Multispecific antibodies and their use

Abstract

The present invention provides anti-CC motif chemokine receptor 8 (CCR8) antigen-binding molecules (e.g., bispecific antigen-binding molecules) and compositions thereof. The present invention also features polynucleotides, vectors, host cells, production methods, pharmaceutical compositions, methods for treating diseases or disorders such as cancer, methods for depleting regulatory T cells, compositions for related applications and uses, and kits for use in conjunction with one or more methods.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy, created on 17 January 2024, is named 50474-313WO2_Sequence_Listing_01_17_24 and has a size of 124,330 bytes.

[0002] Technical field The present invention relates to an antigen-binding molecule that binds to CC motif chemokine receptor 8 (CCR8), comprising a multispecific antibody, a composition thereof, and a method for treating diseases such as cancer. [Background technology]

[0003] Cancer and other cell proliferation disorders are characterized by the uncontrolled growth of cell subpopulations. They are the leading cause of death in developed countries and the second leading cause of death in developing countries. As of 2018, it is estimated that more than 17 million people are newly diagnosed with cancer each year, and 9.5 million people die from it. The probability of developing cancer more than doubles after the age of 70, so as the elderly population increases, the incidence of cancer also rises. Therefore, cancer care represents a significant and ever-increasing social burden.

[0004] Regulatory T (Treg) cells expressing the transcription factor Foxp3 are crucial for maintaining peripheral immune tolerance and preventing autoimmunity. Treg cells also constitute a major component of immune infiltration in solid tumors, promoting tumor development and progression by establishing an immunosuppressive tumor microenvironment and attenuating the anti-tumor immune response. Treg cells also hinder the effectiveness of immunotherapy. An increased proportion of Treg cells in tumor-infiltrating lymphocytes is associated with poorer outcomes in several cancer indications.

[0005] Several strategies targeting Treg cell depletion or inhibition have been shown to enhance antitumor immunity and lead to tumor growth inhibition in preclinical breast, melanoma, and colon cancer models. However, strategies targeting surface receptors expressed on both Treg cells and effector T cells have shown limited efficacy in established tumors, possibly due to the accompanying depletion of effector T cells, which are crucial for antitumor immunity.

[0006] Therefore, there is an unmet need in the field of developing effective therapeutic agents for the depletion of Treg cells for use in cancer treatment. [Overview of the project]

[0007] The present invention provides, in particular, antigen-binding molecules, compositions containing bispecific antigen-binding molecules (e.g., pharmaceutical compositions), polynucleotides encoding antigen-binding molecules, vectors, host cells, methods for production, and methods and uses thereof, including, but more specifically, bispecific antigen-binding molecules that bind to multispecific antigen-binding molecules, such as CC motif chemokine receptor 8 (CCR8) and activated T cell antigens (e.g., differentiation antigen group 3 (CD3)) (e.g., bispecific antibodies; including 2+1 T cell-dependent bispecific antibodies (TDBs); e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies).

[0008] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CC motif chemokine receptor 8 (CCR8), comprising the following six complementarity-determining regions (CDRs): (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) C comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6). (b) A first antigen-binding domain comprising DR-L3; and a second antigen-binding domain that binds to differentiated antigen group 3 (CD3), comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22).

[0009] In some embodiments, the first antigen-binding domain comprises a light chain variable region (VL) domain and a heavy chain variable region (VH) domain, where (a) the VL domain contains a proline residue at position 12 (numbering according to Kabat); and / or (b) the VL domain contains a lysine residue at position 38 and the VH domain contains a glutamate residue at position 39 (numbering according to Kabat). In some embodiments, the second antigen-binding domain comprises a VL domain and a VH domain, where the VL domain contains a glutamate residue at position 38 and the VH domain contains a lysine residue at position 39 (numbering according to Kabat). In some embodiments, (a) the first antigen-binding domain comprises one or more of the following eight framework regions (FRs): (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 14; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and / or (viii) comprising the amino acid sequence of SEQ ID NO: 16. (b) The second antigen-binding domain includes one or more of the following eight FRs: (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 25; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 26; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 27; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 28; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 29; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 30; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 31; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 32.

[0010] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24.

[0011] In some embodiments, the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and / or the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain. In some embodiments, the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, where (a) the Fab light chain of the first antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the first antigen-binding domain contains a lysine residue at position 183 (numbering follows Kabat); and / or (b) the Fab light chain of the second antigen-binding domain contains a lysine residue at position 133 and the Fab heavy chain of the second antigen-binding domain contains a glutamate residue at position 183 (numbering follows Kabat).

[0012] In some embodiments, the bispecific antigen-binding molecule further comprises an Fc domain containing a first subunit and a second subunit. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain includes modifications that facilitate association between the first and second subunits of the Fc domain.

[0013] In some embodiments, the bispecific antigen-binding molecule comprises one or more heavy chain constant domains, the one or more heavy chain constant domains selected from a first CH1(CH11) domain, a first CH2(CH21) domain, a first CH3(CH31) domain, a second CH1(CH12) domain, a second CH2(CH22) domain, and a second CH3(CH32) domain. In some embodiments, the first subunit comprises one or more heavy chain constant domains selected from the first CH2(CH21) domain and / or the first CH3(CH31) domain; the second subunit comprises one or more heavy chain constant domains selected from the second CH2(CH22) domain and / or the second CH3(CH32) domain. In some embodiments, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some embodiments, the CH31 domain and the CH32 domain each include a projection or cavity, and the projection or cavity of the CH31 domain can be located in the cavity or projection of the CH32 domain, respectively. In some embodiments, the CH31 and CH32 domains meet at the interface between the projection and the cavity. In some embodiments, the CH21 domain and the CH22 domain each include a projection or cavity, and the projection or cavity of the CH21 domain can be located in the cavity or projection of the CH22 domain, respectively. In some embodiments, the CH21 and CH22 domains meet at the interface between the projection and the cavity.

[0014] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and the bispecific antigen-binding molecule comprises an Fc domain containing a first subunit and a second subunit; the first antigen-binding domain is fused to the N-terminus of the first subunit at the C-terminus of the Fab heavy chain, and the second antigen-binding domain is fused to the N-terminus of the second subunit at the C-terminus of the Fab heavy chain.

[0015] In some embodiments, the first subunit contains a tryptophan residue at position 366; the second subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index).

[0016] In some embodiments, each of the first and second subunits includes an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering follows the Kabat EU index).

[0017] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6).

[0018] In some embodiments, the third antigen-binding domain comprises a VL domain and a VH domain, where (a) the VL domain contains a proline residue at position 12 (numbering according to Kabat); and / or (b) the VL domain contains a lysine residue at position 38 and the VH domain contains a glutamic acid residue at position 39 (numbering according to Kabat). In some embodiments, the third antigen-binding domain comprises one or more of the following eight FRs: (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 10; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 14; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 15; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 16.

[0019] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8.

[0020] In some embodiments, the third antigen-binding domain is a Fab molecule. In some embodiments, the third antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, the Fab light chain of the third antigen-binding domain containing a glutamic acid residue at position 133, and the Fab heavy chain of the third antigen-binding domain containing a lysine residue at position 183 (numbering follows Kabat).

[0021] In some embodiments, the second antigen-binding domain and the third antigen-binding domain are fused to each other. In some embodiments, the second antigen-binding domain and the third antigen-binding domain are fused to each other via a peptide linker. In some embodiments, the peptide linker contains the amino acid sequence of SEQ ID NO: 37. In some embodiments, the second antigen-binding domain and the third antigen-binding domain are each Fab molecules, and the third antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain.

[0022] In some embodiments, the bispecific antigen-binding molecule comprises an Fc domain including a first subunit and a second subunit; the first antigen-binding domain, the second antigen-binding domain and the third antigen-binding domain are each Fab molecules; the first antigen-binding domain is fused to the N-terminus of the first subunit at the C-terminus of the Fab heavy chain; the second antigen-binding domain is fused to the N-terminus of the second subunit at the C-terminus of the Fab heavy chain; and the third antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain.

[0023] In some embodiments, the bispecific antigen-binding molecule includes a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 33; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 35; and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 33, a first polypeptide and a second polypeptide each containing the amino acid sequence of SEQ ID NO: 34, a polypeptide containing the amino acid sequence of SEQ ID NO: 35, and a polypeptide containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, (i) the polypeptide containing the amino acid sequence of SEQ ID NO: 33 is linked to the first polypeptide containing the amino acid sequence of SEQ ID NO: 34 via interactions between the Fab heavy chain and the Fab light chain; (ii) the polypeptide containing the amino acid sequence of SEQ ID NO: 35 is linked to the second polypeptide containing the amino acid sequence of SEQ ID NO: 34 via interactions between the Fab heavy chain and the Fab light chain; (iii) the polypeptide containing the amino acid sequence of SEQ ID NO: 35 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 36 via interactions between the Fab heavy chain and the Fab light chain; and (iv) the polypeptide containing the amino acid sequence of SEQ ID NO: 33 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 35 via first and second subunits of the Fc domain.

[0024] In one embodiment, the present invention provides an isolated polynucleotide or a set of isolated polynucleotides encoding any one of the bispecific antigen-binding molecules described herein.

[0025] In one embodiment, the present invention provides an isolated polynucleotide or a set of isolated polynucleotides comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of the nucleic acid sequences of SEQ ID NOs. 85 to 89.

[0026] In one embodiment, the present invention provides an isolated polynucleotide or a set of isolated polynucleotides comprising any one nucleic acid sequence of sequence numbers 85 to 89.

[0027] In one embodiment, the present invention provides a set of isolated polynucleotides comprising an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO: 85, an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO: 86, an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO: 87, and an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO: 88.

[0028] In one embodiment, the present invention provides a vector or set of vectors comprising any one of the isolated polynucleotides described herein or any one of the sets of isolated polynucleotides.

[0029] In one embodiment, the present invention provides a host cell or a set of host cells comprising (i) any one of the isolated polynucleotides or any one of the sets of isolated polynucleotides described herein, or (ii) any one of the vectors or any one of the sets of vectors described herein.

[0030] In one embodiment, the present invention provides a method for producing a bispecific antigen-binding molecule that binds to CCR8 and CD3, comprising the step of (a) culturing one of any of the host cells or any of the set of host cells described herein under conditions suitable for the expression of the bispecific antigen-binding molecule. In some embodiments, the method further comprises recovering the bispecific antigen-binding molecule.

[0031] In one embodiment, the present invention provides a bispecific antigen-binding molecule that binds to CCR8 and CD3 produced by any one of the methods described herein.

[0032] In one embodiment, the present invention provides a pharmaceutical composition comprising one of the bispecific antigen-binding molecules described herein and a pharmaceutically acceptable carrier.

[0033] In one embodiment, the present invention provides any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein for use as a pharmaceutical.

[0034] In one embodiment, the present invention provides the use of any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein in the manufacture of a pharmaceutical.

[0035] In one embodiment, the present invention provides any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein for use in the treatment of cancer.

[0036] In one embodiment, the present invention provides the use of any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein for the treatment of cancer in subjects requiring treatment of cancer.

[0037] In one embodiment, the present invention provides the use of any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein for treating cancer in subjects requiring treatment of cancer.

[0038] In one embodiment, the present invention provides a method for treating cancer in a subject, comprising administering an effective amount of any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein to the subject.

[0039] In some embodiments, cancer is selected from the group consisting of bladder cancer, blastoma, hematological cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer.

[0040] In one embodiment, the present invention provides the use of any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein for depleting regulatory T cells.

[0041] In one embodiment, the present invention provides a method for depleting regulatory T cells in the tumor microenvironment of a subject having cancer, comprising administering to the subject a sufficient amount to deplete regulatory T cells in the tumor microenvironment of any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein.

[0042] In one embodiment, the present invention provides a method for depleting regulatory T cells outside the tumor microenvironment in a subject having cancer, comprising administering to the subject a sufficient amount to deplete regulatory T cells outside the tumor microenvironment, one of the bispecific antigen-binding molecules described herein or one of the pharmaceutical compositions described herein.

[0043] In some embodiments, regulatory T cells present in the tumor microenvironment of cancer are depleted. In some embodiments, regulatory T cells outside the tumor microenvironment of cancer are depleted.

[0044] In one embodiment, the present invention provides an in vitro method for depleting regulatory T cells from a cancer cell population, comprising contacting the cell population with any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein in an amount sufficient to deplete regulatory T cells from the cell population.

[0045] In some embodiments, the subjects exhibit reduced CCR8 mRNA expression.

[0046] In one embodiment, the present invention provides a method for reducing CCR8 mRNA, comprising contacting a cell population with any one of the bispecific antigen-binding molecules described herein or any one of the pharmaceutical compositions described herein in an amount sufficient to reduce CCR8 mRNA. In some embodiments, the method reduces CCR8 mRNA expression in subjects having cancer.

[0047] In some embodiments, any one of the bispecific antigen-binding molecules for use, pharmaceutical compositions for use, or methods of use described herein further includes administering an additional therapeutic agent to a target.

[0048] In some embodiments, the additional therapeutic agent is an anticancer agent. In some embodiments, the anticancer agent is selected from the group consisting of microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapies, kinase inhibitors, receptor antagonists, tumor cell apoptosis activators, anti-angiogenic agents, immunomodulators, cell adhesion inhibitors, cytotoxic or cell proliferation inhibitors, cell apoptosis activators, agents that increase the sensitivity of cells to apoptosis inducers, cytokines, anticancer vaccines or oncolytic viruses, Toll-like receptor (TLR) agents, bispecific antibodies, cell therapies, and immune cell engagers. In some embodiments, the anticancer agent is a PD-L1 conjugated antagonist. In some embodiments, the PD-L1 conjugated antagonist is atezolizumab.

[0049] In some embodiments, the additional therapeutic agent is tocilizumab or a corticosteroid.

[0050] In one embodiment, the present invention provides a bispecific antigen-binding molecule for depleting regulatory T cells or a pharmaceutical composition comprising the bispecific antigen-binding molecule, the bispecific antigen-binding molecule comprising (a) a first antigen-binding domain that binds to CCR8 and comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) a second antigen-binding domain that binds to an activated T cell antigen.

[0051] In one embodiment, the present invention provides a method for depleting regulatory T cells in the tumor microenvironment of a subject having cancer, comprising administering to the subject a sufficient amount of a bispecific antigen-binding molecule or a pharmaceutical composition containing the bispecific antigen-binding molecule to deplete regulatory T cells in the tumor microenvironment, wherein the bispecific antigen-binding molecule comprises (a) a first antigen-binding domain that binds to CCR8 and includes the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) a second antigen-binding domain that binds to an activated T cell antigen.

[0052] In one embodiment, the present invention provides a method for depleting regulatory T cells outside the tumor microenvironment in a subject having cancer, comprising administering to the subject an effective amount sufficient to deplete regulatory T cells outside the tumor microenvironment, wherein the bispecific antigen-binding molecule comprises (a) a first antigen-binding domain that binds to CCR8 and includes the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) a second antigen-binding domain that binds to an activated T cell antigen.

[0053] In some embodiments, regulatory T cells present in the tumor microenvironment of cancer are depleted. In some embodiments, regulatory T cells outside the tumor microenvironment of cancer are depleted.

