoft).

[0154] Essentially following the procedures described above, the following data was obtained.

[0155] Table 14 shows the mean median PE-signal (with standard deviation) and the ratio of the mean median PE in CCL1-treated conditions to CCL1-untreated conditions. Identifiers of CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules are shown in the left column.

[0156] "I2E" represents a scFv that binds to CD3. The amino acid sequences of TCE1 TCE molecules (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 113 to 128. The amino acid sequences of TCE8 TCE molecules (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 97 to 112. The amino acid sequences of TCE2 TCE molecules (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 49 to 64.

[0157] [Table 15]

[0158] These data demonstrate that the presence of CCL1 did not affect the binding of the CCR8-binding TCE molecule TCE1 or the scFab-containing CCR8-binding TCE molecule TCE1, whereas CCL1 blocked the binding of the TCE8 and TCE2 TCE molecules.

[0159] Example: Evaluation of surface hydrophobicity of CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules To determine the surface hydrophobicity of scFab-containing CCR8-binding TCE molecules, the isolated and prepared CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecule monomers adjusted to a defined protein concentration are transferred into an autosampler fitting sample vial and measured by FPLC system. A hydrophobic interaction chromatography (HIC) column is equilibrated with preparation buffer, and a defined volume of protein solution is applied at a constant preparation buffer flow rate. Detection is performed by optical absorption at OD280nm. The elution behavior is determined by peak shape, and the downward sloping signal peak slope is mathematically calculated respectively. A steeper slope / higher slope value indicates less hydrophobic interaction of the protein surface compared to constructs with flatter elution behavior and lower slope value. Essentially following the procedure described above, the following data was obtained.

[0160] The amino acid sequences of the TCE4 TCE molecule (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 17 to 32. The amino acid sequences of the TCE1 TCE molecule (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 113 to 128. The amino acid sequences of the TCE8 TCE molecule (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 97 to 112. The amino acid sequences of the TCE2 TCE molecule (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 49 to 64. The amino acid sequences of the TCE7 TCE molecule (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 81 to 96. The amino acid sequences of the TCE5 TCE molecules (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 33 to 48. The amino acid sequences of the TCE6 TCE molecules (scFab-I2E-scFc or scFv CC x I2E x scFc) are shown by SEQ ID NOs: 65 to 80. "CC" stands for an engineered cysteine ​​clamp between the cysteine ​​at residue 44 of the heavy chain variable region and residue 100 of the light chain variable region of the anti-target scFv.

[0161] [Table 16]

[0162] These data demonstrate that the HIC elution peak slope for scFab-containing CCR8-binding TCE molecules is steeper and has a mathematically higher value compared to CCR8-binding TCE molecules having an scFv that binds to CCR8.

[0163] Example: CCR8 TCE molecule affinity The cell-based affinity of CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules is determined by nonlinear regression (single-site specific binding) analysis. CHO cells expressing human CCR8 (SEQ ID NO: 131), cynomolgus CCR8 (SEQ ID NO: 129), or cynomolgus CCR8 (T4R; SEQ ID NO: 130) are incubated with decreasing concentrations of CCR8 bispecific constructs (50-3200 nM, step 1:2, 11 steps) for 16 hours at 4°C. Bound CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules are detected with Alexa Fluor 488-conjugated AffiniPure Fab Fragment Goat Anti-Human IgG (H+L). Fixed cells are stained with DRAQ5, a Far-Red fluorescent live cell permeant DNA dye, and signals are detected by fluorescence cytometry. The equilibrium dissociation constant (Kd) value is calculated by the site-specific binding evaluation tool of GraphPad Prism software. The average Kd value and affinity gap are calculated by Microsoft Excel. The average Kd value is calculated from two or three independent experiments. The affinity gap is determined by dividing the cynomolgus monkey Kd by the human Kd. The following data was obtained essentially according to the procedure described above.

[0164] [Table 17]

[0165] These cell-based affinity measurements demonstrate that TCE molecules such as TCE1, with or without scFab, have high affinity for both human CCR8 and cynomolgus CCR8 without T4R mutations. The affinity of the TCE1 TCE molecule was reduced for cynomolgus cells carrying T4R mutations.

[0166] In addition, the TCE molecule CCR8 TCE2 could not be produced in sufficient quantities. However, a TCE molecule with an scFab portion (CCR8 TCE2 scFab) could be produced, demonstrating that the scFab portion provides an advantage in molecule production.

[0167] Example: Evaluation of aggregation temperature of CCR8-bound TCE molecule and scFab-containing CCR8-bound TCE molecule To measure the aggregation temperature, the isolated and prepared CCR8-binding TCE molecule and scFab-containing CCR8-binding TCE molecule monomers adjusted to a specified protein concentration are pipetted in duplicate into a 96-well plate and overlaid with paraffin oil. The 96-well plate is transferred to a dynamic light scattering DLS reader that can heat the plate at a specified rate within the indicated temperature range. Measurements are performed from 40°C to 70°C with a defined rate of temperature increase. Detection is performed by dynamic light scattering, which determines the hydrodynamic radius of the construct across a temperature gradient. The temperature at the onset of the increase in hydrodynamic radius is defined as the aggregation temperature. Essentially following the procedure described above, the following data was obtained.

[0168] [Table 18]

[0169] Essentially following the procedures described above, scFab-containing CCR8-binding TCE molecules demonstrated higher aggregation temperatures compared to CCR8-binding TCE molecules having scFvs that bind to CCR8. As described above, the TCE molecule CCR8 TCE2 could not be produced in sufficient quantities, but a TCE molecule having an scFab portion (CCR8 TCE2 scFab) could be produced.

[0170] Example: CCR8 molecule cytotoxicity assay using unstimulated human PBMCs Human peripheral blood mononuclear cells (PBMCs) are prepared by Ficoll density gradient centrifugation from enriched lymphocyte preparations (buffy coats), a by-product of blood banks that collect blood for transfusion. Buffy coats are provided by peripheral blood banks, and PBMCs are prepared on the same day of blood collection. After Ficoll density centrifugation and extensive washing with Dulbecco's PBS (Gibco), residual red blood cells are removed from the PBMCs via incubation with red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 100 μM EDTA). Centrifugation of the PBMCs at 100×g removes platelets via the supernatant. Residual lymphocytes mainly include B and T lymphocytes, NK cells, and monocytes. PBMCs are maintained in culture at 37° C. / 5% CO2 in RPMI medium (Gibco) with 10% FCS (Gibco).

[0171] CD14 + For cell depletion, human CD14 microbeads (Milteny Biotec, MACS, #130-050-201) were used. For NK cell depletion, human CD56 microbeads (MACS, #130-050-401) were used. PBMCs were counted and centrifuged at 300×g for 10 min at room temperature. The supernatant was discarded and the cell pellet was resuspended in MACS isolation buffer [80 μL / 10 7 Cells; resuspended in PBS (Invitrogen, #20012-043), 0.5% (v / v) FBS (Gibco, #10270-106), 2 mM EDTA (Sigma-Aldrich, #E-6511). CD14 microbeads and CD56 microbeads (20 μl / 107 Add MACS isolation buffer (1-2 ml / 10 7 After centrifugation (see above), discard the supernatant and resuspend the cells in MACS isolation buffer (500 μL / 10 8 CD14 / CD56 negative cells are isolated using LS columns (Miltenyi Biotec, #130-042-401). PBMCs devoid of CD14+ / CD56+ cells are cultured at 37°C in RPMI complete medium, i.e. RPMI1640 (Biochrom AG, #FG1215) supplemented with 10% FBS (Biochrom AG, #S0115), 1x non-essential amino acids (Biochrom AG, #K0293), 10 mM Hepes buffer (Biochrom AG, #L1613), 1 mM sodium pyruvate (Biochrom AG, #L0473), and 100 U / mL penicillin / streptomycin (Biochrom AG, #A2213).