[0054] In one embodiment, the present invention provides an in vitro method for depleting regulatory T cells from a cancer cell population, comprising contacting the cell population with a bispecific antigen-binding molecule or a pharmaceutical composition containing the bispecific antigen-binding molecule in an amount sufficient to deplete regulatory T cells from the cell population, wherein the bispecific antigen-binding molecule is (a) a first antigen-binding domain that binds to CCR8 and comprises the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) amino acid sequence LIHRSG A first antigen-binding domain comprising (iii) CDR-H2 containing RTYYATWAKG (SEQ ID NO: 2); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) a second antigen-binding domain that binds to an activated T cell antigen.

[0055] In some embodiments, the activated T cell antigen is CD3.

[0056] In one embodiment, the present invention provides a method for reducing CCR8 mRNA expression in the blood of a subject, comprising administering to the subject a bispecific antigen-binding molecule or a pharmaceutical composition comprising a sufficient amount to reduce CCR8 mRNA expression, wherein the bispecific antigen-binding molecule comprises (a) a first antigen-binding domain that binds to CCR8 and comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) a second antigen-binding domain that binds to an activated T cell antigen. In some embodiments, the method reduces CCR8 mRNA expression in subjects with cancer. [Brief explanation of the drawing]

[0057] [Figure 1] Figures 1A to 1E show different structural formats of bispecific antigen-binding molecules that bind to CCR8 and CD3. Figure 1A shows a 1+1 A(CCR8) / B(CD3) bispecific antigen-binding molecule (e.g., TDB). Figure 1B shows a 2+0 AB bispecific antigen-binding molecule (e.g., TDB). Figure 1C shows a 2+1 A / AB bispecific antigen-binding molecule (e.g., TDB). Figure 1D shows a 2+1 A / BA bispecific antigen-binding molecule (e.g., TDB). Figure 1E shows a 2+1 B / AA bispecific antigen-binding molecule (e.g., TDB). [Figure 2] Figure 2 is a graph comparing the hCCR8+ CHO cell-killing efficacy of 1+1, 2+1, and 2+0 bispecific antigen-binding molecule (e.g., TDB) formats. All TDB formats compared used the 1889 CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) CD3 antigen-binding domain. [Figure 3] Figures 3A–3E are a series of graphs comparing the ability of various 1+1 and 2+1 bispecific antigen-binding molecule (e.g., TDB) formats to deplete CCR8+ Treg cells. All TDB formats compared used the 1889 CCR8 antigen-binding domain and either the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. Figures 3A–3C show the absolute number of Treg cell depletions, while Figures 3D and 3E show the percentage of Treg cell depletions in Figures 3B and 3C, respectively. [Figure 4] Figures 4A–4E compare two different orientations of 2+1 bispecific antigen-binding molecule (e.g., TDB) formats, A / AB and A / BA. Figures 4A–4D are a series of graphs comparing the ability of the A / AB format versus the A / BA format to deplete Treg cells in donor 1 (Figures 4A and 4C) and donor 2 (Figures 4B and 4D). Figures 4A and 4B show Treg cell depletion in CCR8+ Treg cells. Figures 4C and 4D show Treg cell depletion in non-CCR8+ Treg cells. Figure 4E shows the orientations of A / AB and A / BA format TDBs. [Figure 5] Figures 5A–5F are a series of graphs comparing the ability of A / AB 2+1 bispecific antigen-binding molecule (e.g., TDB) formats having either the high-affinity CD3-binding agent MD1 or the low-affinity CD3-binding agent 40G5c to deplete CCR8+ Treg cells. All TDB formats compared used the 1889 CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. Figures 5A, 5C, and 5E show the absolute number of Treg cell depletions, while Figures 5B, 5D, and 5F show the percentage of Treg cell depletion for Figures 5A, 5C, and 5E, respectively. [Figure 6]Figures 6A–6C are a series of graphs comparing the ability of A / AB 2+1 bispecific antigen-binding molecule (e.g., TDB) formats with either the high-affinity CD3 conjugate MD1 or the low-affinity CD3 conjugate 40G5c to reduce tumor size in either a mouse model of breast cancer (E0771 breast cancer model) or colorectal cancer (MD-38 colorectal model). Experiments were performed in hu.CD3.tg.B6N mice. Values ​​in parentheses report dose levels in mg / kg. All TDB formats compared used the 1356 mouse CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. [Figure 7] Figure 7 includes a series of graphs showing in vivo cytokine release induced by different CCR8 antigen-binding molecules. Experiments were performed in MD-38 colorectal model mice. 1+1 format TDB was administered at a dose of 0.5 mg / kg, and 2+1 format TDB was administered at 0.67 mg / kg. Within each panel, bars from left to right represent the vehicle, anti-CCR8(1294), anti-CCR8 / CD3(38E4), anti-CCR8 / CD3(40G5c), anti-CCR8-CD3(38E4) / CCR8, anti-CCR8-CD3(40G5c) / CCR8, and anti-CCR8-CD3(2C11) / CCR8 treatment groups, respectively. [Figure 8] Figure 8 is a graph comparing the off-rates of single-specific anti-CCR8 antibodies on human CCR8+ CHO cells, as determined by flow cytometry, for affinity-matured variants of the anti-CCR8.1889 antibody. In particular, five variants showed improved off-rates compared to WT 1889. [Figure 9A] This is a series of graphs comparing the ability of various CCRB TDB formats with different CCR8 and CD3 conjugates to deplete CCR8+ Treg cells. Both 1+1 and 2+1 formats are tested. The TDB formats compared used the 1889 WT or 1889 PVS variant CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. [Figure 9B] This is a series of graphs comparing the ability of various CCRB TDB formats with different CCR8 and CD3 conjugates to deplete CCR8+ Treg cells. Both 1+1 and 2+1 formats are tested. The TDB formats compared used the 1889 WT or 1889 PVS variant CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. [Figure 9C] This is a series of graphs comparing the ability of various CCRB TDB formats with different CCR8 and CD3 conjugates to deplete CCR8+ Treg cells. Both 1+1 and 2+1 formats are tested. The TDB formats compared used the 1889 WT or 1889 PVS variant CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. [Figure 9D] This is a series of graphs comparing the ability of various CCRB TDB formats with different CCR8 and CD3 conjugates to deplete CCR8+ Treg cells. Both 1+1 and 2+1 formats are tested. The TDB formats compared used the 1889 WT or 1889 PVS variant CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. [Figure 9E] This is a series of graphs comparing the ability of various CCRB TDB formats with different CCR8 and CD3 conjugates to deplete CCR8+ Treg cells. Both 1+1 and 2+1 formats are tested. The TDB formats compared used the 1889 WT or 1889 PVS variant CCR8 antigen-binding domain and the 38E4v1.MD1 (i.e., MD1) or 40G5c CD3 antigen-binding domain. [Figure 10]Figure 10 is a graph comparing the pharmacokinetics (PK) of various CCR8 TDB formats having either the 1889 WT or 1889 PVS variant CCR8 antigen-binding domain. Severely immunocompromised (SCID) mice were administered a single intravenous (IV) dose of 5 mg / kg of the test CCR8 TDB. Serum concentration-time profiles are shown. [Figure 11A] This is a series of graphs showing the results of three experiments (runs 1-3) evaluating the use of protein L affinity chromatography to purify the generated S12P variant 2+1 1889WT / 1889WT. For each experiment (runs 1-3 corresponding to Figures 11A, 11B, and 11C, respectively), the effect of protein L purification was determined by evaluating the initial protein fraction (i.e., before protein L affinity chromatography; "protein L load"), the flow-through fraction ("flow-through"), and the bound pool fraction ("pool") using size exclusion ultrahigh performance liquid chromatography (SE-UPLC) elution chromatograms. The two peaks evaluated by SE-UPLC were the main peak and the low molecular weight species fraction (LMWS). [Figure 11B] This is a series of graphs showing the results of three experiments (runs 1-3) evaluating the use of protein L affinity chromatography to purify the generated S12P variant 2+1 1889WT / 1889WT. For each experiment (runs 1-3 corresponding to Figures 11A, 11B, and 11C, respectively), the effect of protein L purification was determined by evaluating the initial protein fraction (i.e., before protein L affinity chromatography; "protein L load"), the flow-through fraction ("flow-through"), and the bound pool fraction ("pool") using size exclusion ultrahigh performance liquid chromatography (SE-UPLC) elution chromatograms. The two peaks evaluated by SE-UPLC were the main peak and the low molecular weight species fraction (LMWS). [Figure 11C]This is a series of graphs showing the results of three experiments (runs 1-3) evaluating the use of protein L affinity chromatography to purify the generated S12P variant 2+1 1889WT / 1889WT. For each experiment (runs 1-3 corresponding to Figures 11A, 11B, and 11C, respectively), the effect of protein L purification was determined by evaluating the initial protein fraction (i.e., before protein L affinity chromatography; "protein L load"), the flow-through fraction ("flow-through"), and the bound pool fraction ("pool") using size exclusion ultrahigh performance liquid chromatography (SE-UPLC) elution chromatograms. The two peaks evaluated by SE-UPLC were the main peak and the low molecular weight species fraction (LMWS). [Figure 12A] This figure shows the structural components of 2+1 1889WT / 1889WT.40G5c 2+1 CCR8 TDB. Figure 12A shows the charge mutations on each domain of the monovalent and bivalent arms, as well as which arms contain knob or hole mutations. [Figure 12B] This figure shows the structural components of 2+1 1889WT / 1889WT.40G5c 2+1 CCR8 TDB. Figure 12B further provides the molecular weight of each chain, as well as amino acid mutations including the charge mutations shown in Figure 12A, and domains including LALA-PG mutations and knob ("K") and hole ("H") mutations. [Figure 13A] Figure 13A is a series of graphs showing the depletion of CCR8+ Treg cells incubated with human dissociated tumor cells for 72 hours in the presence of 2+1 1889WT / 1889WT.40G5c 2+1 CCR8 TDBs. [Figure 13B] Figure 13B is a series of graphs showing the depletion of CCR8+ Treg cells incubated with human dissociated tumor cells for 72 hours in the presence of 2+1 1889WT / 1889WT.40G5c 2+1 CCR8 TDBs. [Figure 13C]Figure 13C is a series of graphs showing the depletion of CCR8+ Treg cells incubated with human dissociated tumor cells for 72 hours in the presence of 2+1 1889WT / 1889WT.40G5c 2+1 CCR8 TDBs. [Figure 14A] The amino acid sequence alignment of anti-CCR8 1889 antibody variants compared to the 1889 wild-type (WT) antibody is shown. Figure 14A shows the amino acid sequence alignment of the light chain variable region (VL domain) of the 1889 P, PV, PI, PS, PVI, PVS, PIS, and PVIS variants relative to the VL domain of the 1889 WT antibody. [Figure 14B] The amino acid sequence alignment of the anti-CCR8 1889 antibody variant compared to the 1889 wild-type (WT) antibody is shown. Figure 14B shows the amino acid sequence alignment of the heavy chain variable region (VH domain) of the 1889 A and DA variants relative to the 1889 WT antibody. Amino acid substitutions are highlighted. Underlining indicates the corresponding CDR region as defined by Kabat. [Figure 15] Figures 15A and 15B show the amino acid sequence alignments of the anti-CD3 40G5c antibody and 38E4v1.MD1 (MD1). Figure 15A shows the amino acid sequence alignment of the light chain variable region (VL domain) of the 40G5c antibody and the MD1 antibody. Figure 15B shows the amino acid sequence alignment of the heavy chain variable region (VH domain) of the 40G5c antibody and the MD1 antibody. [Figure 16A] Figure 16A is a series of graphs showing the binding of CCR8 TDB to sulfated G protein-coupled receptors (GPCRs) CCR2, CCR3, CCR4, CCR5, CXCR4, ACKR2, ACKR4, and CCR8, as evaluated by flow cytometry. The light gray curves show binding evaluated using Myc-negative cells, and the dark gray curves show binding evaluated using Myc-positive cells. [Figure 16B]Figure 16B is a series of graphs showing the binding of CCR8 TDB to sulfated G protein-coupled receptors (GPCRs) CCR2, CCR3, CCR4, CCR5, CXCR4, ACKR2, ACKR4, and CCR8, as evaluated by flow cytometry. The light gray curves show binding evaluated using Myc-negative cells, and the dark gray curves show binding evaluated using Myc-positive cells. [Figure 17A] Figure 17A shows the monitoring of CCR8 activation by Ca2+ influx using a fluorescence imaging plate reader (FLIPR®). CCR8 is activated by treating cells with CCL1, and the Ca2+ release induced by CCR8 activation is visualized by a fluorescent Ca2+ chelate dye. [Figure 17A] Figure 17B is a line graph showing the blockade rate of CCL1-mediated activation of CCR8 by CCR8 TDB (1889 / 1889.40G5c) or the control antibody (BD 433H). 433H inhibited CCL1-mediated CCR8 activation with an IC50 of 8.4 nM, while CCR8 TDB did not affect CCR8 activation. [Figure 18] Figure 18 is a line graph showing the percentage of CCR8+ Treg cells in the peripheral blood of cynomolgus monkeys treated with either a control or CCR8 TDB over time. A7, A8, and A9 received the vehicle control, while A10, A11, and A12 received CCR8 TDB. Either the vehicle control or CCR8 TDB was administered intravenously on days 1 and 11. [Figure 19] Figure 19 is a dot plot showing the mean mRNA expression levels of CCR8 in the blood of cynomolgus monkeys treated with CCR8 TDB or vehicle control at various time points. The horizontal line represents the mean of three animals. * indicates p<0.05, determined by one-way ANOVA followed by Sidak's multiple comparison test. [Figure 20]Figures 20A–20D are a series of line graphs showing the levels of cytokines IL-1 RA (Figure 20A), IL-8 (Figure 20B), IL-6 (Figure 20C), and MCP-1 (Figure 20D) in cynomolgus monkeys treated with CCR8 TDB or vehicle control at various time points. The symbols represent individual animals (circle: control; square: 10 mg / kg CCR8 TDB), and the lines lie on the mean values. [Modes for carrying out the invention]

[0058] The present invention is at least in part based on the applicant's discovery that anti-CCR8 / anti-CD3 multispecific (e.g., bispecific) antigen-binding molecules described herein (e.g., T cell-dependent bispecific antibodies (TDBs)) result in unexpectedly efficient Treg depletion and tumor cell killing with an acceptable safety profile. See, for example, Examples 1-6 disclosed herein.

[0059] While we do not wish to be bound by any particular theory, the multispecific antigen-binding molecules disclosed herein are expected to deplete Treg cells through the formation of immune synapses between Treg and T effector (Teff) cells expressing CCR8. Prior to our findings, it was unexpected whether the bispecific antigen-binding formats disclosed herein would deplete Treg cells, given that Treg cells repress Teff cells and both CCR8 and CD3 are expressed on Treg cells. In particular, while we do not wish to be bound by any particular theory, the expression of both CCR8 and CD3 on Treg cells presents a potential challenge for the bispecific antigen-binding molecules: they must avoid binding only to Treg in cis by binding to both CCR8 and CD3 present on the Treg, which would prevent the CD3 arm from binding to and activating CD3+ Teff cells. Therefore, the present invention provides Treg depletion using multispecific antigen-binding molecules that bind to Treg cells (e.g., via binding to CCR8 or other Treg markers) and Teff cells (e.g., via binding to activated T cell antigens (e.g., CD3)).