[0172] Fluorescent membrane dye DiOC for analysis of cell lysis by flow cytometry assay 18 Human CCR8 or macaque CCR8 transfected CHO cells are labeled as target cells to distinguish them from effector cells using (DiO) (Molecular Probes, #V22886). Briefly, cells are harvested, washed once with PBS, and resuspended in 2% (v / v) FBS and membrane dye DiO (5 μL / 10 6 in PBS containing 10 6 After 3 min incubation at 37°C, cells were washed twice in complete RPMI medium and the cell number was adjusted to 1.25 x 10 5 Adjust to cells / mL. Determine cell viability using a NC-250 cell counter (Chemometec).

[0173] To quantify the lysis of cyno or human CCR8-transfected CHO cells in the presence of serial dilutions of CCR8-binding TCE molecules or scFab-containing CCR8-binding TCE molecules, equal volumes of DiO-labeled target cells and effector cells (i.e., CD14 + PBMCs (cell-free) are mixed to give a 10:1 E:T cell ratio. 80 μl of this suspension is transferred to each well of a 96-well plate. 20 μl of serial dilutions of CCR8-binding TCE molecules or scFab-containing CCR8-binding TCE molecules and a negative control (CD3-based TCE molecule recognizing an irrelevant target antigen) or RPMI complete medium as an additional negative control are added. The TCE molecule or scFab-containing TCE molecule-mediated cytotoxic reaction is allowed to proceed for 48 hours in a humidified incubator at 7% CO2. The cells are transferred to a new 96-well plate and loss of target cell membrane integrity is monitored by adding propidium iodide (PI) to a final concentration of 1 μg / mL. PI is a membrane-impermeable dye that is normally excluded from live cells, but dead cells take it up and can be identified by fluorescence emission.

[0174] Samples are measured by flow cytometry on an iQue Plus instrument and analyzed by Forecyt software (both Intellicyt). Target cells are identified as DiO positive cells. PI negative target cells are classified as viable target cells. The percentage of cytotoxicity is calculated as the number of dead target cells / number of target cells x 100. The percentage of cytotoxicity is plotted against the corresponding TCE molecule or TCE molecule containing scFab concentration using GraphPad Prism 5 software (Graph Pad Software, San Diego). Dose-response curves were analyzed by a four parametric logistic regression model for the evaluation of sigmoidal dose-response curves bordered by a fixed Hill slope to determine EC 50 Calculate the value.

[0175] Essentially following the procedures described above, data from a 48 hour FACS-based cytotoxicity assay of scFab-containing CCR8-binding TCE molecules with human CCR8 transfected CHO cells (clone #A2) and human CCR8 isoform A27G (SEQ ID NO:132) transfected CHO cells as target cells and unstimulated human PBMCs (CD14- / CD56-) as effector cells (E:T ratio 10:1) are shown below in Table 18.

[0176] [Table 19]

[0177] These data demonstrate that binders TCE1 and TCE8 exhibit comparable biological activity in the one- to two-digit pM range.

[0178] Data from a 48 hour FACS-based cytotoxicity assay of the bispecific constructs with Chinese cyno CCR8 (SEQ ID NO: 129) transfected CHO cells (clone #G4) and mauritian cyno CCR8 isoform T4R (SEQ ID NO: 130) transfected CHO cells as target cells and unstimulated human PBMCs (CD14- / CD56-) as effector cells (E:T ratio 10:1) are shown below in Table 19.

[0179] [Table 20]

[0180] These data demonstrate that only the TCE1 TCE molecule tested (with a scFab targeted to CCR8) exhibited pM bioactivity on the Chinese cynomolgus monkey CCR8 transfected CHO cell line. The other molecules tested did not demonstrate activity (indicated by an X in the table).

[0181] Data from a 48 hour FACS-based cytotoxicity assay of CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules with HUT-78 (CD3+) as target cells and unstimulated human PBMCs (CD14- / CD56-) as effector cells (E:T ratio 10:1) are shown below in Table 20. A four parametric logistic regression model for evaluation of sigmoidal dose-response curves bordered by a fixed Hill slope yielded EC 50 Determine the value.

[0182] [Table 21]

[0183] These data demonstrate that the binder TCE1 exhibits high biological activity on the endogenous cell line HUT-78 (CD3+).

[0184] Example: Luciferase-based cytotoxicity assay with unstimulated human PBMCs Isolation of effector cells and CD14 + and CD56 + Cell depletion is performed as described above. Target cells (described below) are harvested, spun down and diluted to 1.2×10 in complete RPMI medium. 5 Adjust to cells / mL. Determine cell viability using a Nucleocounter NC-250 (Chemometec) and Solution 18 Dye containing acridine orange and DAPI (Chemometec).

[0185] To quantify target cell lysis in the presence of serial dilutions of CCR8-binding TCE molecules or scFab-containing CCR8-binding TCE molecules, equal volumes of luciferase-positive target cells and effector cells (i.e., CD14 + ;CD56 +PBMCs (cell-free) are mixed to give a 10:1 E:T cell ratio. 42 μL of this suspension is transferred to each well of a 384-well plate. 8 μL of the corresponding serial dilutions of CCR8-binding TCE molecules or scFab-containing CCR8-binding TCE molecules, as well as a negative control (CD3-based TCE molecule recognizing an unrelated target antigen) or RPMI complete medium as an additional negative control are added. The TCE molecule cytotoxicity reaction is allowed to proceed for 48 hours in a humidified incubator at 5% CO2. Then, 25 μL of substrate (Steady-Glo® Reagent, Promega) is transferred to the 384-well plate. Only live luciferase-positive cells are allowed to react with the substrate to produce a luminescent signal. Samples are measured by a SPARK microplate reader (TECAN) and analyzed by Spark Control Magellan software (TECAN). The percentage of cytotoxicity is calculated as (1-RLU Sample / RLU Negative-Control ) × 100. RLU means relative light units. "Negative control" means cells without TCE molecules.

[0186] The percentage of cytotoxicity is plotted against the corresponding polyspecific TCE molecule concentration using GraphPad Prism 7.04 software (Graph Pad Software, San Diego). Dose-response curves were analyzed by a four parametric logistic regression model for the evaluation of sigmoidal dose-response curves with a fixed Hill slope to determine EC 50 Calculate the value.

[0187] Essentially following the procedures described above, the following data was obtained: Data shown is for CCR8-binding TCE molecules containing CCR8 scFab against a human CCR8-positive HUT-78 CD3ε-ko cell line (parental cells and clones as shown below) or a human CCR8-negative (ko) HUT-78 (CD3ε+) cell line (monoplex clone 2E3; negative control) as target cells and unstimulated human PBMCs (CD14− / CD56−) as effector cells (E:T ratio 10:1).

[0188] [Table 22]

[0189] These data demonstrate that TCE1 scFab-containing TCE molecules exhibit superior biological activity on the human CCR8-positive HUT-78 (CD3ε-) cell line compared to TCE8 and TCE2.

[0190] Examples: dual specific binding and species cross-reactivity For confirmation of binding to human CCR8 and CD3 and to cyno CCR8 and CD3, TCE1 scFab-containing CCR8-binding TCE molecules, control TCE molecules (CD3-based TCE molecules recognizing an unrelated target antigen), or anti-CCR8 antibody clones L263G8 (BioLegend) and 433H (BD) are tested by flow cytometry using CHO cells transfected with human CCR8 and / or macaque CCR8, human CCR8 and CD3 positive human cell line HUT-78, human CCR8 positive and CD3 negative HUT-78 cell line, CD3 expressing human T cell leukemia cell line HPB-all (DSMZ, Braunschweig, ACC483), and cynomolgus monkey CD3 expressing T cell line LnPx 4119.

[0191] Essentially following the procedures described above, the following data was obtained. "-" indicates no signal was detected. Data represents the mean BL2 of the sample (channel in which signal was detected) / mean BL2 of the second antibody control.

[0192] [Table 23]

[0193] These data demonstrate that the scFab-containing TCE1 TCE molecule bound to human and cynomolgus CCR8 in human or cyno-expressing CHO cells, but not to cynomolgus CCR8 carrying T4R mutations. These data also demonstrate that TCE1 binds to CCR8 expressed on a naturally expressing CCR8 cell line (HUT-78 CCR8+ / CD3e-).

[0194] Example: Epitope clustering of CCR8 TCE molecules The extracellular domain of human CCR8 contains three loops of 35 amino acids and an N-terminal peptide. For epitope mapping, the N-terminal peptide of human CCR8 (designated P_1-35 (SEQ ID NO: 133)) is divided into three consecutive segments (designated P_1-12 (SEQ ID NO: 134), P_13-24 (SEQ ID NO: 135), P_25-35 (SEQ ID NO: 136)). Two additional overlapping fragments (designated P_7-18 (SEQ ID NO: 137) and P_19-30 (SEQ ID NO: 138)) are formed to cover adjacent N- or C-terminal regions of the consecutive segments. A V5 tag is fused to the C-terminus of the full-length N-terminal peptide and all the truncated N-terminal peptides of human CCR8 mentioned above via a G4S linker. The V5 tag is followed by chicken albumin fused via an additional G4S linker, followed by a FLAG tag, BAP (biotin acceptor protein) for in vivo biotinylation, and H3G (each fused via an SG linker). All the above constructs are cloned into pEFDHFR vector and transiently transfected into HEK 293 cells.

[0195] HEK 293 cells (1x10E8) are resuspended in 100ml FreeStyle expression medium (Gibco 12338-018) and transfected with 4ml OptiMEM (Gibco 31985-047), 100μl 293fectin (Invitrogen 12347-019), and 50μg DNA encoding either full-length or truncated N-terminal CCR8 constructs according to the manufacturer's protocol. Cells are grown in FreeStyle expression medium for 72 hours at 130 rpm in a humidified incubator with 8% CO2. Cells are centrifuged at 1500 rpm for 10 minutes and the supernatant is collected. 10ml of supernatant from each transfected cell or 9ml of HEK 293 cells as a negative control is concentrated 20x to 500μL with Amicon Ultra-15 tubes (UFC901008). 18 × 10 for each full-length and truncated N-terminal CCR8 construct, as well as the HEK 293 negative control. E Six washed streptavidin-beads (Streptavidin Microspheres, 6 μm; Polysciences 24172-1) are resuspended in 500 μL of concentrated supernatant and incubated for 1 h with gentle shaking. Beads coupled with each antigen or negative control are washed and stored overnight at 4° C.

[0196] To confirm expression of full-length and truncated N-terminal CCR8 constructs and binding to streptavidin-beads, 2 × 10 cells were stained. EFive beads are incubated with 5 μg / mL of anti-FLAG antibody (clone M2, Sigma F3165 / F1804) and 5 μg / mL of anti-V5 antibody (clone SV5-Pk1; AbD Serotec, MCA 1360), as well as a 1:100 dilution of PE-labeled anti-mouse Fcy secondary antibody (Jackson 115-116-071). The antigen-bound beads are incubated with three different anti-human CCR8 antibodies. Binding of two of the anti-human CCR8 antibodies (clone L263G8; BioLegend, 360602 and clone 433H; BD 747578; each at 5 μg / ml) is detected with a 1:100 dilution of PE-labeled anti-mouse Fcy secondary antibody (Jackson 115-116-071). Binding of anti-human CCR8 antibody (polyclonal; Abcam, ab140796) is detected with a 1:50 dilution of PE-labeled anti-goat Fcy secondary antibody (Jackson 109-116-098).

[0197] To evaluate the binding of CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules to the full-length and truncated N-terminal CCR8 constructs bound to streptavidin-beads, the beads are incubated with 5 μg / mL of each TCE molecule. The binding of these CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules is detected using 2 μg / ml of anti-histidine antibody (clone AD1.1.10; AbD Serotec MCA 1396) and a 1:100 dilution of PE-labeled anti-mouse Fcy secondary antibody (Jackson 115-116-071). All antibodies, CCR8-binding TCE molecules, and scFab-containing CCR8-binding TCE molecules are diluted in PBS with 2% FBS, and all incubations are performed at 4° C. for 45 minutes (primary antibody) or 30 minutes (secondary antibody). Washing is performed with PBS with 2% FBS. The final suspension buffer before FACS analysis is also PBS with 2% FBS. Antibody and TCE binding is detected using Intellicyte IQue. Changes in mean fluorescence are analyzed by Intellicyte IQue and FlowJo. Binding to the various full-length and truncated N-terminal CCR8 constructs is reflected as a positive signal detected by flow cytometry.

[0198] Essentially following the procedures described above, expression and binding of full-length and various truncated N-terminal CCR8 constructs to streptavidin-beads can be confirmed by flow cytometry.

[0199] [Table 24]

[0200] These data demonstrate that the anti-human CCR8 antibodies bound to the full-length N-terminal peptide of human CCR8 P_1-35, indicating that the anti-human CCR8 antibodies recognized the N-terminal peptide of human CCR8. Neither antibody showed binding to streptavidin beads alone or to the HEK 293 control. The anti-human CCR8 antibodies (clone L263G8 and clone 433H) showed the same binding pattern, while the polyclonal anti-human CCR8 antibody showed additional binding to the overlapping fragment P_7-18.

[0201] As shown in Table 23, when the binding to the truncated N-terminal peptides of CCR8 was evaluated, the CCR8-binding TCE molecules and the scFab-containing CCR8-binding TCE molecules showed two different binding patterns. For example, TCE4, TCE3, and TCE8 each bound to the truncated N-terminal peptide P_13-24. However, TCE1 bound to the truncated N-terminal peptide P_1-12.

[0202] [Table 25]

[0203] [Table 26]

[0204] These data demonstrate that CCR8-binding TCE molecules and scFab-containing CCR8-binding TCE molecules bound to the full-length N-terminal CCR8 peptide P_1-35. All molecules except TCE1 bound to the truncated N-terminal CCR8 peptide P_13-24. Interestingly, TCE1 binds to the truncated N-terminal CCR8 peptide P_1-12. This suggests that TCE1 binds to a unique epitope on CCR8, which may contribute to the high affinity and biological activity of TCE1.

[0205] Additional scFvs representing new sequence families binding in the 1-12 amino acid epitope cluster were generated and screened by phage display.