[0060] The present invention is also, at least in part, based on the applicant's findings that the bispecific 2+1 TDB molecules having the “A / AB” orientation disclosed herein are unexpectedly superior to other tested orientations of TDB cells, for example in terms of Treg depletion, and enable the use of relatively low affinity anti-CD3 arms (e.g., 40G5c) with acceptable levels of cytokine release, and are more likely to have, for example, an acceptable safety profile and a more beneficial risk-benefit profile.

[0061] I. Definition For the purposes of this specification, “acceptor human framework” means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0062] As used herein, “T cell activating antigen” refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, that can induce T cell activation through interaction with an antigen-binding molecule. Specifically, the interaction of an antigen-binding molecule with a T cell activating antigen can induce T cell activation by triggering a cascade of signaling in the T cell receptor complex. In certain embodiments, the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt number P07766 (version 130), NCBI RefSeq number NP_000724.1 for the human sequence; or UniProt number Q95LI5 (version 49), NCBI GENBANK® number BAB71849.1 for the cynomolgus monkey (Macaca fascicularis) sequence).

[0063] As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The T cell activation multispecific (e.g., bispecific) antigen-binding molecules disclosed herein can induce T cell activation. Appropriate assays for measuring T cell activation are known in the art and are described herein.

[0064] "Administer" means a method of giving a target a dosage of a compound (e.g., an anti-CCR8 / anti-CD3 multispecific antigen-binding molecule disclosed herein or a nucleic acid encoding an anti-CCR8 / anti-CD3 multispecific antigen-binding molecule disclosed herein) or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an anti-CCR8 / anti-CD3 multispecific antigen-binding molecule disclosed herein). Compositions used in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarterially, intranasally, intravitreously, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesically, intramucosa, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, directly by local perfusion bath target cells, by catheter, by perfusion, in cream, or in lipid composition. The method of administration may vary depending on various factors (e.g., the compound or composition being administered, and the severity of the symptoms, disease, or disorder being treated).

[0065] "Affinity" refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D) can be expressed by. Affinity can be measured by methods common in the art, including those described herein. Specific descriptive and exemplary methods for measuring binding affinity are also described herein.

[0066] A "affinity-matured" antibody refers to an antibody that has one or more modifications in one or more complementarity-determining regions (CDRs), and compared to a parent antibody that does not have such modifications, such modifications result in improved affinity of the antibody to the antigen.

[0067] For the purposes of this specification, “atezolizumab” is a PD-L1-binding Fc-modified humanized nonglycosylated IgG1 kappa immunoglobulin. Atezolizumab contains a single amino acid substitution (asparagine to alanine) (N297A) at position 297 on the heavy chain using EU numbering of Fc region amino acid residues, resulting in a nonglycosylated antibody with minimal binding to the Fc receptor. Atezolizumab is also listed in WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances (proposed INN)) List 112, Vol.28, No.4, 2014, p.488.

[0068] The terms “anti-CCR8 antibody” and “antibody that binds to CCR8” refer to an antibody that can bind to CCR8 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent in targeting CCR8. In one embodiment, the degree of binding of an anti-CCR8 antibody to unrelated non-CCR8 proteins is less than about 10% of the antibody’s binding to CCR8, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to CCR8 has an affinity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -13 M~10 -8 M, for example, 10 -13 M~10 -9The dissociation constant (K D ) of M). In certain embodiments, an antibody that binds CCR8 has a K -12 of about 1×10 -10 M to about 1×10 -12 M, about 1×10 -11 M to about 1×10 -11 M, or about 1×10 -11 M to about 5×10 D M. In certain embodiments, an antibody that binds CCR8 has a K -11 of about 2×10 D M. In certain embodiments, an antibody that binds CCR8 has a K -12 of about 5×10 D M. An antibody is said to "specifically bind" to CCR8 if the antibody has a K D of 1 μM or less. In certain embodiments, the anti-CCR8 antibody binds to epitopes of CCR8 from at least two different species (e.g., human and cynomolgus CCR8).

[0069] The terms "anti-CD3 antibody" and "antibody that binds CD3" refer to an antibody that can bind CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3. In one embodiment, the degree of binding of an anti-CD3 antibody to an unrelated non-CD3 protein is less than about 10% of the binding of the antibody to CD3, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds CD3 has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM or ≦0.001 nM (e.g., 10 -8 or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). An antibody is said to "specifically bind" to CD3 if the antibody has a K D of 1 μM or less. In certain embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3s from different species.

[0070] The term "antibody" is used herein in its broadest sense and encompasses, but is not limited to, a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0071] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology (2005) 23:1126-1136.

[0072] An "antigen-binding domain" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Molecules characterized by an antigen-binding portion include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIP, domain antibodies, bispecific antibodies, small molecules, scFv-Fc, aphibodies, nanobodies, and the VH and / or VL domains of antibodies), receptors, ligands, aptamers, and other molecules having identified binding partners.

[0073] As used herein, for example, in relation to the monovalent arm of a bispecific antigen-binding molecule, the term "monovalent" means a molecule or part thereof having one antigen-binding domain (e.g., a part of an antigen-binding molecule, e.g., one of the two arms of a bispecific antigen-binding molecule). Therefore, a monovalent molecule or part thereof is specifically capable of binding to exactly one antigen. The "monovalent binding affinity" or "monovalent K" refers to one of the two antigen-binding moieties of the bivalent arm of a bispecific antibody. D "(For example, one of the CCR8 or CD3 antigen-binding regions of a 2+1 TDB)" refers to the binding affinity of the antigen-binding domain in monovalent form, i.e., as a monovalent arm of a bispecific antibody capable of specifically binding to two different antigens, or as a Fab molecule.

[0074] As used herein, for example, in relation to the bivalent arms of a bispecific antigen-binding molecule, the term “bivalent” means a molecule or part thereof that precisely has two antigen-binding moieties, each capable of specifically binding to one antigen (e.g., a part of an antigen-binding molecule, e.g., one of the two arms of a bispecific antigen-binding molecule). Thus, a bivalent molecule or part thereof can specifically bind to two antigens or two different epitopes on the same antigen.

[0075] The term "epitope" refers to a site on a proteinaceous or non-proteinaceous antigen to which the binding domain of an anti-CCR8 antibody, anti-CD3 antibody, or an anti-CCR8 / anti-CD3 multispecific antigen-binding molecule described herein (e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule) binds. Epitopes may be formed from a continuous amino acid stretch site (linear epitopes) or from discontinuous amino acids (structural epitopes), and may be formed in spatial proximity, for example, due to the folding of the antigen (i.e., by the tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound by the binding domain of an anti-CCR8 antibody, anti-CD3 antibody, or anti-CCR8 / anti-CD3 multispecific antigen-binding molecule described herein (e.g., a bispecific antigen-binding molecule) after exposure of a proteinaceous antigen to a denaturing agent, while structural epitopes are typically destroyed upon treatment with a denaturing agent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 30, or at least 35, or 3-25, 3-20, 3-15, 3-10, 3-5, 30-40, 35-40, or 5-10 amino acids in its unique spatial conformation.

[0076] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) may be performed using methods commonly used in the art, such as, but not limited to, alanine scanning, peptide blotting (see, e.g., Kobeissy et al., Meth.Mol.Biol.(2004)248:443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Hochleitner et al., Prot.Sci.9(2000)487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0077] Competitive binding can be used to easily determine whether an antibody binds to the same CCR8 or CD3 epitope as a reference anti-CCR8 or anti-CD3 antibody, or whether it competes for binding. For example, an antibody that "binds to the same epitope" as a reference anti-CCR8 or anti-CD3 antibody is one that inhibits the binding of the reference anti-CCR8 or anti-CD3 antibody to the antigen by 50% or more in a competitive assay, and conversely, the reference antibody inhibits the binding of the antibody to the antigen by 50% or more in a competitive assay. Alternatively, for example, to determine whether an antibody binds to the same epitope as a reference anti-CCR8 or anti-CD3 antibody, the reference antibody can be bound to CCR8 or CD3 under saturated conditions. After removing excess reference antibody, the ability of the anti-CCR8 or anti-CD3 antibody in question to bind to CCR8 or CD3, respectively, is evaluated. If the anti-CCR8 or anti-CD3 antibody can bind to CCR8 or CD3, respectively, after saturated binding of the reference anti-CCR8 or anti-CD3 antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question is unable to bind to the target epitope after saturation binding of the reference antibody, the anti-CCR8 or anti-CD3 antibody in question may bind to the same epitope as the one bound by the reference anti-CCR8 or anti-CD3 antibody. Conventional experiments can be used to determine whether the antibody in question is binding to the same epitope or whether binding is simply being hindered for steric reasons (e.g., peptide mutation, and binding analysis using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay should be performed in two setups, i.e., with both antibodies being saturated antibodies. If, in both setups, only the first (saturated) antibody can bind to CCR8 (or CD3), then it can be concluded that the anti-CCR8 antibody (or anti-CD3 antibody) in question and the reference anti-CCR8 antibody (or reference anti-CD3 antibody) compete for binding to CCR8 (or CD3).

[0078] In some embodiments, two antibodies are considered to bind to the same or overlapping epitopes if, as measured by competitive binding assays, a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even more than 99% (see, for example, Junghans et al., Cancer Res. 50(1990) 1495-1502).

[0079] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies are considered to have an "overlapping epitope" if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0080] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0081] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0082] As used herein, the terms “human-derived constant region” or “human constant region” refer to the constant heavy chain region and / or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are publicly known in the art and are described, for example, in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G., and Wu, TT, Nucleic Acids Res. 28(2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72(1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant region follows the EU numbering system, also known as the Kabat EU index (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242).

[0083] "Effector function" refers to the biological activity resulting from the Fc region of an antibody (e.g., a bispecific antigen-binding molecule, e.g., 2+1 TDB) that varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0084] An "effective amount" of a compound, such as a bispecific antigen-binding molecule or a composition thereof (e.g., a pharmaceutical composition) disclosed herein, is present in at least the minimum amount necessary to achieve the desired therapeutic or prophylactic outcome, such as measurable improvement or prevention of a particular disorder (e.g., cell proliferation disorder, e.g., cancer). The effective amount as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the antibody's ability to induce the desired response in the individual. The effective amount is also such that the therapeutically beneficial effect outweighs any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desired outcomes include the elimination or reduction of the risk of disease, reduction of disease severity, or delay of disease onset, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the onset of the disease. In therapeutic use, beneficial or desired outcomes include clinical results such as a reduction in one or more symptoms caused by the disease, an improvement in the quality of life of the person affected, a reduction in the dose of other drugs required to treat the disease, an enhancement of the effect of another drug (e.g., by targeting), a delay in disease progression, and / or an extension of survival. In the case of cancer or tumors, an effective dose of the drug may have the effect of reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., delaying to some extent, or preferably stopping) the invasion of cancer cells into peripheral organs, inhibiting (i.e., delaying to some extent, or preferably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more of the symptoms associated with the disorder. An effective dose may be administered in one or more doses. In this invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic measure. As understood in the clinical field, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Therefore, the “effective dose” may be considered in relation to the administration of one or more therapeutic agents, and a monotherapy agent may be considered to be given in an effective dose if, when combined with one or more other agents, the desired outcome can be achieved or is achieved.

[0085] As used herein, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids from the C-terminus of the heavy chain, particularly one or two amino acids. Thus, by expression of certain nucleic acid molecules encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or a cleaved variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In one embodiment, the heavy chain containing the Fc region specified herein, as included in the antibody according to the present invention, includes a further C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one embodiment, the heavy chain containing the Fc region specified herein, as included in the antibody according to the present invention, includes a further C-terminal glycine residue (G446, numbering according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also called the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0086] The "framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0087] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein synonymously to refer to antibodies having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region as defined herein. It should be understood that a full-length antibody includes the heavy chain variable domain and light chain variable domain as defined herein, as well as the Fc region as defined herein.

[0088] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may not have exactly the same nucleic acid content as the parent cells and may contain mutations. Hereinafter, mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included.

[0089] A "human antibody" is an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody, such as the human antibody repertoire. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0090] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. (above). In one embodiment, for VH, the subgroup is subgroup III, as described in Kabat et al. (above).

[0091] A "humanized" antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody substantially contains all of at least one, typically two, variable domains, within which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0092] As used herein, the terms “hypervariable region” or “HVR” refer to each region of the antibody variable domain that is sequence-hypervariable and determines antigen-binding specificity, such as “complementarity-determining region” (CDR).

[0093] In certain embodiments, the antibody contains six CDRs, three of which are in the VH region (CDR-H1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). In certain embodiments, the antibody containing six CDRs is a full-length antibody. In certain embodiments, the antibody containing six CDRs is an antibody fragment.

[0094] Examples of CDRs described herein include: (a) Hypervariable loops present at amino acid residues 26-32 (l1), 50-52 (l2), 91-96 (l3), 26-32 (h1), 53-55 (h2), and 96-101 (h3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact occurring at amino acid residues 27c-36(l1), 46-55(l2), 89-96(l3), 30-35b(h1), 47-58(h2), and 93-101(h3) (MacCallum et al. J.Mol.Biol.262:732-745(1996)).

[0095] Unless otherwise specified, CDRs are determined according to Kabat et al. (above). Those skilled in the art will understand that CDR designations may also be determined according to McCallum (above), Chothia (above), or other scientifically recognized nomenclature.

[0096] In one exemplary embodiment, the CDR residues of the bispecific antigen-binding molecules disclosed herein include those identified in Table 2.

[0097] The "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the subject is a human.

[0098] An "isolated" antibody is one that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity of 95% or higher than 99%, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0099] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a sequence of bases, thereby representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of antibodies described herein in vitro and / or in vivo, for example, in a host or subject. Such DNA vectors (e.g., cDNA) or RNA vectors (e.g., mRNA) may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject in vivo to produce an antibody. (See, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823B1).

[0100] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained in cells that contain nucleic acid molecules, but these nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0101] "Isolated nucleic acid encoding a bispecific antigen-binding molecule," e.g., "isolated nucleic acid encoding an anti-CCR8 antibody," or "isolated nucleic acid encoding an anti-CCR8 bispecific antigen-binding molecule," refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of a bispecific antigen-binding molecule (e.g., an anti-CCR8 bispecific antigen-binding molecule, e.g., anti-CCR8 / anti-CD3 TDB), and includes such nucleic acid molecules in a single vector or separate vectors, where such nucleic acid molecules are located at one or more positions within a host cell.

[0102] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bound to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or variant antibodies that may arise during the production of a monoclonal antibody preparation, such variants generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on one antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies according to this disclosure may be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0103] A "naked antibody" refers to an antibody that is not conjugated with a heterogeneous portion (e.g., a cytotoxic portion) or a radiolabel. Naked antibodies may be present in a pharmaceutical composition.

[0104] "Native antibodies" refer to naturally occurring immunoglobulin molecules with diverse structures. For example, a native IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0105] The term “package insert” is used to refer to instructions that are typically included in the market packaging of therapeutic products and that contain information about indications, usage, dosage, administration, combination therapy, contraindications and / or warnings relating to the use of such therapeutic products.

[0106] The term “PD-L1-binding antagonist” refers to a molecule that reduces, blocks, inhibits, represses, or interferes with signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In some cases, a PD-L1-binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In specific embodiments, a PD-L1-binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some cases, a PD-L1-binding antagonist includes anti-PD-L1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, repress, or interfere with signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In one case, a PD-L1-binding antagonist reduces negative costimulatory signaling mediated by or via cell surface proteins expressed on T lymphocytes via PD-L1-mediated signaling, thereby preventing dysfunctional T cells from becoming dysfunctional (e.g., enhancing the effector response to antigen recognition). In some cases, a PD-L1-binding antagonist binds to PD-L1. In some cases, a PD-L1-binding antagonist is an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). Examples of anti-PD-L1 antagonist antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, emvafolimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cosivelimab, rhodapolimab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636.In some embodiments, the anti-PD-L1 antibody is atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). In one specific embodiment, the PD-L1-binding antagonist is MDX-1105. In another specific embodiment, the PD-L1-binding antagonist is MEDI4736 (durvalumab). In yet another specific embodiment, the PD-L1-binding antagonist is MSB0010718C (avelumab). In other embodiments, the PD-L1-binding antagonist may be a small molecule, e.g., GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041, which may be administered orally in some cases. Other exemplary PD-L1-binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003, and JS-003. In a preferred embodiment, the PD-L1-binding antagonist is atezolizumab.

[0107] The term “PD-1 binding antagonist” refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with signaling resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PD-L2. PD-1 (programmed cell death 1) is also known in the art as “programmed cell death 1,” “PDCD1,” “CD279,” and “SLEB2.” An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot accession number Q15116. In some cases, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In specific embodiments, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1-binding antagonists include anti-PD-1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signaling resulting from the interaction between PD-1 and PD-L1 and / or PD-L2. In one case, a PD-1-binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes via PD-1 signaling, so as to prevent dysfunctional T cells from becoming dysfunctional (e.g., enhancing the effector response to antigen recognition). In some cases, PD-1-binding antagonists bind to PD-1. In some cases, PD-1-binding antagonists are anti-PD-1 antibodies (e.g., anti-PD-1 antagonist antibodies).Examples of anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartalizumab), REGN2810 (semiprimab), BGB-108, prorugolimab, canrelizumab, cintilimab, tislerizumab, tripalimab, dostalimab, retifanlimab, sasanlimab, pemplimab, CS1003, HLX10, SCT-I10A, zinbererimab, valstilimab, genolimusumab, and BI. Examples include 754091, cetrerimab, YBL-006, BAT1306, HX008, budicalimab, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21. In a specific embodiment, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific embodiment, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In yet another specific embodiment, the PD-1 binding antagonist is a PD-L2 Fc fusion protein, such as AMP-224. In another specific embodiment, the PD-1 binding antagonist is MED1-0680. In another specific embodiment, the PD-1 binding antagonist is PDR001 (spartalizumab). In another specific embodiment, the PD-1 binding antagonist is REGN2810 (semiprimab). In another specific embodiment, the PD-1 binding antagonist is BGB-108. In another specific embodiment, the PD-1 binding antagonist is prorugolimab. In another specific embodiment, the PD-1 binding antagonist is canrelizumab. In another specific embodiment, the PD-1 binding antagonist is cintilimab. In another specific embodiment, the PD-1 binding antagonist is ticererizumab. In another specific embodiment, the PD-1 binding antagonist is tripalimab. Other example PD-1-binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.

[0108] The term “PD-L2-binding antagonist” refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. PD-L2 (programmed cell death ligand 2) is also referred to in the art as “programmed cell death ligand 12,” “PDCD1LG2,” “CD273,” “B7-DC,” “Btdc,” and “PDL2.” An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot accession number Q9BQ51. In some cases, a PD-L2-binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In specific embodiments, a PD-L2-binding antagonist inhibits the binding of PD-L2 to PD-1. Exemplary PD-L2 antagonists include anti-PD-L2 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2-binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes via PD-L2-mediated signaling, so as to prevent dysfunction of T cells (e.g., by enhancing the effector response to antigen recognition). In some embodiments, the PD-L2-binding antagonist binds to PD-L2. In some embodiments, the PD-L2-binding antagonist is an immunoadhesin. In other embodiments, the PD-L2-binding antagonist is an anti-PD-L2 antagonist antibody.

[0109] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences for alignment purposes and introducing gaps if necessary to achieve the maximum sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, MegAlign® (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to obtain the maximum alignment over the full length of the sequences to be compared. Alternatively, the percentage identity value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the US Copyright Office (Washington DC, 20559), registered under US Copyright Registration No. TXU510087, and published in International Publication No. 2001 / 007611.

[0110] Unless otherwise specified, for the purposes of this specification, amino acid sequence identity percentage values ​​are generated using the ggsearch program and BLOSUM50 comparison matrix in FASTA package version 36.3.8c or later. The FASTA program package is described by WRPearson and DJLipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WRPearson (1996), "Effective protein sequence comparison", Meth.Enzymol.266:227-258; and Pearson et al. (1997), Genomics 46:24-36, and is publicly available at fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or ebi.ac.uk / Tools / sss / fasta. Alternatively, you can use the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2) to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, ensuring that a global rather than local alignment is performed. The amino acid identity percentage is shown in the output alignment header.

[0111] The terms “pharmaceutical composition” and “pharmaceutical preparation” are used interchangeably herein and refer to preparations in which the biological activity of the active ingredients contained herein is effective and which do not contain additional components that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0112] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient that is non-toxic to the target. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0113] As used herein, the term “CCR8” refers to any natural CCR8 from any vertebrate source, including mammals such as primates (e.g., humans, monkeys (cyno)) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses “full-length” unprocessed CCR8 and any form of CCR8 resulting from intracellular processing. The term also encompasses naturally occurring variants of CCR8, such as splice variants or allele variants. In certain embodiments, CCR8 is human CCR8 ("hCCR8" or "huCCR8"). The amino acid sequence of an exemplary human CCR8 is shown in SEQ ID NO: 79, as shown in Table 1 below. In certain embodiments, CCR8 is cynomolgus monkey ("cyno") CCR8. The amino acid sequence of an exemplary cyno CCR8 is shown in SEQ ID NO: 80, as shown in Table 1 below. In certain embodiments, CCR8 is mouse CCR8 ("mCCR8"). The amino acid sequence of an exemplary mouse CCR8 is shown in SEQ ID NO: 87, as shown in Table 1 below. [Table 1]

[0114] The term “differentiation antigen group 3” or “CD3” refers, as used herein, to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated, and includes, for example, CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses “full-length” unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 obtained from intracellular processing. The term also encompasses spontaneously occurring variants of CD3, including, for example, splice variants or allele variants. CD3 includes, for example, the human CD3ε protein with a length of 207 amino acids (NCBI reference sequence number NP_000724) and the human CD3γ protein with a length of 182 amino acids (NCBI reference sequence number NP_000064).

[0115] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease (e.g., cancer) in the subject being treated, and may be performed for prevention (“preventive treatment” or “prophylactically treating”) or during the course of clinicopathology (“therapeutic treatment” or “therapeutic treating”). Desired effects of therapeutic treatment include, but are not limited to, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of cancer metastasis, reduction of the rate of disease progression, recovery or remission of the condition, and remission or improvement of prognosis. Desired effects of preventive treatment include, but are not limited to, prevention of the onset or recurrence of the disease. In some embodiments, the antibodies described herein are used to delay the onset of a disease or to slow the progression of a disease.

[0116] The term "variable region" or "variable domain" refers to a domain of the antibody heavy chain or antibody light chain involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (e.g., Kindt et al. Kuby Immunology, 6) th See ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain of the antibody that binds to that antigen, and complementary libraries of VL or VH domains can be screened. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0117] The term “vector” as used herein refers to a nucleic acid molecule capable of replicating another nucleic acid it is linked to. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors that have been incorporated into the genome of a host cell into which they have been introduced. Certain vectors can direct the expression of a functionally linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0118] II. Compositions and Methods In one embodiment, the disclosure is based in part on multispecific (e.g., bispecific) antigen-binding molecules (e.g., bispecific antibodies; e.g., bispecific antibodies that bind to CCR8 and CD3). In some embodiments, the bispecific antigen-binding molecule has a monovalent arm that can specifically bind to a first antigen (e.g., CCR8) and a bivalent arm that can specifically bind to two additional antigens (e.g., one antigen-binding domain that specifically binds to CD3 and one antigen-binding domain that specifically binds to CCR8). For example, the bivalent arm may include two antigen-binding moieties, each capable of specifically binding to a target antigen (e.g., CCR8 or CD3). Anti-CCR8 antibodies are also provided herein. The multispecific (e.g., bispecific) antigen-binding molecules and antibodies described herein are useful, for example, in the treatment of cancer.

[0119] A. Bispecific antigen-binding molecules In one embodiment, the disclosure provides a bispecific antigen-binding molecule that binds to CCR8 and CD3 (e.g., a bispecific antibody; e.g., a 2+1 TDB; e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., an anti-CCR8 / anti-CD3 bispecific antibody). In one embodiment, the provided bispecific antigen-binding molecule is an isolated bispecific antigen-binding molecule that binds to CD3 and CCR8. In one embodiment, the disclosure provides a bispecific antigen-binding molecule comprising one or more antigen-binding moieties that specifically bind to a Treg cell antigen (e.g., CCR8) and one or more antigen-binding moieties that specifically bind to an activated T cell antigen (e.g., CD3). In one embodiment, the disclosure provides a bispecific antigen-binding molecule comprising, for example, one or more antigen-binding moieties that specifically bind to CCR8 and one or more antigen-binding moieties that specifically bind to CD3. In one embodiment, the disclosure provides a bispecific antigen-binding molecule comprising, for example, two antigen-binding moieties that specifically bind to CCR8 and one antigen-binding domain that specifically binds to CD3. In certain embodiments, CCR8 is human CCR8. In certain embodiments, CD3 is human CD3 or cynomolgus monkey (cyno) CD3.

[0120] In one embodiment, the present invention provides isolated bispecific antigen-binding molecules that bind to CCR8 and CD3. In some embodiments, the antigen-binding domain of the bispecific antigen-binding molecule of the present invention includes at least one, at least two, at least three, at least four, at least five, or all six CDRs exemplified in Table 2 (Kabat) (e.g., including 1, 2, 3, 4, 5, or 6 CDRs). In some cases, the antigen-binding molecule includes VH and / or VL as shown in Table 2. [Table 2]

[0121] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6). (b) a first antigen-binding domain comprising (i) CCR8 1889 S12P(P) and (ii) a second antigen-binding domain that binds to differentiated antigen group 3 (CD3), comprising the following six CDRs: (i) CDR-H1 comprising amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P(P) and the second antigen-binding domain is CD3 40G5c.

[0122] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 WT and the second antigen-binding domain is CD3 40G5c.

[0123] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 41); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 42); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 43). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V(PV) and the second antigen-binding domain is CD3 40G5c.

[0124] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 44); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 45); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 46). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.A32I(PI) and the second antigen-binding domain is CD3 40G5c.

[0125] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 47); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 48); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 49). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.E95dS(PS) and the second antigen-binding domain is CD3 40G5c.

[0126] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 50); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 51); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 52). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I (PVI), and the second antigen-binding domain is CD3 40G5c.

[0127] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 53); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 54); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 55). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.E95dS (PVS), and the second antigen-binding domain is CD3 40G5c.

[0128] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 56); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 57); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 58). (b) a first antigen-binding domain; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 CCR8 1889 S12P.A32I.E95dS(PIS), and the second antigen-binding domain is CD3 40G5c.

[0129] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 59); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 60); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 61). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I.E95dS (PVIS), and the second antigen-binding domain is CD3 40G5c.

[0130] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 62); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTAYATWAKG (SEQ ID NO: 63); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 64); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L2 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). (b) a first antigen-binding domain comprising 3; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 Y58A(A) and the second antigen-binding domain is CD3 40G5c.

[0131] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 65); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRDAYATWAKG (SEQ ID NO: 66); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 67); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L2 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). (b) a first antigen-binding domain comprising 3; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 22). In some embodiments, the first antigen-binding domain is CCR8 1889 T57D.Y58A(DA) and the second antigen-binding domain is CD3 40G5c.

[0132] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six complementarity-determining regions (CDRs): (i) heavy chain complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6). (b) A first antigen-binding domain comprising CDR-L3 containing ) and (b) a second antigen-binding domain that binds to differentiated antigen group 3 (CD3), comprising the following six CDRs: (i) CDR-H1 comprising amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P(P), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0133] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 WT and the second antigen-binding domain is CD3 38E4v1.MD1 (MD1).

[0134] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 41); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 42); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 43). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V (PV), and the second antigen-binding domain is CD3 38E4v1.MD1 (MD1).

[0135] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 44); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 45); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 46). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.A32I (PI), and the second antigen-binding domain is CD3 38E4v1.MD1 (MD1).

[0136] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 47); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 48); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 49). (b) a first antigen-binding domain; and (b) a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.E95dS(PS) and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0137] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 50); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 51); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 52). (b) a first antigen-binding domain; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I(PVI), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0138] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 53); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 54); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 55). (b) a first antigen-binding domain; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.E95dS(PVS), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0139] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 56); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 57); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 58). (b) a first antigen-binding domain; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 CCR8 1889 S12P.A32I.E95dS(PIS), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0140] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 59); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 60); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 61). (b) a first antigen-binding domain; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I.E95dS(PVIS), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0141] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 62); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTAYATWAKG (SEQ ID NO: 63); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 64); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L2 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). (b) a first antigen-binding domain comprising 3; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 Y58A(A) and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0142] In some embodiments, the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 65); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRDAYATWAKG (SEQ ID NO: 66); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 67); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L2 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). (b) a first antigen-binding domain comprising 3; and a second antigen-binding domain that binds to CD3, comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence SYYIH (SEQ ID NO: 91); (ii) CDR-H2 comprising the amino acid sequence WIYPENDNTKYNEKFKD (SEQ ID NO: 92); (iii) CDR-H3 comprising the amino acid sequence DGYSRYYFDY (SEQ ID NO: 93); (iv) CDR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 94); (v) CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 95); and (vi) CDR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 96). In some embodiments, the first antigen-binding domain is CCR8 1889 T57D.Y58A (DA), and the second antigen-binding domain is CD3 38E4v1.MD1 (MD1).

[0143] In some embodiments, the first antigen-binding domain comprises a light chain variable region (VL) domain and a heavy chain variable region (VH) domain, where (a) the VL domain contains a proline residue at position 12 (numbering according to Kabat); and / or (b) the VL domain contains a lysine residue at position 38 and the VH domain contains a glutamate residue at position 39; or the VL domain contains a glutamate residue at position 38 and the VH domain contains a lysine residue at position 39 (numbering according to Kabat). In some embodiments, the second antigen-binding domain comprises a VL domain and a VH domain, where the VL domain contains a glutamate residue at position 38 and the VH domain contains a lysine residue at position 39; or the VL domain contains a lysine residue at position 38 and the VH domain contains a glutamate residue at position 39 (numbering according to Kabat).

[0144] In some embodiments, (a) the first antigen-binding domain comprises one or more of the following eight framework regions (FRs): (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 10 or 113; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 14 or 114; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and / or (viii) the amino acid sequence of SEQ ID NO: 16 (b) The second antigen-binding domain includes FR-L4; and / or (b) the second antigen-binding domain includes one or more of the following eight FRs: (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 25; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 115; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 27; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 28; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 29; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 116; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 31; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 32.