[0206] array TCE3 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 1) SYVMH TCE3 CCR8 scFv and scFab HCDR2 (SEQ ID NO:2) VISYDGSSQYYTDSVKG TCE3 CCR8 scFv and scFab HCDR3 (SEQ ID NO:3) GRLATAILFDY TCE3 CCR8 scFv and scFab LCDR1 (SEQ ID NO: 4) KSSQSLLYSDGKTYLF TCE3 CCR8 scFv and scFab LCDR2 (SEQ ID NO:5) EVSNRFS TCE3 CCR8 scFv and scFab LCDR3 (SEQ ID NO: 6) MQSIKLPLT TCE3 CCR8 scFv VH (SEQ ID NO: 7) [ka] TCE3 CCR8 scFv VL (SEQ ID NO: 8) EILMTQTPLSLSVTPGQPASISCKSSQSLLYSDGKTYLFWYLQRPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYYCMQSIKLPLTFGCGTKVEIK TCE3 CCR8 scFv (SEQ ID NO: 9) [ka] TCE3 scFv (CCR8) x scFv (CD3) TCE (SEQ ID NO: 10) [ka] TCE3 scFv (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 11) [ka] TCE3 CCR8 scFab VH and CH1 (SEQ ID NO: 12) [ka] TCE3 CCR8 scFab VL and Ck (SEQ ID NO: 13) [ka] TCE3 CCR8 scFab (SEQ ID NO: 14) [ka] TCE3 scFab (CCR8) x scFv (CD3) TCE (SEQ ID NO: 15) [ka] TCE3 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 16) [ka] TCE4 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 17) SYGMH TCE4 CCR8 scFv and scFab HCDR2 (SEQ ID NO: 18) VISYDGSNKYYADSVKG TCE4 CCR8 scFv and scFab HCDR3 (SEQ ID NO: 19) GRYFDWFLFDY TCE4 CCR8 scFv and scFab LCDR1 (SEQ ID NO: 20) KSSQSLLHSDGKTYLF TCE4 CCR8 scFv and scFab LCDR2 (SEQ ID NO: 21) EVSNRFS TCE4 CCR8 scFv and scFab LCDR3 (SEQ ID NO: 22) MQSLRLPLT TCE4 CCR8 scFv VH (CCR8) (SEQ ID NO: 23) [ka] TCE4 CCR8 scFv VL (CCR8) (SEQ ID NO: 24) DTVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLFWYLQKPGQPPQLLISEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQSLRLPLTFGCGTKVEIK TCE4 CCR8 scFv (SEQ ID NO: 25) [ka] TCE4 scFv (CCR8) x scFv (CD3) TCE (SEQ ID NO: 26) [ka] TCE4 CCR8 scFv (CCR8) x scFv (CD3) TCE x scFc (SEQ ID NO: 27) [ka] TCE4 CCR8 scFab VH and CH1 (SEQ ID NO:28) [ka] TCE4 CCR8 scFab VL and Ck (SEQ ID NO:29) [ka] TCE4 CCR8 scFab (SEQ ID NO: 30) [ka] TCE4 scFab (CCR8) x scFv (CD3) TCE (SEQ ID NO: 31) [ka] TCE4 CCR8 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 32) [ka] TCE5 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 33) NAWMS TCE5 CCR8 scFv and scFab HCDR2 (SEQ ID NO: 34) RIKRKTDGGTTDYAAPVKG TCE5 CCR8 scFv and scFab HCDR3 (SEQ ID NO: 35) VTLVRGVIFDY TCE5 CCR8 scFv and scFab LCDR1 (SEQ ID NO: 36) RVSQSVSSSQLA TCE5 CCR8 scFv and scFab LCDR2 (SEQ ID NO: 37) GASSRAT TCE5 CCR8 scFv and scFab LCDR3 (SEQ ID NO: 38) QQYGNSRT TCE5 CCR8 scFv VH (CCR8) (SEQ ID NO: 39) [ka] TCE5 CCR8 scFv VL (CCR8) (SEQ ID NO: 40) EIVLTQFPGTLSLSPGESATLSCRVSQSVSSSQLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLIISRLEPEDFAVYYCQQYGNSRTFGCGTKVEIK TCE5 CCR8 scFv (SEQ ID NO: 41) [ka] TCE5 scFv (CCR8) x scFv (CD3) TCE (SEQ ID NO: 42) [ka] TCE5 scFv (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 43) [ka] TCE5 CCR8 scFab VH and CH1 (SEQ ID NO: 44) [ka] TCE5 CCR8 scFab VL and Ck (SEQ ID NO: 45) [ka] TCE5 CCR8 scFab (SEQ ID NO: 46) [ka] TCE5 scFab (CCR8) x scFv (CD3) (SEQ ID NO: 47) [ka] TCE5 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 48) [ka] TCE2 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 49) NYGMH TCE2 CCR8 scFv and scFab HCDR2 (SEQ ID NO:50) VISYDGSNKFYADSVKG TCE2 CCR8 scFv and scFab HCDR3 (SEQ ID NO:51) AGGIGRFDY TCE2 CCR8 scFv and scFab LCDR1 (SEQ ID NO:52) KYSQSLLHSDGKTYLF TCE2 CCR8 scFv and scFab LCDR2 (SEQ ID NO:53) EVSNRFS TCE2 CCR8 scFv and scFab LCDR3 (SEQ ID NO:54) MQTLKLPLT TCE2 CCR8 scFv VH (SEQ ID NO: 55) [ka] TCE2 CCR8 scFv VL (SEQ ID NO: 56) DFVMTQTPLSLSVTPGQPASISCKYSQSLLHSDGKTYLFWYLQKPGQPPHLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCMQTLKLPLTFGCGTKVEIN TCE2 CCR8 scFv (SEQ ID NO: 57) [ka] TCE2 scFv (CCR8) x scFv (CD3) (SEQ ID NO: 58) [ka] TCE2 scFv (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 59) [ka] TCE2 CCR8 scFab VH and CH1 (SEQ ID NO:60) [ka] TCE2 scFab CCR8 VL and Ck (SEQ ID NO: 61) [ka] TCE2 CCR8 scFab (SEQ ID NO: 62) [ka] TCE2 scFab (CCR8) x scFv (CD3) (SEQ ID NO: 63) [ka] TCE2 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 64) [ka] TCE6 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 65) NAWMS TCE6 CCR8 scFv and scFab HCDR2 (SEQ ID NO: 66) RIKRKTDGGTTDYAAPVKG TCE6 CCR8 scFv and scFab HCDR3 (SEQ ID NO: 67) VTLVRGIIFDY TCE6 CCR8 scFv and scFab LCDR1 (SEQ ID NO: 68) RVSQSVSSSQLA TCE6 CCR8 scFv and scFab LCDR2 (SEQ ID NO: 69) GASSRAT TCE6 CCR8 scFv and scFab LCDR3 (SEQ ID NO: 70) QQYGNSRT TCE6 CCR8 scFv VH (SEQ ID NO: 71) [ka] TCE6 CCR8 scFv VL (SEQ ID NO: 72) EIVLTQSPGTLSLSPGESATLSCRVSQSVSSSQLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSRTFGCGTKVEIK TCE6 CCR8 scFv (SEQ ID NO: 73) [ka] TCE6 scFv (CCR8) x scFv (CD3) (SEQ ID NO: 74) [ka] TCE6 scFv (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 75) [ka] TCE6 CCR8 scFab VH and CH1 (SEQ ID NO:76) [ka] TCE6 CCR8 scFab VL and Ck (SEQ ID NO: 77) [ka] TCE6 CCR8 scFab (SEQ ID NO: 78) [ka] TCE6 scFab (CCR8) x scFv (CD3) (SEQ ID NO: 79) [ka] TCE6 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 80) [ka] TCE7 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 81) NAWMS TCE7 CCR8 scFv and scFab HCDR2 (SEQ ID NO: 82) RIKRKTDGGTTDYAAPVKG TCE7 CCR8 scFv and scFab HCDR3 (SEQ ID NO: 83) VTLVRGVIFDY TCE7 CCR8 scFv and scFab LCDR1 (SEQ ID NO: 84) RASQSVSSSQLA TCE7 CCR8 scFv and scFab LCDR2 (SEQ ID NO: 85) GASSRAT TCE7 CCR8 scFv and scFab LCDR3 (SEQ ID NO: 86) QQYGNSRT TCE7 CCR8 scFv VH (SEQ ID NO: 87) [ka] TCE7 CCR8 scFv VL (SEQ ID NO: 88) EIVLTQSPGTLSLSPGESATLSCRASQSVSSSQLAWYQQKPGQTPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSRTFGCGTKVEIK TCE7 CCR8 scFv (SEQ ID NO: 89) [ka] TCE7 scFv (CCR8) x scFv (CD3) (SEQ ID NO: 90) [ka] TCE7 scFv (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 91) [ka] TCE7 CCR8 scFab VH and CH1 (SEQ ID NO:92) [ka] TCE7 CCR8 scFab VL and Ck (SEQ ID NO: 93) [ka] TCE7 CCR8 scFab (SEQ ID NO: 94) [ka] TCE7 scFab (CCR8) x scFv (CD3) (SEQ ID NO: 95) [ka] TCE7 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 96) [ka] TCE8 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 97) NAWMS TCE8 CCR8 scFv and scFab HCDR2 (SEQ ID NO: 98) RIKRRTDGGTTDYAAPVKD TCE8 CCR8 scFv and scFab HCDR3 (SEQ ID NO: 99) VTMVRGVIADY TCE8 CCR8 scFv and scFab LCDR1 (SEQ ID NO: 100) RASQSVSSGSLA TCE8 CCR8 scFv and scFab LCDR2 (SEQ ID NO: 101) GASSRAT TCE8 CCR8 scFv and scFab LCDR3 (SEQ ID NO: 102) QQYGSSRT TCE8 CCR8 scFv VH (SEQ ID NO: 103) [ka] TCE8 CCR8 scFv VL (SEQ ID NO: 104) EIVLTQSPGTLSLSPGERATLSCRASQSVSSGSLAWYQQKLGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSRTFGCGTKVELK TCE8 CCR8 scFv (SEQ ID