[0145] In some embodiments, (a) the first antigen-binding domain comprises one or more of the following eight framework regions (FRs): (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 14; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and (viii) the amino acid sequence of SEQ ID NO: 16 (b) The FR-L4 contains the sequence of (i) SEQ ID NO: 25; (ii) SEQ ID NO: 26; (iii) SEQ ID NO: 27; (iv) SEQ ID NO: 28; (v) SEQ ID NO: 29; (vi) SEQ ID NO: 30; (vii) SEQ ID NO: 31; and (viii) SEQ ID NO: 32.

[0146] In some embodiments, (a) the first antigen-binding domain comprises one or more of the following eight framework regions (FRs): (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 113; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 114; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and (viii) the amino acid sequence of SEQ ID NO: 16 (b) The FR-L4 contains the sequence of (i) SEQ ID NO: 25; (ii) SEQ ID NO: 115; (iii) SEQ ID NO: 27; (iv) SEQ ID NO: 28; (v) SEQ ID NO: 29; (vi) SEQ ID NO: 116; (vii) SEQ ID NO: 31; and (viii) SEQ ID NO: 32.

[0147] In some embodiments, (a) the first antigen-binding domain comprises one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 10 or 113; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 14 or 114; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 15; and / or (viii) FR containing the amino acid sequence of SEQ ID NO: 16 -L4; and / or (b) the second antigen-binding domain comprises one or more of the following eight FRs: (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 99; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 100 or 115; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 101; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 102; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 103; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 104 or 116; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 105; and / or (viii) FR-L4 comprising the amino acid sequence of SEQ ID NO: 106.

[0148] In some embodiments, (a) the first antigen-binding domain comprises one or more of the following eight framework regions (FRs): (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 14; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and (viii) comprising the amino acid sequence of SEQ ID NO: 16 FR-L4; and (b) the second antigen-binding domain includes one or more of the following eight FRs: (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 99; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 100; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 101; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 102; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 103; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 104; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 105; and (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 106.

[0149] In some embodiments, (a) the first antigen-binding domain comprises one or more of the following eight framework regions (FRs): (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 113; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 114; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and (viii) comprising the amino acid sequence of SEQ ID NO: 16. (b) The second antigen-binding domain includes one or more of the following eight FRs: (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 99; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 115; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 101; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 102; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 103; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 116; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 105; and (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 106.

[0150] In some embodiments, the first antigen-binding domain comprises: (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) a domain comprising the VH domain described in (a) and the VL domain described in (b). ; and / or the second antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P(P) and the second antigen-binding domain is CD3 40G5c.

[0151] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 69; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 WT and the second antigen-binding domain is CD3 40G5c.

[0152] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 70; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 70; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 70; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V(PV) and the second antigen-binding domain is CD3 40G5c.

[0153] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 71; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 71; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 71; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.A32I(PI) and the second antigen-binding domain is CD3 40G5c.

[0154] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 72; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 72; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 72; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.E95dS(PS), and the second antigen-binding domain is CD3 40G5c.

[0155] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 73; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 73; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 73; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I(PVI), and the second antigen-binding domain is CD3 40G5c.

[0156] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 74; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 74; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.E95dS(PVS), and the second antigen-binding domain is CD3 40G5c.

[0157] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 75; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 75; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.A32I.E95dS(PIS), and the second antigen-binding domain is CD3 40G5c.

[0158] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 76; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 76; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I.E95dS(PVIS), and the second antigen-binding domain is CD3 40G5c.

[0159] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 77; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 77; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 77 and a VL domain containing the amino acid sequence of SEQ ID NO: 69; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 Y58A(A) and the second antigen-binding domain is CD3 40G5c.

[0160] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 78 and a VL domain containing the amino acid sequence of SEQ ID NO: 69; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first antigen-binding domain is CCR8 1889 T57D.Y58A(DA) and the second antigen-binding domain is CD3 40G5c.

[0161] In some embodiments, the first antigen-binding domain comprises: (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) a domain comprising the VH domain described in (a) and the VL domain described in (b). ; and / or the second antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P(P) and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0162] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 69; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 WT and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0163] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 70; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 70; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 70; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V(PV), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0164] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 71; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b).

[0165] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 71; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 71; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.A32I(PI), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0166] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 72; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 72; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 72; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.E95dS(PS), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0167] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 73; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 73; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 73; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I(PVI), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 74; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 74; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.E95dS(PVS), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0168] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 75; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 75; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.A32I.E95dS(PIS), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0169] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 76; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 76; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 S12P.I29V.A32I.E95dS(PVIS), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0170] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 77; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 77; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 77 and a VL domain containing the amino acid sequence of SEQ ID NO: 69; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 Y58A(A), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0171] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 98; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 97; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 98; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 78 and a VL domain containing the amino acid sequence of SEQ ID NO: 69; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain is CCR8 1889 T57D.Y58A(DA), and the second antigen-binding domain is CD3 38E4v1.MD1(MD1).

[0172] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 107; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 108; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 109; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 110; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 107; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 108; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 109; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 110; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 107 and a VL domain containing the amino acid sequence of SEQ ID NO: 108; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 109 and a VL domain containing the amino acid sequence of SEQ ID NO: 110.In some embodiments, the first antigen-binding domain is CCR8 1889 S12P(P), the second antigen-binding domain is CD3 40G5c, and the bispecific antigen-binding molecule further includes reverse charge modification, i.e., the charge modification in the antibody is reversed compared to the bispecific antigen-binding molecule containing the amino acid sequence set of SEQ ID NOs. 7, 8, 23, and 24.

[0173] In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 107; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 108; or (c) comprising the VH domain described in (a) and the VL domain described in (b). (a) a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 111; (b) a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 112; or (c) comprising the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 107; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 108; or (c) the VH domain described in (a) and the VL domain described in (b); and / or the second antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 111; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 112; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the first antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 107 and a VL domain containing the amino acid sequence of SEQ ID NO: 108; and the second antigen-binding domain comprises (b) a VH domain containing the amino acid sequence of SEQ ID NO: 111 and a VL domain containing the amino acid sequence of SEQ ID NO: 112.In some embodiments, the first antigen-binding domain is CCR8 1889 S12P(P), the second antigen-binding domain is CD3 38E4v1.MD1(MD1), and the bispecific antigen-binding molecule further includes reverse charge modification; that is, the charge modification in the antibody is reversed compared to anti-CCR8 TDB, which contains the amino acid sequence set of SEQ ID NOs. 7, 8, 97, and 98.

[0174] In some embodiments, the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and / or the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain. In some embodiments, (a) the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain; (b) the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain; or (c) the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain. In some embodiments, the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, wherein (a) the Fab light chain of the first antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the first antigen-binding domain contains a lysine residue at position 183; or the Fab light chain of the first antigen-binding domain contains a lysine residue at position 133 and the Fab heavy chain of the first antigen-binding domain contains a glutamate residue at position 183 (numbering follows Kabat), and / or (b) the Fab light chain of the second antigen-binding domain contains a lysine residue at position 133 and the Fab heavy chain of the second antigen-binding domain contains a glutamate residue at position 183; or the Fab light chain of the second antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the second antigen-binding domain contains a lysine residue at position 183 (numbering follows Kabat).In some embodiments, the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, (a) the Fab light chain of the first antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the first antigen-binding domain contains a lysine residue at position 183; or the Fab light chain of the first antigen-binding domain contains a lysine residue at position 133 and the Fab heavy chain of the first antigen-binding domain contains a glutamate residue at position 183 (numbering follows Kabat); (b) the Fab light chain of the second antigen-binding domain contains a lysine residue at position 133 and the Fab heavy chain of the second antigen-binding domain contains a glutamate residue at position 183; or the Fab light chain of the second antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the second antigen-binding domain The heavy chain contains a lysine residue at position 183 (numbering follows Kabat); or (c) the Fab light chain of the first antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the first antigen-binding domain contains a lysine residue at position 183; or the Fab light chain of the first antigen-binding domain contains a lysine residue at position 133, the Fab heavy chain of the first antigen-binding domain contains a glutamate residue at position 183, the Fab light chain of the second antigen-binding domain contains a lysine residue at position 133, the Fab light chain of the second antigen-binding domain contains a lysine residue at position 133, and the Fab heavy chain of the second antigen-binding domain contains a glutamate residue at position 183; or the Fab light chain of the second antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the second antigen-binding domain contains a lysine residue at position 183 (numbering follows Kabat).

[0175] In some embodiments, the bispecific antigen-binding molecule further comprises an Fc domain containing a first subunit and a second subunit. In some embodiments, the Fc domain is an IgG Fc domain. Any suitable IgG Fc domain, e.g., an IgG1Fc domain, an IgG2Fc domain, an IgG3Fc domain, or an IgG4Fc domain may be used. In some embodiments, the Fc domain is an IgG1Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain includes modifications that facilitate association between the first and second subunits of the Fc domain.

[0176] In some embodiments, the bispecific antigen-binding molecule comprises one or more heavy chain constant domains, the one or more heavy chain constant domains being selected from a first CH1(CH11) domain, a first CH2(CH21) domain, a first CH3(CH31) domain, a second CH1(CH12) domain, a second CH2(CH22) domain, and a second CH3(CH32) domain. In some embodiments, the first subunit comprises one or more heavy chain constant domains selected from a first CH2(CH21) domain and / or a first CH3(CH31) domain; the second subunit comprises one or more heavy chain constant domains selected from a second CH2(CH22) domain and / or a second CH3(CH32) domain. In some embodiments, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some embodiments, the CH31 domain and the CH32 domain each include a projection or cavity, and the projection or cavity of the CH31 domain can be located in the cavity or projection of the CH32 domain, respectively. In some embodiments, the CH31 and CH32 domains meet at the interface between the projection and the cavity. In some embodiments, the CH21 domain and the CH22 domain each include a projection or cavity, and the projection or cavity of the CH21 domain can be located in the cavity or projection of the CH22 domain, respectively. In some embodiments, the CH21 and CH22 domains meet at the interface between the projection and the cavity.

[0177] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and the bispecific antigen-binding molecule comprises an Fc domain containing the first subunit and the second subunit; the first antigen-binding domain is fused to the N-terminus of the first subunit at the C-terminus of the Fab heavy chain, and the second antigen-binding domain is fused to the N-terminus of the second subunit at the C-terminus of the Fab heavy chain.

[0178] In some embodiments, the first subunit contains a tryptophan residue at position 366; the second subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index).

[0179] In some embodiments, each of the first and second subunits includes an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering follows the Kabat EU index).

[0180] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P(P).

[0181] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). In some embodiments, the third antigen-binding domain is CCR8 1889 WT.

[0182] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 41); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 42); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 43). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V(PV).

[0183] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 44); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 45); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 46). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.A32I(PI).

[0184] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 47); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 48); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 49). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.E95dS(PS).

[0185] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 50); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 51); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 52). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V.A32I(PVI).

[0186] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 53); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 54); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 55). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V.E95dS (PVS).

[0187] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 56); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 57); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 58). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.A32I.E95dS(PIS).

[0188] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 59); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 60); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 61). In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V.A32I.E95dS (PVIS).

[0189] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 62); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTAYATWAKG (SEQ ID NO: 63); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 64); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). In some embodiments, the third antigen-binding domain is CCR8 1889 Y58A(A).

[0190] In some embodiments, the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8. In some embodiments, the third antigen-binding domain comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 65); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRDAYATWAKG (SEQ ID NO: 66); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 67); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). In some embodiments, the third antigen-binding domain is CCR8 1889 T57D.Y58A(DA).

[0191] In some embodiments, the third antigen-binding domain comprises a VL domain and a VH domain, where (a) the VL domain contains a proline residue at position 12 (numbering according to Kabat); and / or (b) the VL domain contains a lysine residue at position 38 and the VH domain contains a glutamate residue at position 39; or the VL domain contains a glutamate residue at position 38 and the VH domain contains a lysine residue at position 39 (numbering according to Kabat). In some embodiments, the third antigen-binding domain comprises a VL domain and a VH domain, where (a) the VL domain contains a proline residue at position 12 (numbering according to Kabat); (b) the VL domain contains a lysine residue at position 38 and the VH domain contains a glutamate residue at position 39; or the VL domain contains a glutamate residue at position 38 and the VH domain contains a lysine residue at position 39 (numbering according to Kabat); or (c) the VL domain contains a proline residue at position 12 (numbering according to Kabat), the VL domain contains a lysine residue at position 38 and the VH domain contains a glutamate residue at position 39; or the VL domain contains a proline residue at position 12 (numbering according to Kabat), the VL domain contains a glutamate residue at position 38 and the VH domain contains a lysine residue at position 39 (numbering according to Kabat).

[0192] In some embodiments, the third antigen-binding domain comprises one or more of the following eight FRs: (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 10 or 113; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 14 or 114; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and / or (viii) FR-L4 comprising the amino acid sequence of SEQ ID NO: 16.

[0193] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P(P).

[0194] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, the third antigen-binding domain is CCR8 1889 WT.

[0195] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 70; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 70; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 70. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V(PV).

[0196] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 71; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 71; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.A32I(PI).

[0197] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 72; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 72; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 72. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.E95dS(PS).

[0198] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 73; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 73; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V.A32I(PVI).

[0199] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 74; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V.E95dS(PVS).

[0200] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 75; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 75. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.A32I.E95dS(PIS).

[0201] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 76; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 76. In some embodiments, the third antigen-binding domain is CCR8 1889 S12P.I29V.A32I.E95dS(PVIS).

[0202] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 77; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 77; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 77 and a VL domain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, the third antigen-binding domain is CCR8 1889 Y58A(A).

[0203] In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the third antigen-binding domain includes a VH domain containing the amino acid sequence of SEQ ID NO: 78 and a VL domain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, the third antigen-binding domain is CCR8 1889 T57D.Y58A(DA).

[0204] In some embodiments, the third antigen-binding domain is a Fab molecule. In some embodiments, the third antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, wherein the Fab light chain of the third antigen-binding domain contains a glutamate residue at position 133 and the Fab heavy chain of the third antigen-binding domain contains a lysine residue at position 183; or the Fab light chain of the third antigen-binding domain contains a lysine residue at position 133 and the Fab heavy chain of the third antigen-binding domain contains a glutamate residue at position 183 (numbering follows Kabat).

[0205] In some embodiments, the second antigen-binding domain and the third antigen-binding domain are fused to each other. In some embodiments, the second antigen-binding domain and the third antigen-binding domain are fused to each other via a peptide linker. In some embodiments, the peptide linker contains the amino acid sequence of SEQ ID NO: 37. In some embodiments, the second antigen-binding domain and the third antigen-binding domain are each Fab molecules, and the third antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain.

[0206] In some embodiments, the bispecific antigen-binding molecule comprises an Fc domain containing a first subunit and a second subunit; the first antigen-binding domain, the second antigen-binding domain and the third antigen-binding domain are each Fab molecules; the first antigen-binding domain is fused to the N-terminus of the first subunit at the C-terminus of the Fab heavy chain; the second antigen-binding domain is fused to the N-terminus of the second subunit at the C-terminus of the Fab heavy chain; and the third antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain. In some embodiments, the bispecific antigen-binding molecule is an anti-CCR8 TDB in a 2+1 A / AB format (A: CCR8-binding domain, B: CD3-binding domain).

[0207] In some embodiments, the multispecific antigen-binding molecule described herein comprises a first Fab molecule (Fab) that specifically binds to CCR8, each containing Q39E (Kabat numbering) and S183K (EU numbering) substitutions in the heavy chain and Q38K (Kabat numbering) and V133E (EU numbering) substitutions in the light chain. A ) and the third Fab molecule (Fab B2 ); and a second Fab molecule that specifically binds to CD3, including the substitution of Q39K (Kabat numbering) and S183E (EU numbering) in the heavy chain and the substitution of Q38E (Kabat numbering) and V133K (EU numbering) in the light chain. B1 ) includes.

[0208] In some embodiments, the multispecific antigen-binding molecule described herein comprises a first Fab molecule (Fab) that specifically binds to CCR8, each containing Q39K (Kabat numbering) and S183E (EU numbering) substitutions in the heavy chain and Q38E (Kabat numbering) and V133K (EU numbering) substitutions in the light chain. A ) and the third Fab molecule (Fab B2 ); and a second Fab molecule that specifically binds to CD3, including the substitution of Q39E (Kabat numbering) and S183K (EU numbering) in the heavy chain and the substitution of Q38K (Kabat numbering) and V133E (EU numbering) in the light chain. B1 ) includes.

[0209] In some embodiments, the bispecific antigen-binding molecule includes a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 33; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 35; and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 33, a first polypeptide and a second polypeptide each containing the amino acid sequence of SEQ ID NO: 34, a polypeptide containing the amino acid sequence of SEQ ID NO: 35, and a polypeptide containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, (i) the polypeptide containing the amino acid sequence of SEQ ID NO: 33 is linked to the first polypeptide containing the amino acid sequence of SEQ ID NO: 34 via interactions between the Fab heavy chain and the Fab light chain; (ii) the polypeptide containing the amino acid sequence of SEQ ID NO: 35 is linked to the second polypeptide containing the amino acid sequence of SEQ ID NO: 34 via interactions between the Fab heavy chain and the Fab light chain; (iii) the polypeptide containing the amino acid sequence of SEQ ID NO: 35 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 36 via interactions between the Fab heavy chain and the Fab light chain; and (iv) the polypeptide containing the amino acid sequence of SEQ ID NO: 33 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 35 via first and second subunits of the Fc domain. In some embodiments, the bispecific antigen-binding molecule is a 2+1 A / AB format (A: 1889 P, B: 40G5c) 1889 / 1889:40G5c anti-CCR8 TDB.

[0210] In some embodiments, the bispecific antigen-binding molecule includes a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 117; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 118; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 119; and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 117, a first polypeptide and a second polypeptide each containing the amino acid sequence of SEQ ID NO: 118, a polypeptide containing the amino acid sequence of SEQ ID NO: 119, and a polypeptide containing the amino acid sequence of SEQ ID NO: 120. In some embodiments, (i) the polypeptide containing the amino acid sequence of SEQ ID NO: 117 is linked to the first polypeptide containing the amino acid sequence of SEQ ID NO: 118 via interactions between Fab heavy chains and Fab light chains; (ii) the polypeptide containing the amino acid sequence of SEQ ID NO: 119 is linked to the second polypeptide containing the amino acid sequence of SEQ ID NO: 118 via interactions between Fab heavy chains and Fab light chains; (iii) the polypeptide containing the amino acid sequence of SEQ ID NO: 119 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 120 via interactions between Fab heavy chains and Fab light chains; and (iv) the polypeptide containing the amino acid sequence of SEQ ID NO: 117 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 119 via first and second subunits of the Fc domain.In some embodiments, the bispecific antigen-binding molecule is a 2+1 A / AB format (A: 1889 P, B: 40G5c) 1889 / 1889:40G5c anti-CCR8 TDB, and the bispecific antigen-binding molecule further includes reverse charge modification, i.e., the charge modification of the antibody is reversed compared to the anti-CCR8 TDB containing the set of amino acid sequences of SEQ ID NOs. 33-36.

[0211] In some embodiments, the bispecific antigen-binding molecule includes a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 33; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 90; and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 33, a first polypeptide and a second polypeptide each containing the amino acid sequence of SEQ ID NO: 34, a polypeptide containing the amino acid sequence of SEQ ID NO: 90, and a polypeptide containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, (i) the polypeptide containing the amino acid sequence of SEQ ID NO: 33 is linked to the first polypeptide containing the amino acid sequence of SEQ ID NO: 34 via interactions between Fab heavy chains and Fab light chains; (ii) the polypeptide containing the amino acid sequence of SEQ ID NO: 90 is linked to the second polypeptide containing the amino acid sequence of SEQ ID NO: 34 via interactions between Fab heavy chains and Fab light chains; (iii) the polypeptide containing the amino acid sequence of SEQ ID NO: 90 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 36 via interactions between Fab heavy chains and Fab light chains; and (iv) the polypeptide containing the amino acid sequence of SEQ ID NO: 33 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 90 via first and second subunits of the Fc domain. In some embodiments, the bispecific antigen-binding molecule is a 2+1 A / AB format (A: 1889 P, B: MD1) 1889 / 1889: MD1 anti-CCR8 TDB.

[0212] In some embodiments, the bispecific antigen-binding molecule includes a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 117; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 118; a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 121; and a polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 117, a first polypeptide and a second polypeptide each containing the amino acid sequence of SEQ ID NO: 118, a polypeptide containing the amino acid sequence of SEQ ID NO: 121, and a polypeptide containing the amino acid sequence of SEQ ID NO: 120. In some embodiments, (i) the polypeptide containing the amino acid sequence of SEQ ID NO: 117 is linked to the first polypeptide containing the amino acid sequence of SEQ ID NO: 118 via interactions between Fab heavy chains and Fab light chains; (ii) the polypeptide containing the amino acid sequence of SEQ ID NO: 121 is linked to the second polypeptide containing the amino acid sequence of SEQ ID NO: 118 via interactions between Fab heavy chains and Fab light chains; (iii) the polypeptide containing the amino acid sequence of SEQ ID NO: 121 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 120 via interactions between Fab heavy chains and Fab light chains; and (iv) the polypeptide containing the amino acid sequence of SEQ ID NO: 117 is linked to the polypeptide containing the amino acid sequence of SEQ ID NO: 121 via first and second subunits of the Fc domain.In some embodiments, the bispecific antigen-binding molecule is a 2+1 A / AB format (A: 1889P, B: MD1) 1889 / 1889:MD1 anti-CCR8 TDB, and the bispecific antigen-binding molecule further comprises an opposite charge modification, i.e., the charge modification of the antibody is opposite compared to the anti-CCR8 TDB comprising the set of amino acid sequences of SEQ ID NO: 33, 34, 36, and 90.

[0213] The bispecific antigen-binding molecules described herein may include any one or combination of the properties further described in Section C below.

[0214] B. Anti-CCR8 Antibodies In other embodiments, anti-CCR8 antibodies are provided herein. Any of the anti-CCR8 antibodies disclosed herein can be used in a multispecific (e.g., bispecific) antigen-binding molecule as described herein.

[0215] In some embodiments, the invention provides an isolated anti-CCR8 antibody. In some embodiments, the anti-CCR8 antibody of the invention comprises at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 CDRs exemplified in Table 2 (Kabat) (e.g., comprises 1, 2, 3, 4, 5, or 6 CDRs). In some cases, the anti-CCR8 antibody comprises VH and / or VL as shown in Table 2.

[0216] In some embodiments, the present invention provides an anti-CCR8 antibody comprising the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRX1X2YATWAKG (SEQ ID NO: 82), wherein X1 is T or D and X2 is Y or A; (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENX3ANX4LA (SEQ ID NO: 83), wherein X3 is I or V and X4 is A or I; (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVX5GT (SEQ ID NO: 84), wherein X5 is E or S. In some examples, the anti-CCR8 antibody comprises a VL domain that does not contain a serine residue at position 12 (Kabat numbering). For example, in some examples, the anti-CCR8 antibody does not comprise a VL domain having the amino acid sequence of SEQ ID NO: 69.

[0217] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6). In some embodiments, the anti-CCR8 antibody is CCR8 188,9 S12P (P).

[0218] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). In some embodiments, the anti-CCR8 antibody is CCR8 1889 WT.

[0219] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 41); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 42); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 43). In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V(PV).

[0220] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 44); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 45); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 46). In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.A32I(PI).

[0221] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 47); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 48); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 49). In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.E95dS(PS).

[0222] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 50); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 51); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 52). In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V.A32I (PVI).

[0223] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANALA (SEQ ID NO: 53); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 54); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 55). In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V.E95dS (PVS).

[0224] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENIANILA (SEQ ID NO: 56); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 57); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 58). In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.A32I.E95dS(PIS).

[0225] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 comprising the amino acid sequence QASENVANILA (SEQ ID NO: 59); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 60); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVSGT (SEQ ID NO: 61). In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V.A32I.E95dS (PVIS).

[0226] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 62); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRTAYATWAKG (SEQ ID NO: 63); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 64); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). In some embodiments, the anti-CCR8 antibody is CCR8 1889 Y58A(A).

[0227] In some embodiments, the anti-CCR8 antibody comprises the following six CDRs: (i) CDR-H1 comprising the amino acid sequence TYAMG (SEQ ID NO: 65); (ii) CDR-H2 comprising the amino acid sequence LIHRSGRDAYATWAKG (SEQ ID NO: 66); (iii) CDR-H3 comprising the amino acid sequence SYPDYSATASI (SEQ ID NO: 67); (iv) CDR-L1 comprising the amino acid sequence QASENIANALA (SEQ ID NO: 38); (v) CDR-L2 comprising the amino acid sequence GASNLAS (SEQ ID NO: 39); and (vi) CDR-L3 comprising the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 40). In some embodiments, the anti-CCR8 antibody is CCR8 1889 T57D.Y58A(DA).

[0228] In some embodiments, the anti-CCR8 antibody comprises one or more of the following eight FRs: (i) FR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) FR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 comprising the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 comprising the amino acid sequence of SEQ ID NO: 14; (vii) FR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and / or (viii) FR-L4 comprising the amino acid sequence of SEQ ID NO: 16.

[0229] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P(P).

[0230] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-CCR8 antibody is CCR8 1889 WT.

[0231] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 70; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 70; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V(PV).

[0232] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 71; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 71; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.A32I(PI).

[0233] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 72; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 72; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.E95dS(PS).

[0234] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 73; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 73; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V.A32I(PVI).

[0235] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 74; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 74; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V.E95dS(PVS).

[0236] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 75; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 75; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.A32I.E95dS(PIS).

[0237] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 68; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 76; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 68 and a VL domain containing the amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-CCR8 antibody is CCR8 1889 S12P.I29V.A32I.E95dS(PVIS).

[0238] In some embodiments, the anti-CCR8 antibody comprises a VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77; a VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 77; (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR VIII antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VL domain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-CCR8 antibody is CCR8 1889 Y58A (A).

[0239] In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 78; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 69; or (c) the VH domain described in (a) and the VL domain described in (b). In some embodiments, the anti-CCR8 antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 78 and a VL domain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-CCR8 antibody is CCR8 1889 T57D.Y58A(DA).

[0240] The antibodies described herein may include any one or a combination of the properties further described in Section C below.

[0241] C. Characteristics of multispecific antigen-binding molecules and / or antibodies 1. Antibody fragment In certain embodiments, the multispecific antigen-binding molecules (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) or antibodies (e.g., anti-CCR8 antibodies) provided herein are antibody fragments. In certain embodiments, the multispecific (e.g., bispecific) antigen-binding molecules provided herein include antibody fragments.

[0242] Any suitable antibody fragment may be used. In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each containing the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, in addition to the variable domains of the heavy and light chains (VH and VL, respectively). Thus, the term "Fab fragment" refers to an antibody fragment containing a light chain with the VL and CL domains, and a heavy chain fragment containing the VH and CH1 domains, while a "Fab' fragment" differs from a Fab fragment by the addition of one or more cysteine-containing residues at the carboxyl terminus of the CH1 domain from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domain retain a free thiol group. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of the Fab and F(ab')2 fragments that constitute salvage receptor-binding epitope residues and increase in vivo half-life, see U.S. Patent No. 5,869,046.

[0243] In another embodiment, the antibody fragment is a bispecific, triplicate, or quadruplicate antibody. A diabody is an antibody fragment having two antigen-binding sites that may be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0244] In a further embodiment, the antibody fragment is a single-chain Fab fragment. The "single-chain Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab fragments will be further stabilized by the formation of interchain disulfide bonds through the insertion of cysteine ​​residues (for example, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0245] In another embodiment, the antibody fragment is a single-stranded variable fragment (scFv). A "single-stranded variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. In particular, the linker is a short polypeptide of typically 10-25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also International Publication No. 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0246] In another embodiment, the antibody fragment is a single-domain antibody. A "single-domain antibody" is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In a particular embodiment, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; e.g., U.S. Patent No. 6,248,516B1).

[0247] Antibody fragments can be prepared by various techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., Escherichia coli), as described herein.

[0248] 2. Chimeric and humanized antigen-binding molecules In certain embodiments, the multispecific antigen-binding molecules provided herein (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) are chimeric multispecific antigen-binding molecules or multispecific antibodies. In certain embodiments, the multispecific (e.g., bispecific) antigen-binding molecules or antibodies provided herein (e.g., anti-CCR8 antibodies) include chimeric antigen-binding molecules or antibodies.

[0249] Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody contains a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from those of the parent antibody. A chimeric antibody contains its antigen-binding fragment.

[0250] In certain embodiments, a chimeric antigen-binding molecule or antibody is a humanized antigen-binding molecule or antibody. Typically, a non-human antigen-binding molecule or antibody is humanized to retain the specificity and affinity of the parent non-human antibody while reducing its immunogenicity to humans. Generally, a humanized antigen-binding molecule or antibody contains one or more variable domains, where the CDR (or a portion thereof) is derived from a non-human antigen-binding molecule or antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antigen-binding molecule or antibody optionally also contains at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antigen-binding molecule or antibody are replaced with corresponding residues from a non-human antigen-binding molecule or antibody (e.g., an antibody from which the CDR residue is derived) to restore or improve antibody specificity or affinity, for example.

[0251] Humanized antigen-binding molecules and antibodies, as well as methods for producing them, are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods. This is described in 36:25-34 (2005) (graft junction of the specificity determination region (SDR)); Padlan, Mol.Immunol.28:489-498 (1991) ("Resurfacing" is described); Dall'Acqua et al., Methods 36:43-60 (2005) ("FR shuffling" is described); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) ("Guided selection" method for FR shuffling is described).