NO: 105) [ka] TCE8 scFv (CCR8) x scFv (CD3) (SEQ ID NO: 106) [ka] TCE8 scFv (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 107) [ka] TCE8 CCR8 scFab VH and CH1 (SEQ ID NO: 108) [ka] TCE8 CCR8 scFab VL and Cκ (SEQ ID NO: 109) [ka] TCE8 CCR8 scFab (SEQ ID NO: 110) [ka] TCE8 scFab (CCR8) x scFv (CD3) (SEQ ID NO: 111) [ka] TCE8 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 112) [ka] TCE1 CCR8 scFv and scFab HCDR1 (SEQ ID NO: 113) NARMG TCE1 CCR8 scFv and scFab HCDR2 (SEQ ID NO: 114) RIKSKTEGGTRDYAAPVKG TCE1 CCR8 scFv and scFab HCDR3 (SEQ ID NO: 115) YSGV TCE1 CCR8 scFv and scFab LCDR1 (SEQ ID NO: 116) KSSQSVLYSSNNKNYLA TCE1 CCR8 scFv and scFab LCDR2 (SEQ ID NO: 117) WASTRES TCE1 CCR8 scFv and scFab LCDR3 (SEQ ID NO: 118) QQYYSIPIT TCE1 CCR8 scFv VH (SEQ ID NO: 119) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNARMGWVRQAPGKCLEWVGRIKSKTEGGTRDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTSYSGVWGQGTMVTVSS TCE1 CCR8 scFv VL (SEQ ID NO: 120) EIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYHQKPGQSPKLLISWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYYCQQYYSIPITFGCGTKVEIK TCE1 CCR8 scFv (SEQ ID NO: 121) [ka] TCE1 scFv (CCR8) x scFv (CD3) (SEQ ID NO: 122) [ka] TCE1 scFv (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 123) [ka] TCE1 CCR8 scFab VH and CH1 (SEQ ID NO: 124) [ka] TCE1 CCR8 scFab VL and Ck (SEQ ID NO: 125) [ka] TCE1 CCR8 scFab (SEQ ID NO: 126) [ka] TCE1 scFab (CCR8) x scFv (CD3) (SEQ ID NO: 127) [ka] TCE1 scFab (CCR8) x scFv (CD3) x scFc (SEQ ID NO: 128) [ka] Cynomolgus monkey China origin CCR8 (SEQ ID NO: 129) [ka] Cynomolgus monkey Mauritius origin T4R CCR8 (SEQ ID NO: 130) [ka] Human CCR8 (SEQ ID NO: 131) [ka] Human A27G CCR8 (SEQ ID NO: 132) [ka] CCR8 P_1-35 peptide (SEQ ID NO: 133) MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGK CCR8 P_1-12 peptide (SEQ ID NO: 134) MDYTLDLSVTTV CCR8 P_13-24 peptide (SEQ ID NO: 135) TDYYYPDIFSSP CCR8 P_25-35 peptide (SEQ ID NO: 136) CDAELIQTNGK CCR8 P_7-18 peptide (SEQ ID NO: 137) LSVTTVTDYYYP CCR8 P_19-30 peptide (SEQ ID NO: 138) DIFSSPCDAELI Antibody 20C1.009 LCDR1 (SEQ ID NO: 139) RASQGISNWLA Antibody 20C1.009 LCDR2 (SEQ ID NO: 140) AASSLQS Antibody 20C1.009 LCDR3 (SEQ ID NO: 141) QQAESFPHT Antibody 20C1.009 HCDR1 (SEQ ID NO: 142) SYDMS Antibody 20C1.009 HCDR2 (SEQ ID NO: 143) LISGGGSQTYYAESVKG Antibody 20C1.009 HCDR3 (SEQ ID NO: 144) PSGHYFYAMDV Antibody 20C1.009 VL (SEQ ID NO: 145) DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAESFPHTFGGGTKVEIK Antibody 20C1.009 VH (SEQ ID NO: 146) [ka] Antibody 20C1.009 LC (SEQ ID NO: 147) [ka] Antibody 20C1.009 HC (SEQ ID NO: 148) [ka] Antibody 20A2.3 LCDR1 (SEQ ID NO: 149) SGDKLGDKYAS Antibody 20A2.3 LCDR2 (SEQ ID NO: 150) QDRKRPS Antibody 20A2.3 LCDR3 (SEQ ID NO: 151) QAFESSTEV Antibody 20A2.3 HCDR1 (SEQ ID NO: 152) NYGMH Antibody 20A2.3 HCDR2 (SEQ ID NO: 153) LIWYDASKKYYAESVKG Antibody 20A2.3 HCDR3 (SEQ ID NO: 154) DPSSLTGSTGYYGMDV Antibody 20A2.3 VL (SEQ ID NO: 155) SYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDRKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAFESSTEVFGGGTKLTVL Antibody 20A2.3 VH (SEQ ID NO: 156) [ka] Antibody 20A2.3 LC (SEQ ID NO: 157) [ka] Antibody 20A2.3 HC (SEQ ID NO: 158) [ka] Antibody 20D4.6 LCDR1 (SEQ ID NO: 159) SGDALPKKYAY Antibody 20D4.6 LCDR2 (SEQ ID NO: 160) EDAKRPS Antibody 20D4.6 LCDR3 (SEQ ID NO: 161) YSTDASGNHRV Antibody 20D4.6 HCDR1 (SEQ ID NO: 162) DYSMS Antibody 20D4.6 HCDR2 (SEQ ID NO: 163) GINWNGGRTYADAVKG Antibody 20D4.6 HCDR3 (SEQ ID NO: 164) EFNNFESNWFDP Antibody 20D4.6 VL (SEQ ID NO: 165) SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKPGQAPVLVISEDAKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDASGNHRVFGGGTKLTVL Antibody 20D4.6 VH (SEQ ID NO: 166) [ka] Antibody 20D4.6 LC (SEQ ID NO: 167) [ka] Antibody 20D4.6 HC (SEQ ID NO: 168) [ka] Antibody 20D4.17 LCDR1 (SEQ ID NO: 169) SGDALPKKYAY Antibody 20D4.17 LCDR2 (SEQ ID NO: 170) EDAKRPS Antibody 20D4.17 LCDR3 (SEQ ID NO: 171) YSTDASGNHRV Antibody 20D4.17 HCDR1 (SEQ ID NO: 172) DYSMS Antibody 20D4.17 HCDR2 (SEQ ID NO: 173) GINWNAGRTYADAVKG Antibody 20D4.17 HCDR3 (SEQ ID NO: 174) EFNNFESNWFDP Antibody 20D4.17 VL (SEQ ID NO: 175) SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKPGQAPVLVISEDAKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDASGNHRVFGGGTKLTVL Antibody 20D4.17 VH (SEQ ID NO: 176) [ka] Antibody 20D4.17 LC (SEQ ID NO: 177) [ka] Antibody 20D4.17 HC (SEQ ID NO: 178) [ka] Antibody 20C1.006 LCDR1 (SEQ ID NO: 179) RASQGISNWLA Antibody 20C1.006 LCDR2 (SEQ ID NO: 180) AASSLQS Antibody 20C1.006 LCDR3 (SEQ ID NO: 181) QQAESFPHT Antibody 20C1.006 HCDR1 (SEQ ID NO: 182) SYDMS Antibody 20C1.006 HCDR2 (SEQ ID NO: 183) LISGGGSNTYYAESVKG Antibody 20C1.006 HCDR3 (SEQ ID NO: 184) PSGHYFYAMDV Antibody 20C1.006 VL (SEQ ID NO: 185) DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAESFPHTFGGGTKVEIK Antibody 20C1.006 VH (SEQ ID NO: 186) [ka] Antibody 20C1.006 LC (SEQ ID NO: 187) [ka] Antibody 20C1.006 HC (SEQ ID NO: 188) [ka] G4S linker (SEQ ID NO: 189) GGGGS (G4S)4 linker (SEQ ID NO: 190) GGGGSGGGGSGGGGSGGGGS I2C-HCDR1 (SEQ ID NO: 191) KYAMN I2C-HCDR2 (SEQ ID NO: 192) RIRSKYNNYATYYADSVKD I2C-HCDR3 (SEQ ID NO: 193) HGNFGNSYISYWAY I2C-LCDR1 (SEQ ID NO: 194) GSSTGAVTSGNYPN I2C-LCDR2 (SEQ ID NO: 195) GTKFLAP I2C-LCDR3 (SEQ ID NO: 196) VLWYSNRWV I2C-VH (SEQ ID NO: 197) [ka] I2C-VL (SEQ ID NO: 198) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL I2E-HCDR1 (SEQ ID NO: 199) KYAIN I2E-HCDR2 (SEQ ID NO: 200) RIRSKYNNYATYYADAVKD I2E-HCDR3 (SEQ ID NO: 201) AGNFGSSYISYWAY I2E-LCDR1 (SEQ ID NO: 202) GSSTGAVTSGNYPN I2E-LCDR2 (SEQ ID NO: 203) GTKFLAP I2E-LCDR3 (SEQ ID NO: 204) VLWYSNRWV I2E-VH (SEQ ID NO: 205) [ka] I2E-VL (SEQ ID NO: 206) QTVVTQEPSLTVSPGGTVTITCGSSTGAVTSGNYPNWVQKKPGQAPRGLIGGTKFLAPGTPARFSGSLSGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGSGTKLTVL G4Q linker (SEQ ID NO: 207) GGGGQ (G4Q)4 linker (SEQ ID NO: 208) GGGGQGGGGQGGGGQGGGGQ mCCR8 (SEQ ID NO: 209) [ka] Rat CCR8 (SEQ ID NO: 210) [ka] hCCR4 (SEQ ID NO: 211) [ka] Antibody 20C1.009 HC (SEQ ID NO: 212) without the C-terminal lysine [ka] Leader sequence (SEQ ID NO:213) MDMRVPAQLL GLLLLWLRGA RC DNA encoding the leader sequence of SEQ ID NO:213 (SEQ ID NO:214) atggacatga gagtgcctgc acagctgctg ggcctgctgc tgctgtggct gagaggcgcc agatgc. Leader sequence (SEQ ID NO:215) MAWALLLLTL LTQGTGSWA DNA encoding the leader sequence of SEQ ID NO:215 (SEQ ID NO:216) atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcc

[0207] [Table 27]

[0208] [Table 28]

[0209] [Table 29]

[0210] [Table 30]

[0211]

Table 31

[0212]

Table 32

[0213]

Table 33

[0214]

Table 34

[0215]

Table 35

[0216]

Table 36

[0217]

Table 37

[0218]

Table 38

[0219]

Table 39

[0220]

Table 40

[0221]

Table 41

[0222]

Table 42

[0223]

Table 43

[0224]

Table 44

[0225]

Table 45

[0226]

Table 46

[0227]

Table 47

[0228]

Table 48

[0229]

Table 49

[0230]

Table 50

[0231]

Table 51

[0232]

Table 52

[0233]

Table 53

[0234]

Table 54

[0235]

Table 55

[0236]

Table 56

[0237]

Table 57

[0238]

Table 58

[0239]

Table 59

[0240]

Table 60

[0241]

Table 61

[0242]

Table 62

[0243]

Table 63

[0244]

Table 64

[0245]

Table 65

[0246]

Table 66

[0247]

Table 67

[0248]

Table 68

[0249]

Table 69

[0250]

Table 70

[0251]

Table 71

[0252]

Table 72

[0253]

Table 73

[0254]

Table 74

[0255]

Table 75

[0256]

Table 76

[0257]

Table 77

[0258]

Table 78

[0259]

Table 79

[0260]

Table 80

[0261]

Table 81

[0262]

Table 82

[0263] [Table 83]

[0264] [Table 84]

[0265] [Table 85]

[0266] [Table 86]

[0267] [Table 87]

[0268] [Table 88]

[0269] [Table 89]

[0270] DNA encoding CCR8 TCE 1.1HLE of SEQ ID NO: 227 (SEQ ID NO: 590) [ka] [ka]

[0271] DNA encoding CCR8 TCE 1.2HLE of SEQ ID NO: 238 (SEQ ID NO: 591) [ka] [ka]

[0272] DNA encoding CCR8 TCE 1.3HLE of SEQ ID NO: 249 (SEQ ID NO: 592) [ka] [ka]

[0273] DNA encoding CCR8 TCE 1.4HLE of SEQ ID NO: 260 (SEQ ID NO: 593) [ka] [ka]

[0274] Leader polynucleotide sequence (SEQ ID NO:594) ATGGACATGAGAGTGCCTGCACAGCTGCTGGGCCTGCTGCTGCTGTGGCTGAGAGGCGCCAGATG

[0275] [Table 90]

[0276] [Table 91]

[0277] [Table 92]

[0278] [Table 93]

[0279]

Table 94

[0280]

Table 95

[0281]

Table 96

[0282]

Table 97

[0283]

Table 98

[0284]

Table 99

[0285]

Table 100

[0286]

Table 101

[0287]

Table 102

[0288]

Table 103

[0289]

Table 104

[0290]

Table 105

[0291]

Table 106

[0292]

Table 107

[0293]

Table 108

[0294]

Table 109

[0295]

Table 110

[0296]

Table 111

[0297]

Table 112

[0298]

Table 113

[0299]

Table 114

[0300]

Table 115

[0301]

Table 116

[0302]

Table 117

[0303]

Table 118

[0304]

Table 119

[0305]

Table 120

[0306]

Table 121

[0307]

Table 122

[0308]

Table 123

[0309]

Table 124

[0310]

Table 125

[0311]

Table 126

[0312]

Table 127

[0313]

Table 128

[0314]

Table 129

[0315]

Table 130

[0316]

Table 131

[0317]

Table 132

[0318]

Table 133

[0319]

Table 134

[0320]

Table 135

[0321]

Table 136

[0322]

Table 137

[0323]

Table 138

[0324]

Table 139

[0325]

Table 140

[0326]

Table 141

[0327]

Table 142

[0328]

Table 143

[0329]

Table 144

[0330]

Table 145

[0331]

Table 146

[0332]

Table 147

[0333]

Table 148

[0334]

Table 149

[0335]

Table 150

[0336]

Table 151

[0337]

Table 152

[0338]

Table 153

[0339]

Table 154

[0340]

Table 155

[0341]

Table 156

[0342]

Table 157

[0343]

Table 158

[0344]

Table 159

[0345]

Table 160

[0346]

Table 161

[0347]

Table 162

[0348]

Table 163

[0349]

Table 164

[0350]

Table 165

[0351]

Table 166

[0352]

Table 167

[0353]

Table 168

[0354]

Table 169

[0355]

Table 170

[0356]

Table 171

[0357]

Table 172

[0358]

Table 173

[0359]

Table 174

[0360]

Table 175

[0361]

Table 176

[0362]

Table 177

[0363]

Table 178

[0364]

Table 179

[0365]

Table 180

[0366]

Table 181

[0367]

Table 182

[0368]

Table 183

[0369]

Table 184

[0370]

Table 185

[0371]

Table 186

[0372]

Table 187

[0373]

Table 188

[0374]

Table 189

[0375]

Table 190

[0376]

Table 191

[0377]

Table 192

[0378]

Table 193

[0379]

Table 194

[0380]

Table 195

[0381]

Table 196

[0382]

Table 197

[0383]

Table 198

Claims

1. A T cell engager (TCE) molecule comprising: (i) an scFab that binds to a tumor antigen, the scFab comprising a first heavy chain variable region (scFab VH), a CH1 domain, a first light chain variable region (scFab VL), and a Cκ or Cλ domain; and (ii) an scFv that binds to CD3 and comprises a second VL and a second VH, the TCE molecule being single-chain.

2. The TCE molecule according to claim 1, wherein the scFab comprises a C-terminal portion linked to the N-terminal portion of the scFv by a linker.

3. The TCE molecule according to claim 1 or 2, wherein the TCE molecule further comprises an scFc.

4. The TCE molecule according to claim 3, wherein the scFc comprises an N-terminal portion linked to the C-terminal portion of the scFv by a linker.

5. The TCE molecule according to claim 1, wherein the scFv binds to human CD3.

6. The TCE molecule according to claim 1, wherein the scFab has the following order from the N-terminus to the C-terminus: VH, CH1, VL, and Cκ or Cλ arrangement.