[0252] Human framework regions that may be used for humanization include, but are not limited to, the following: framework regions selected using the “best fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. See al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0253] 3. Human antigen-binding molecules In certain embodiments, the multispecific antigen-binding molecules (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) or antibodies (e.g., anti-CCR8 antibodies) provided herein are human multispecific antigen-binding molecules (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) or antibodies (e.g., anti-CCR8 antibodies). Human antigen-binding molecules or antibodies can be produced using various techniques known in the art. Human antigen-binding molecules or antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0254] Human antigen-binding molecules or antibodies may be prepared by administering an immunogen to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or that is located extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antigen-binding molecules or antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VelociMouse® technology. Human variable regions derived from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0255] Human antigen-binding molecules or antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human xenomyeloma cell lines for producing human monoclonal antigen-binding molecules or antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antigen-binding molecules or antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0256] Human antigen-binding molecules or antibodies can also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0257] 4. Multispecific antigen binding molecules In certain embodiments, multispecific antigen-binding molecules (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) or antibodies (e.g., anti-CCR8 antibodies) are disclosed herein. “Multispecific antigen-binding molecules” and “multispecific antibodies” are, respectively, monoclonal antigen-binding molecules or antibodies having binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, the multispecific antigen-binding molecule or antibody has three or more binding specificities. In certain embodiments, one binding specificity is for CCR8, and another specificity is for any other antigen. In certain embodiments, the bispecific antigen-binding molecule or antibody may bind to two (or more) different epitopes of CCR8. Multispecific (e.g., bispecific) antigen-binding molecules or antibodies may also be used as cytotoxic agents or to localize cells to express CCR8. Multispecific antigen-binding molecules or antibodies may be prepared as full-length antigen-binding molecules or antibodies or antibody fragments. In certain embodiments, the other antigen is an activated T cell antigen. In some embodiments, the activated T cell antigen is CD3. In some embodiments, CD3 is human or cyno-CD3.

[0258] Techniques for producing multispecific antigen-binding molecules or antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" operations (see, for example, U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies also involve manipulating the electrostatic steering effect to produce antibody Fc heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992) and International Publication No. 2011 / 034605); using common light chain techniques to avoid light chain mispairing problems (see, e.g., International Publication No. 98 / 50431); and using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al.) They may also be prepared by using single-stranded Fv(sFv) dimers (see, for example, Gruber et al., J.Immunol., 152:5368 (1994)); and by preparing trispecific antibodies as described, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0259] 5. Antibody variants In certain embodiments, amino acid sequence variants of multispecific antigen-binding molecules or antibodies provided herein are intended. For example, it may be desirable to alter the binding affinity and / or other biological properties of a multispecific antigen-binding molecule or antibody (e.g., an anti-CCR8 antibody). Amino acid sequence variants of multispecific antigen-binding molecules or antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antigen-binding molecule or antibody, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Deletions, insertions, and substitutions can be arbitrarily combined to arrive at the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0260] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and FRs.

[0261] In one embodiment, the VL sequence of the antigen-binding molecule or antibody disclosed herein includes the V4M mutation, P43A mutation, F46L mutation, C90Q mutation, or a combination thereof. In one embodiment, the VH sequence of the antibody disclosed herein includes the G49S mutation, K71R mutation, S73N mutation, or a combination thereof. In one embodiment, the VL sequence of the antigen-binding molecule or antibody disclosed herein includes the Y2I mutation. In one embodiment, the VH sequence of the antigen-binding molecule or antibody disclosed herein includes the S73N mutation, V78L mutation, T76N mutation, F91Y mutation, and P105Q mutation, or a combination thereof (e.g., numbered according to Kabat).

[0262] Conservative substitutions are shown in Table 3 under the heading "Conservative Substitutions." More substantial substitutions are provided in Table 3 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antigen-binding molecule or antibody of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 3]

[0263] Amino acids can be classified according to their general side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0264] Non-conservative substitution involves exchanging a member of one class with a member of another class.

[0265] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antigen-binding molecule or antibody (e.g., a humanized or human antigen-binding molecule or antibody). Generally, selected for further study, the resulting variant will have alterations (e.g., improvements) to specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antigen-binding molecule or antibody, and / or will substantially retain the specific biological properties of the parent antigen-binding molecule or antibody. Exemplary substitution variants are affinity-matured antigen-binding molecules or antibodies, which can be readily generated using affinity maturation techniques based on phage display, for example, as described herein. Briefly, one or more CDR residues are mutated, the variant antigen-binding molecule or antibody is displayed on a phage, and screened for specific biological activity (e.g., binding affinity).

[0266] For example, modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and then re-selecting from it is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity is the CDR-directed approach, which randomizes several CDR residues (e.g., 4-6 residues at a time). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0267] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within a CDR. Such alterations may, for example, be outside the antigen-contact residue in the CDR. In the specific variant VH and VL sequences described above, each CDR is either unaltered or has one, two, or three or fewer amino acid substitutions.

[0268] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at the positions of amino acids that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues may be targeted as candidate substitutions or removed. Variants may be screened to determine whether they contain the desired properties.

[0269] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme (e.g., ADEPT (for antibody-directed enzyme prodrug therapy)) or polypeptide, which increases the serum half-life of the antibody.

[0270] b) Glycosylated variants In certain embodiments, the multispecific antigen-binding molecules (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) or antibodies (e.g., anti-CCR8 antibodies) provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to multispecific antigen-binding molecules or antibodies can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0271] If a multispecific antigen-binding molecule or antibody contains an Fc region, the oligosaccharide attached to the antibody may be modified. Native antigen-binding molecules or antibodies produced by mammalian cells typically contain branched or bifurcated oligosaccharides that are commonly bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of a bifurcated oligosaccharide structure. In some embodiments, the modification of oligosaccharides in antibodies described herein may be carried out to produce antibody variants having specific improved properties.

[0272] In one embodiment, an antibody variant is provided having an oligosaccharide structure lacking a non-fucosylated oligosaccharide, i.e., fucose binding (direct or indirect) to the Fc region. Such a non-fucosylated oligosaccharide (also called a “defucosylated” oligosaccharide) is in particular an N-linked oligosaccharide lacking a fucose residue to which a first GlcNAc is bound at the stem of a branched oligosaccharide structure, and such an antibody is further referred to herein as a “defucosylated antibody.” In one embodiment, an antibody variant is provided in which the proportion of non-fucosylated oligosaccharides in the Fc region is increased compared to the natural antibody or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). In certain embodiments, the defucosylation rate is approximately 65% ​​to 100%, 80% to 100%, or 80% to 95%. The rate of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides bound to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Fc region residue in EU numbering), although Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300 (e.g., Asn299), due to slight sequence variations in the antibody. Antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publications 2003 / 0157108 and 2004 / 0093621.

[0273] In one embodiment, the disclosure provides a defucosylated antibody variant having enhanced FcγRIIIa receptor binding. In one embodiment, the disclosure provides a defucosylated antibody variant having enhanced antibody-dependent cytotoxicity (ADCC). In one embodiment, the disclosure provides a defucosylated antibody variant having antibody-dependent phagocytic activity (ADCP).

[0274] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11), and knockout cell lines, e.g., alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al.) This includes cells in which GDP-fucose synthesis or transport protein is reduced or absent (see, for example, U.S. Patent Publications 2004259150, 2005031613, 2004132140, and 2004110282). See also Pereira et al., MABS (2018) 693-711.

[0275] In a further embodiment, antibody variants having bifid oligosaccharides are provided, for example, antibody variants in which a bifid oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); International Publication No. 99 / 54342, International Publication No. 2004 / 065540, and International Publication No. 2003 / 011878.

[0276] Antibody variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publications 1997 / 30087; 1998 / 58964; and 1999 / 22764.

[0277] c) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of a multispecific antigen-binding molecule (e.g., a multispecific antibody; e.g., a bispecific antigen-binding molecule (e.g., a bispecific antibody; 2+1 TDB; e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., an anti-CCR8 / anti-CD3 bispecific antibody; e.g., an anti-CCR8 / anti-CD3 TDB)) or antibody (e.g., an anti-CCR8 antibody) provided herein, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing one or more amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0278] In certain embodiments, the present invention conceives antigen-binding molecules or antibody variants that, by possessing some, but not all, effector functions, are desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (e.g., complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that an antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); and U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the desired ADCC activity can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Furthermore, a C1q binding assay may be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol. 18(12):1759-1769 (2006); International Publication No. 2013 / 120929).

[0279] Antibodies with reduced effector function include those having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include those having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0280] This document describes specific antigen-binding molecules or antibody variants that exhibit improved or reduced binding to FcR. (See, for example, U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0281] In certain embodiments, the antigen-binding molecule or antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0282] In certain embodiments, the antigen-binding molecule or antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., Fc region positions 234 and 235 (residue EU numbering). In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antigen-binding molecule or antibody variant further includes D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2012 / 130831). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region.

[0283] In certain embodiments, the Fc region includes modifications configured to facilitate association of a first Fc subunit with a second Fc subunit. A “knob-in-hole” operation of a multispecific antigen-binding molecule or antibody may be used to generate a first arm containing a knob and a second arm containing a hole to which the knob of the first arm binds. In one embodiment, the knob of the multispecific antibody of the present invention may be a monovalent arm (e.g., an anti-CCR8 arm). Alternatively, the knob of the multispecific antigen-binding molecule or antibody of the present invention may be a bivalent arm. In one embodiment, the hole of the multispecific antigen-binding molecule or antibody of the present invention may be a monovalent arm. Alternatively, the hole of the multispecific antigen-binding molecule or antibody of the present invention may be a bivalent arm. The multispecific antigen-binding molecules or antibodies may also be manipulated using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) techniques (see, for example, WO2009 / 080253;Schaefer et al., Proc.Natl.Acad.Sci.USA,108:11187-11192(2011)). Multispecific antigen-binding molecules or antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (WO2009 / 089004A1), by crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); or by using a leucine zipper to produce bispecific antibodies (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)).

[0284] By replacing amino acid residues in the CH3 domain of the second Fc subunit with amino acid residues having a larger side-chain volume, a projection (e.g., a knob) can be generated within the CH3 domain of the second Fc subunit that can be located in a cavity (e.g., a hole) within the CH3 domain of the first Fc subunit, and by replacing amino acid residues in the CH3 domain of the first Fc subunit with amino acid residues having a smaller side-chain volume, a cavity (e.g., a hole) can be generated within the CH3 domain of the first Fc subunit that can be located in a projection (e.g., a knob) within the CH3 domain of the second Fc subunit. In some embodiments, the CH3 domain of the second Fc subunit includes an amino acid substitution at T366, and the CH3 domain of the first Fc subunit includes amino acid substitutions at one, two, or all three of T366, L368, and / or Y407. In some embodiments, the CH3 domain of the second Fc subunit contains the amino acid substitution T366W, and the CH3 domain of the first Fc subunit contains one, two, or all three amino acid substitutions T366S, L368A, and / or Y407V.

[0285] In certain embodiments, a multispecific antigen-binding molecule (e.g., a multispecific antibody; e.g., a bispecific antigen-binding molecule (e.g., a bispecific antibody; 2+1 TDB; e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., an anti-CCR8 / anti-CD3 bispecific antibody; e.g., an anti-CCR8 / anti-CD3 TDB)) or an antibody (e.g., an anti-CCR8 antibody) includes an Fc region having one or more amino acid substitutions, e.g., repositional substitutions, that improve FcγR binding (thereby improving effector function). In certain embodiments, the antibody variant includes an Fc region having at least one amino acid substitution of G236A, I332E, S298A, E333A, K334A, S239D, A330L, F243L, R292P, Y300L, V305I, P396L, L235V, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, or K334E (see, for example, Liu et al., Antibodies (Basel) (2020); 9(4):64).

[0286] In some embodiments, modifications are made in the Fc region that result in alterations (i.e., either improvements or reductions) to C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as disclosed, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0287] Antibodies responsible for the transfer of maternal IgG to the fetus, with extended half-lives and improved binding affinity to the neonatal Fc receptor (FcRn), are described in U.S. Patent Publication No. 2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Examples of such Fc variants include those with one or more of the following Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, those with a substitution at fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; dall'acqua, wf, et al. j. biol.chem. 281 (2006) 23514-23524).

[0288] The fc region residues crucial to the mouse-fc-mouse-fcrn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues i253, h310, h433, n434, and h435 (EU numbering of the residues) are involved in the interaction (Medesan, C., et al., EUR. J. Immunol. 26 (1996) 2533; Firan, M., et al., INT. Immunol. 13 (2001) 993; Kim, JK, et al., EUR. J. Immunol. 24 (1994) 542). Residues i253, h310, and h435 have been found to be important for the interaction between human fc and mouse fcrn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human fc-human fcrn complex have shown that residues i253, s254, h435, and y436 are important for this interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, Rl, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J.Immunol.182(2009)7667-7671) reported and investigated various mutants at residues 248-259, 301-317, 376-382, and 424-437.

[0289] In certain embodiments, a multispecific antigen-binding molecule (e.g., a multispecific antibody; e.g., a bispecific antigen-binding molecule (e.g., a bispecific antibody; 2+1 TDB; e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., an anti-CCR8 / anti-CD3 bispecific antibody; e.g., an anti-CCR8 / anti-CD3 TDB)) or antibody (e.g., an anti-CCR8 antibody) includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253 and / or 310 and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antigen-binding molecule or antibody includes an Fc region having amino acid substitutions at positions 253, 310 and 435. In one embodiment, the substitutions are I253A, H310A and H435A within the Fc region derived from the human IgG1 Fc region. For example, see Grevys, A., et al., J.Immunol. 194(2015) 5497-5508.

[0290] In certain embodiments, a multispecific antigen-binding molecule (e.g., a multispecific antibody; e.g., a bispecific antigen-binding molecule (e.g., a bispecific antibody; 2+1 TDB; e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., an anti-CCR8 / anti-CD3 bispecific antibody; e.g., an anti-CCR8 / anti-CD3 TDB)) or antibody (e.g., an anti-CCR8 antibody) includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310 and / or 433 and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antigen-binding molecule or antibody includes an Fc region having amino acid substitutions at positions 310, 433 and 436. In one embodiment, the substitutions are H310A, H433A and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2014 / 177460).

[0291] In certain embodiments, a multispecific antigen-binding molecule (e.g., a multispecific antibody; e.g., a bispecific antigen-binding molecule (e.g., a bispecific antibody; 2+1 TDB; e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., an anti-CCR8 / anti-CD3 bispecific antibody; e.g., an anti-CCR8 / anti-CD3 TDB)) or antibody (e.g., an anti-CCR8 antibody) includes an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252 and / or 254 and / or 256 (EU numbering of residues) of the Fc region. In certain embodiments, the antigen-binding molecule or antibody includes an Fc region having amino acid substitutions at positions 252, 254 and 256. In one embodiment, the substitutions are M252Y, S254T and T256E within the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260, 5,624,821, and International Publication No. 94 / 29351.

[0292] The C-terminus of the heavy chain of a multispecific antigen-binding molecule (e.g., multispecific antibody; e.g., bispecific antigen-binding molecule (e.g., bispecific antibody; 2+1 TDB; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., anti-CCR8 / anti-CD3 bispecific antibody; e.g., anti-CCR8 / anti-CD3 TDB)) or antibody (e.g., anti-CCR8 antibody) described herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus from which one or two of the C-terminal amino acid residues have been removed. In one embodiment, the C-terminus of the heavy chain is PG ending with a shortened C-terminus. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain containing a C-terminal CH3 domain as specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one embodiment of all the embodiments reported herein, a multispecific antigen-binding molecule (e.g., a multispecific antibody; e.g., a bispecific antigen-binding molecule (e.g., a bispecific antibody; 2+1 TDB; e.g., an anti-CCR8 / anti-CD3 bispecific antigen-binding molecule; e.g., an anti-CCR8 / anti-CD3 bispecific antibody; e.g., an anti-CCR8 / anti-CD3 TDB)) or antibody (e.g., an anti-CCR8 antibody) comprising a heavy chain containing a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, EU index numbering of the amino acid position). In one embodiment of all the embodiments reported herein, an antibody comprising a heavy chain containing a C-terminal CH3 domain as specified herein comprises a C-terminal proline residue (P445, EU index numbering of the amino acid position).