7. The TCE molecule according to claim 1, wherein the scFab has the following order from the N-terminus to the C-terminus: VL, Cκ or Cλ, VH, and CH1 arrangement.

8. The TCE molecule according to claim 1, wherein the scFab comprises a linker connecting the CH1 and the VL, and the linker is (G4S)6, (G4S)7, (G4S)8, (G4Q)6, (G4Q)7, or (G4Q)8.

9. The TCE molecule according to claim 1, wherein the TCE molecule comprises a linker connecting the scFab Cκ or Cλ and the scFab VH, and the linker is (G4S)6, (G4S)7, (G4S)8, (G4Q)6, (G4Q)7, or (G4Q)8.

10. The TCE molecule according to claim 1, wherein the CH1, Cκ, and / or Cλ domain is IgG, IgM, IgA, IgD, or IgE.

11. The TCE molecule according to claim 10, wherein the domain is IgG.

12. The TCE molecule according to claim 10, wherein the domain is IgG1.

13. The TCE molecule according to claim 1, wherein the scFab contains a cysteine clamp between CH1 and Cκ or Cλ.

14. The arrangement of the TCE molecule is, from the N-terminus to the C-terminus: VH-CH1-linker-VL-Ck or Cλ-linker-VH-linker-VL-linker-Fc1(CH2-CH3)-linker-Fc2(CH2-CH3), the TCE molecule according to claim 1.

15. The arrangement of the TCE molecule is, from the N-terminus to the C-terminus: VL-CH1-linker-VH-Ck or Cλ-linker-VH-linker-VL-linker-Fc1(CH2-CH3)-linker-Fc2(CH2-CH3), the TCE molecule according to claim 1.

16. The tumor antigen is CCR8, the TCE molecule according to claim 1.

17. The scFv that binds to CD3 is I2E, the TCE molecule according to claim 1.

18. The scFv that binds to CD3 is I2C, the TCE molecule according to claim 1.

19. A TCE molecule comprising: (i) a first scFv that binds to CCR8, the first scFv comprising a first VH region (CCR8 scFv VH) and a first VL region (CCR8 scFv VL); and (ii) a second scFv that binds to CD3, the second scFv comprising a second VH region and a second VL region, the TCE molecule being single-chain.

20. The first VH comprises HCDR1, HCDR2, HCDR3, and the first VL comprises LCDR1, LCDR2, and LCDR3: a. HCDR1 comprises an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 33, SEQ ID NO: 49, SEQ ID NO: 65, SEQ ID NO: 81, SEQ ID NO: 97, or SEQ ID NO: 113; b. HCDR2 comprises an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO: 50, SEQ ID NO: 66, or SEQ ID NO: 82; c. HCDR3 comprises an amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 51, SEQ ID NO: 67, or SEQ ID NO: 83; d. LCDR1 comprises an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68, or SEQ ID NO: 84; e. LCDR2 comprises an amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 37, SEQ ID NO: 53, SEQ ID NO: 69, or SEQ ID NO: 85, f. The LCDR3 contains the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 38, SEQ ID NO: 54, SEQ ID NO: 70, or SEQ ID NO: 86, The TCE molecule according to claim 1.

21. The HCDR1 contains the amino acid sequence represented by SEQ ID NO: 1, the HCDR2 contains the amino acid sequence represented by SEQ ID NO: 2, the HCDR3 contains the amino acid sequence represented by SEQ ID NO: 3, the LCDR1 contains the amino acid sequence represented by SEQ ID NO: 4, the LCDR2 contains the amino acid sequence represented by SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence represented by SEQ ID NO:

6. The TCE molecule according to claim 20.

22. The HCDR1 contains the amino acid sequence represented by SEQ ID NO: 17, the HCDR2 contains the amino acid sequence represented by SEQ ID NO: 18, the HCDR3 contains the amino acid sequence represented by SEQ ID NO: 19, the LCDR1 contains the amino acid sequence represented by SEQ ID NO: 20, the LCDR2 contains the amino acid sequence represented by SEQ ID NO: 21, and the LCDR3 contains the amino acid sequence represented by SEQ ID NO:

22. The TCE molecule according to claim 20.

23. The HCDR1 contains the amino acid sequence represented by SEQ ID NO: 33, the HCDR2 contains the amino acid sequence represented by SEQ ID NO: 34, the HCDR3 contains the amino acid sequence represented by SEQ ID NO: 35, the LCDR1 contains the amino acid sequence represented by SEQ ID NO: 36, the LCDR2 contains the amino acid sequence represented by SEQ ID NO: 37, and the LCDR3 contains the amino acid sequence represented by SEQ ID NO:

38. The TCE molecule according to claim 20.

24. The HCDR1 contains the amino acid sequence represented by SEQ ID NO: 49, the HCDR2 contains the amino acid sequence represented by SEQ ID NO: 50, the HCDR3 contains the amino acid sequence represented by SEQ ID NO: 51, the LCDR1 contains the amino acid sequence represented by SEQ ID NO: 52, the LCDR2 contains the amino acid sequence represented by SEQ ID NO: 53, and the LCDR3 contains the amino acid sequence represented by SEQ ID NO:

54. The TCE molecule according to claim 20.

25. HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 65, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 66, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 67, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 68, LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 69, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 70, the TCE molecule according to claim 20.

26. HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 81, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 82, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 83, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 84, LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 85, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 86, the TCE molecule according to claim 20.

27. HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 97, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 98, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 99, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 100, LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 101, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 102, the TCE molecule according to claim 20.

28. HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 113, HCDR2 comprises the amino acid sequence represented by SEQ ID NO: 114, HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 115, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 116, LCDR2 comprises the amino acid sequence represented by SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 118, the TCE molecule according to claim 20.

29. The first VH contains the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 71, SEQ ID NO: 87, SEQ ID NO: 103, or SEQ ID NO: 119, and the first VL contains the amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 40, SEQ ID NO: 56, SEQ ID NO: 72, SEQ ID NO: 88, SEQ ID NO: 104, or SEQ ID NO:

120. The TCE molecule according to claim 19.

30. The first scFv contains the amino acid sequence represented by SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, or 121. The TCE molecule according to claim 19.

31. Contains the amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 42, SEQ ID NO: 58, SEQ ID NO: 74, SEQ ID NO: 90, SEQ ID NO: 106, or SEQ ID NO:

122. The TCE molecule according to claim 19.

32. Further contains scFc, and the TCE molecule contains the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 59, SEQ ID NO: 75, SEQ ID NO: 91, SEQ ID NO: 107, or SEQ ID NO:

123. The TCE molecule according to claim 19.

33. The first VH and CH1 contain the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, or SEQ ID NO:

124. The TCE molecule according to claim 1.

34. Contains Cκ, and the first VL and Cκ contain the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 29, SEQ ID NO: 45, SEQ ID NO: 61, SEQ ID NO: 77, SEQ ID NO: 93, SEQ ID NO: 109, or SEQ ID NO:

125. The TCE molecule according to claim 1.

35. Contains the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 46, SEQ ID NO: 62, SEQ ID NO: 78, SEQ ID NO: 94, SEQ ID NO: 110, or SEQ ID NO:

126. The TCE molecule according to claim 1.

36. Contains the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 47, SEQ ID NO: 63, SEQ ID NO: 79, SEQ ID NO: 95, SEQ ID NO: 111, or SEQ ID NO:

127. The TCE molecule according to claim 1.

37. Contains the amino acid sequence represented by SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 48, SEQ ID NO: 64, SEQ ID NO: 80, SEQ ID NO: 96, SEQ ID NO: 112, or SEQ ID NO:

128. The TCE molecule according to claim 3.

38. A method of treating cancer in a patient, the method comprising administering to the patient an effective amount of the TCE molecule according to claim 1.

39. The method according to claim 38, wherein the cancer is a solid tumor.

40. The method according to claim 38 or 39, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer.

41. The method according to claim 38, further comprising administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody.

42. The method according to claim 41, wherein the PD-1 antagonist antibody or the PD-L1 antagonist antibody is administered before, simultaneously with, and / or after the administration of the TCE molecule.

43. The method according to claim 41 or 42, wherein the PD-1 antagonist antibody is pembrolizumab, nivolumab, semiprilumab, or antibody 20C1.

009.

44. The method according to claim 41 or 42, wherein the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab.

45. The TCE molecule according to claim 1 for use in therapy.

46. The TCE molecule according to claim 1 for use in treating cancer.

47. The TCE molecule according to claim 46 for use in treating cancer, wherein the cancer is a solid tumor.

48. The TCE molecule according to claim 46 or 47 for use in treating cancer, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer.

49. The TCE molecule according to claim 46 or 47 for use in treating cancer, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer.

50. The use according to claim 46 or 47 for use in treating cancer, further comprising administering to the patient a PD-1 antagonist antibody or a PD-L1 antagonist antibody.

51. The TCE molecule according to claim 50 for use in treating cancer, wherein the PD-1 antagonist antibody or the PD-L1 antagonist antibody is administered before, simultaneously with, and / or after the administration of the TCE molecule.

52. The PD-1 antagonist antibody is pembrolizumab, nivolumab, semiprimumab, or antibody 20C1.009, and the TCE molecule according to claim 50 for use in treating cancer.

53. The PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab, and the TCE molecule according to claim 50 for use in treating cancer.

54. Use of the TCE molecule according to claim 1 for the manufacture of a medicament for treating cancer.

55. The use according to claim 54, wherein the cancer is a solid tumor.

56. The use according to claim 54 or 55, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer.

57. The use according to claim 54 or 55, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer.

58. A pharmaceutical composition comprising the TCE molecule according to claim 1 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

59. A TCE molecule comprising an scFv comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences respectively shown by SEQ ID NOs: 217, 218, 219, 220, 221, and 220.

60. The TCE molecule according to claim 59, comprising VH and VL each comprising the amino acid sequences respectively shown by SEQ ID NOs: 223 and 224.

61. The TCE molecule according to claim 59 or 60, comprising an scFv comprising the amino acid sequence shown by SEQ ID NO:

225.

62. The TCE molecule according to claim 59, comprising the amino acid sequence shown by SEQ ID NO:

226.

63. The TCE molecule according to claim 62, comprising the amino acid sequence shown by SEQ ID NO:

227.

64. A TCE molecule comprising an scFv comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequences respectively shown by SEQ ID NOs: 228, 229, 230, 231, 232, and 233.

65. The TCE molecule according to claim 64, comprising VH and VL each comprising the amino acid sequences respectively shown by SEQ ID NOs: 234 and 235.

66. The TCE molecule according to claim 64 or 65, comprising an scFv comprising the amino acid sequence represented by SEQ ID NO:

236.

67. The TCE molecule according to claim 64, comprising the amino acid sequence represented by SEQ ID NO:

237.

68. The TCE molecule according to claim 67, comprising the amino acid sequence represented by SEQ ID NO:

238.

69. A TCE molecule comprising an scFab comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequence represented by SEQ ID NO: 239, 240, 241, 242, 243, and 244, respectively.

70. The TCE molecule according to claim 69, comprising VH-CH1 comprising the amino acid sequence represented by SEQ ID NO:

245.

71. The TCE molecule according to claim 69 or 70, comprising VL-Cκ comprising the amino acid sequence represented by SEQ ID NO:

246.

72. The TCE molecule according to claim 69, wherein the scFab comprises the amino acid sequence represented by SEQ ID NO:

247.

73. The TCE molecule according to claim 69, comprising the amino acid sequence represented by SEQ ID NO:

248.

74. The TCE molecule according to claim 73, comprising the amino acid sequence represented by SEQ ID NO:

249.

75. A TCE molecule comprising an scFab comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising the amino acid sequence represented by SEQ ID NO: 250, 251, 252, 253, 254, and 255, respectively.

76. The TCE molecule according to claim 75, comprising VH-CH1 comprising the amino acid sequence represented by SEQ ID NO:

256.

77. The TCE molecule according to claim 75 or 76, comprising VL-Cκ comprising the amino acid sequence represented by SEQ ID NO:

257.

78. The TCE molecule according to claim 75, wherein the scFab comprises the amino acid sequence represented by SEQ ID NO:

258.

79. The TCE molecule according to claim 75, comprising the amino acid sequence represented by SEQ ID NO:

259.

80. The TCE molecule according to claim 79, comprising the amino acid sequence represented by SEQ ID NO:

260.

81. A method for treating cancer in a patient, the method comprising administering to the patient an effective amount of the TCE molecule according to claim 59, 64, 69, or 75.

82. The method according to claim 81, wherein the cancer is a solid tumor. **Claim 83** The method according to claim 81, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. **Claim 84** The method according to claim 81, further comprising administering a PD-1 antagonist antibody or a PD-L1 antagonist antibody to the patient. **Claim 85** The method according to claim 84, wherein the PD-1 antagonist antibody or the PD-L1 antagonist antibody is administered before, simultaneously with, and / or after the administration of the TCE molecule. **Claim 86** The method according to claim 84, wherein the PD-1 antagonist antibody is pembrolizumab, nivolumab, semiprimab, or antibody 20C1.

009. **Claim 87** The method according to claim 84, wherein the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab. **Claim 88** The TCE molecule according to any one of claims 59, 64, 69, and 75, for use in therapy. **Claim 89** The TCE molecule according to any one of claims 59, 64, 69, and 75, for use in treating cancer. **Claim 90** The TCE molecule according to any one of claims 59, 64, 69, and 75, for use in treating cancer, wherein the cancer is a solid tumor. **Claim 91** The TCE molecule according to any one of claims 59, 64, 69, and 75, for use in treating cancer, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, triple-negative breast cancer, colorectal cancer, pancreatic cancer, or metastatic castration-resistant prostate cancer. **Claim 92** The TCE molecule according to any one of claims 59, 64, 69, and 75, for use in treating cancer, wherein the cancer is non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, or triple-negative breast cancer. **Claim 93** The use according to claim 93, further comprising administering a PD-1 antagonist antibody or a PD-L1 antagonist antibody to the patient. **Claim 94** The TCE molecule according to claim 93, for use in treating cancer, wherein the PD-1 antagonist antibody is pembrolizumab, nivolumab, semiprimab, or antibody 20C1.

009. **Claim 95** The TCE molecule for use in treating cancer according to claim 93, wherein the PD-L1 antagonist antibody is atezolizumab, avelumab, or durvalumab.

96. Use of a TCE molecule according to any one of claims 59, 64, 69 and 75 for the manufacture of a medicament for treating cancer.

97. Use according to claim 96, wherein the cancer is a solid tumor.

98. A pharmaceutical composition comprising a TCE molecule according to any one of claims 59, 64, 69 and 75, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

99. A method of treating cancer in a patient, comprising administering to the patient an effective amount of a CCR8 TCE molecule that binds to human CCR8 at an epitope, wherein the epitope comprises at least one residue of SEQ ID NO:

134.

100. The method according to claim 99, wherein the epitope comprises at least two residues of SEQ ID NO:

134.

101. The method according to claim 99, wherein the epitope comprises at least three residues of SEQ ID NO:

134.

102. The method according to claim 99, wherein the epitope comprises at least four residues of SEQ ID NO:

134.

103. The method according to claim 99, wherein the epitope comprises at least five residues of SEQ ID NO:

134.

104. The method according to claim 99, wherein the epitope comprises a threonine residue at position 4 of SEQ ID NO:

134.

105. A TCE molecule that binds to human CCR8, comprising the HCDR1 amino acid sequence of SEQ ID NO: 787; the HCDR2 amino acid sequence of SEQ ID NO: 788; the HCDR3 amino acid sequence of SEQ ID NO: 789; the LCDR1 amino acid sequence of SEQ ID NO: 336 (wherein X1 is K or R); the LCDR2 amino acid sequence of SEQ ID NO: 791; and the LCDR3 amino acid sequence of SEQ ID NO:

792.

106. The TCE molecule according to claim 105, wherein the TCE molecule comprises the VH amino acid sequence of SEQ ID NO: 965 and the VL amino acid sequence of SEQ ID NO: 342, X1 is K or R, X2 is H or Q, and / or X3 is S or P.

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