[0293] d) Cysteine-modified antibody variant In certain embodiments, a cysteine-modified antibody is used in which one or more residues of the antibody are replaced with cysteine ​​residues, such as THIOMAB. (商標)It may be desirable to produce such antibodies. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site on the antibody, which can be used to produce an immunoconjugate by conjugating the antibody to other parts, such as a drug moiety or a linker-drug moiety, as further described herein. Cysteine-manipulated antibodies can be produced, for example, as described in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or International Publication No. 2016040856.

[0294] e) Antibody derivative In certain embodiments, the multispecific antigen-binding molecules (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) or antibodies (e.g., anti-CCR8 antibodies) provided herein may be further modified to include additional non-proteinoid moieties known and readily available in the art. Moieties suitable for the derivatization of antigen-binding molecules or antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, the polymers may be the same molecule or different molecules. Generally, the number and / or types of polymers used for derivatization can be determined based on considerations such as the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used therapeutically under defined conditions, but this is not limited to these factors.

[0295] D. Recombination methods and compositions Multispecific antigen-binding molecules (e.g., multispecific antibodies; e.g., bispecific antigen-binding molecules (e.g., bispecific antibodies; 2+1 TDBs; e.g., anti-CCR8 / anti-CD3 bispecific antigen-binding molecules; e.g., anti-CCR8 / anti-CD3 bispecific antibodies; e.g., anti-CCR8 / anti-CD3 TDBs)) or antibodies (e.g., anti-CCR8 antibodies) can be produced using recombinant methods and compositions such as those described in U.S. Patent No. 4,816,567 and U.S. Patent Application Publication No. 2013 / 0078249, which are incorporated herein in their entirety by reference, respectively. In one embodiment, an isolated nucleic acid (e.g., polynucleotide) encoding a bispecific antigen-binding molecule or antibody described herein is provided. In one embodiment, an isolated nucleic acid (e.g., polynucleotide) encoding a bispecific antigen-binding molecule described herein is provided. Such nucleic acids may encode an amino acid sequence containing VL and / or VH of a bispecific antigen-binding molecule or antibody (e.g., the light chain and / or heavy chain of either arm of the bispecific antigen-binding molecule). In further embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided.

[0296] In one aspect, the present invention provides an isolated polynucleotide or set of isolated polynucleotides comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of the nucleic acid sequences of SEQ ID NOs. 85 to 89. In one aspect, the present invention provides an isolated polynucleotide or set of isolated polynucleotides comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleic acid sequence of SEQ ID NOs. 85. In one aspect, the present invention provides an isolated polynucleotide or set of isolated polynucleotides comprising a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleic acid sequence of SEQ ID NOs. 86. In one aspect, the present invention provides an isolated polynucleotide or set...

Claims

1. (a) A first antigen-binding domain that binds to C-C motif chemokine receptor 8 (CCR8), comprising the following six complementarity-determining regions (CDRs): (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) A first antigen-binding domain comprising CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) A second antigen-binding domain that binds to differentiated antigen group 3 (CD3), comprising the following six CDRs: (i) CDR-H1 containing the amino acid sequence NYYIH (SEQ ID NO: 17); (ii) CDR-H2 containing the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 18); (iii) CDR-H3 containing the amino acid sequence DSYSNYYFDY (SEQ ID NO: 19); (iv) CDR-L1 containing the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 20); (v) CDR-L2 containing the amino acid sequence WASTRES (SEQ ID NO: 21); and (vi) A second antigen-binding domain containing CDR-L3 with the amino acid sequence TQSFILRT (SEQ ID NO: 22) A bispecific antigen-binding molecule containing this molecule.

2. The first antigen-binding domain comprises a light chain variable region (VL) domain and a heavy chain variable region (VH) domain, (a) The VL domain contains a proline residue at position 12 (numbering follows Kabat); and / or (b) The VL domain contains a lysine residue at position 38, and the VH domain contains a glutamic acid residue at position 39 (numbering follows Kabat), The bispecific antigen-binding molecule according to claim 1.

3. The bispecific antigen-binding molecule according to claim 1 or 2, wherein the second antigen-binding domain comprises a VL domain and a VH domain, the VL domain comprising a glutamic acid residue at position 38, and the VH domain comprising a lysine residue at position 39 (numbering follows Kabat).

4. (a) The first antigen-binding domain is one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 10; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 14; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 15; and / or (viiii) FR-L4 containing the amino acid sequence of SEQ ID NO: 16 Includes; and / or (b) The second antigen-binding domain is one or more of the following eight FRs: (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 25; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 26; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 27; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 28; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 29; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 30; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 31; and / or (viiii) FR-L4 containing the amino acid sequence of SEQ ID NO: 32 A bispecific antigen-binding molecule according to any one of claims 1 to 3, comprising:

5. The first antigen-binding domain described above is (a) A VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) A VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) comprising the VH domain described in (a) and the VL domain described in (b); and / or The second antigen-binding domain described above is (a) A VH domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23; (b) A VL domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24; or (c) The VH domain described in (a) and the VL domain described in (b). A bispecific antigen-binding molecule according to any one of claims 1 to 4, comprising:

6. The first antigen-binding domain described above is (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) comprising the VH domain described in (a) and the VL domain described in (b); and / or The second antigen-binding domain described above is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 23; (b) A VL domain containing the amino acid sequence of SEQ ID NO: 24; or (c) The VH domain described in (a) and the VL domain described in (b). A bispecific antigen-binding molecule according to any one of claims 1 to 5, comprising:

7. The first antigen-binding domain described above is (a) comprising a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8; and The second antigen-binding domain described above is (b) A VH domain containing the amino acid sequence of SEQ ID NO: 23 and a VL domain containing the amino acid sequence of SEQ ID NO: 24 A bispecific antigen-binding molecule according to claim 6, comprising:

8. A bispecific antigen-binding molecule according to any one of claims 1 to 7, wherein the first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and / or the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain.

9. The first antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, and the second antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain. (a) The Fab light chain of the first antigen-binding domain contains a glutamic acid residue at position 133, and the Fab heavy chain of the first antigen-binding domain contains a lysine residue at position 183 (numbering follows Kabat); and / or (b) The Fab light chain of the second antigen-binding domain contains a lysine residue at position 133, and the Fab heavy chain of the second antigen-binding domain contains a glutamic acid residue at position 183 (numbering follows Kabat), A bispecific antigen-binding molecule according to any one of claims 1 to 8.

10. A bispecific antigen-binding molecule according to any one of claims 1 to 9, further comprising an Fc domain containing a first subunit and a second subunit.

11. The bispecific antigen-binding molecule according to claim 10, wherein the Fc domain is an IgG Fc domain.

12. The aforementioned Fc domain is IgG １ The bispecific antigen-binding molecule according to claim 11, wherein the Fc domain is present.

13. The bispecific antigen-binding molecule according to any one of claims 10 to 12, wherein the Fc domain is a human IgG Fc domain.

14. The bispecific antigen-binding molecule according to any one of claims 10 to 13, comprising a modification in which the Fc domain promotes association between the first subunit and the second subunit of the Fc domain.

15. The bispecific antigen-binding molecule comprises one or more heavy chain constant domains, and the one or more heavy chain constant domains are first CH1(CH1 １ ) Domain, the first CH2 (CH2 １ ) Domain, the first CH3 (CH3 １ ) Domain, second CH1 (CH1 ２ ) Domain, second CH2 (CH2 ２ ) domain, and second CH3 (CH3 ２ A bispecific antigen-binding molecule according to any one of claims 1 to 14, selected from the domains.

16. The first sub-unit comprises one or more heavy chain constant domains selected from a first CH2(CH2 １ ) domain and / or a first CH3(CH3 １ ) domain; the second sub-unit comprises one or more heavy chain constant domains selected from a second CH2(CH2 ２ ) domain and / or a second CH3(CH3 ２ ) domain, the bispecific antigen-binding molecule according to any one of claims 10 to 14.

17. The bispecific antigen-binding molecule according to claim 16, wherein at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain.

18. The aforementioned CH3 １ Domain and the CH3 ２ Each domain includes a protrusion or cavity, and the CH3 １ The protrusion or cavity of the domain is the CH3 ２ The bispecific antigen-binding molecule according to claim 17, which can be positioned in the cavities or protrusions of the domain, respectively.

19. The aforementioned CH3 １ Domain and the CH3 ２ The bispecific antigen-binding molecule according to claim 18, wherein the domains associate at the interface between the protrusion and the cavity.

20. The aforementioned CH2 １ Domain and the CH2 ２ Each domain includes a projection or cavity, and the CH2 １ The protrusion or cavity of the domain is the CH2 ２ A bispecific antigen-binding molecule according to any one of claims 16 to 19, which can be positioned in the cavities or protrusions of the domain, respectively.

21. The aforementioned CH2 １ Domain and the CH2 ２ The bispecific antigen-binding molecule according to claim 20, wherein the domains associate at the interface between the protrusion and the cavity.

22. The bispecific antigen-binding molecule according to any one of claims 8 to 21, wherein the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and the bispecific antigen-binding molecule comprises an Fc domain including a first subunit and a second subunit; the first antigen-binding domain is fused to the N-terminus of the first subunit at the C-terminus of the Fab heavy chain, and the second antigen-binding domain is fused to the N-terminus of the second subunit at the C-terminus of the Fab heavy chain.

23. The bispecific antigen-binding molecule according to claim 22, wherein the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index).

24. A bispecific antigen-binding molecule according to any one of claims 10 to 23, wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).

25. The bispecific antigen-binding molecule according to any one of claims 1 to 24, wherein the bispecific antigen-binding molecule further comprises a third antigen-binding domain that binds to CCR8.

26. The third antigen-binding domain is one of the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6) A bispecific antigen-binding molecule, as specified in claim 25.

27. The third antigen-binding domain comprises a VL domain and a VH domain, (a) The VL domain contains a proline residue at position 12 (numbering follows Kabat); and / or (b) The VL domain contains a lysine residue at position 38, and the VH domain contains a glutamic acid residue at position 39 (numbering follows Kabat), The bispecific antigen-binding molecule according to claim 25 or 26.

28. The third antigen-binding domain is one or more of the following eight FRs: (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 9; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 10; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 11; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 12; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 13; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 14; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 15; and / or (viiii) FR-L4 containing the amino acid sequence of SEQ ID NO: 16 A bispecific antigen-binding molecule according to any one of claims 25 to 27, comprising:

29. A bispecific antigen-binding molecule according to any one of claims 25 to 28, wherein the third antigen-binding domain comprises (a) a VH domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b).

30. The bispecific antigen-binding molecule according to any one of claims 25 to 29, wherein the third antigen-binding domain comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 7; (b) a VL domain containing the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b).

31. The bispecific antigen-binding molecule according to claim 30, wherein the third antigen-binding domain comprises a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO:

8.

32. The bispecific antigen-binding molecule according to any one of claims 25 to 31, wherein the third antigen-binding domain is a Fab molecule.

33. The bispecific antigen-binding molecule according to any one of claims 25 to 32, wherein the third antigen-binding domain is a Fab molecule comprising a Fab light chain and a Fab heavy chain, the Fab light chain of the third antigen-binding domain comprising a glutamic acid residue at position 133, and the Fab heavy chain of the third antigen-binding domain comprising a lysine residue at position 183 (numbering follows Kabat).

34. A bispecific antigen-binding molecule according to any one of claims 25 to 33, wherein the second antigen-binding domain and the third antigen-binding domain are fused to each other.

35. The bispecific antigen-binding molecule according to claim 34, wherein the second antigen-binding domain and the third antigen-binding domain are fused to each other via a peptide linker.

36. The bispecific antigen-binding molecule according to claim 35, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:

37.

37. The bispecific antigen-binding molecule according to any one of claims 34 to 36, wherein the second antigen-binding domain and the third antigen-binding domain are each Fab molecules, and the third antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain.

38. The bispecific antigen-binding molecule according to any one of claims 25 to 37, wherein the bispecific antigen-binding molecule comprises an Fc domain including a first subunit and a second subunit; the first antigen-binding domain, the second antigen-binding domain and the third antigen-binding domain are each Fab molecules; the first antigen-binding domain is fused to the N-terminus of the first subunit at the C-terminus of the Fab heavy chain; the second antigen-binding domain is fused to the N-terminus of the second subunit at the C-terminus of the Fab heavy chain; and the third antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain.

39. A bispecific antigen-binding molecule according to any one of claims 1 to 38, comprising a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 33; a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34; a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 35; and a polypeptide containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO:

36.

40. The bispecific antigen-binding molecule according to claim 39, comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 33, a first polypeptide and a second polypeptide each containing the amino acid sequence of SEQ ID NO: 34, a polypeptide containing the amino acid sequence of SEQ ID NO: 35, and a polypeptide containing the amino acid sequence of SEQ ID NO:

36.

41. (i) The polypeptide comprising the amino acid sequence of SEQ ID NO: 33 is linked to the first polypeptide comprising the amino acid sequence of SEQ ID NO: 34 via the interaction of the Fab heavy chain and the Fab light chain; (ii) The polypeptide comprising the amino acid sequence of SEQ ID NO: 35 is linked to the second polypeptide comprising the amino acid sequence of SEQ ID NO: 34 via the interaction of the Fab heavy chain and the Fab light chain; (iii) The polypeptide comprising the amino acid sequence of SEQ ID NO: 35 is linked to the polypeptide comprising the amino acid sequence of SEQ ID NO: 36 via the interaction of the Fab heavy chain and the Fab light chain; and (iv) The polypeptide comprising the amino acid sequence of SEQ ID NO: 33 is linked to the polypeptide comprising the amino acid sequence of SEQ ID NO: 35 via a first subunit and a second subunit of the Fc domain. The bispecific antigen-binding molecule according to claim 40.

42. An isolated polynucleotide or set of isolated polynucleotides encoding a bispecific antigen-binding molecule according to any one of claims 1 to 41.

43. A vector or set of vectors comprising an isolated polynucleotide or a set of isolated polynucleotides as described in claim 42.

44. (i) a host cell or a set of host cells comprising an isolated polynucleotide or a set of isolated polynucleotides as described in claim 42, or a vector or a set of vectors as described in claim 43.

45. A method for producing a bispecific antigen-binding molecule that binds to CCR8 and CD3, comprising the step of culturing a host cell or set of host cells as described in claim 44 under conditions suitable for the expression of the bispecific antigen-binding molecule.

46. The method according to claim 45, further comprising recovering the bispecific antigen-binding molecule.

47. A bispecific antigen-binding molecule that binds to CCR8 and CD3, produced by the method described in claim 45 or 46.

48. A pharmaceutical composition comprising a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47, and a pharmaceutically acceptable carrier.

49. A bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47, or a pharmaceutical composition according to claim 48, for use as a pharmaceutical.

50. Use of a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or a pharmaceutical composition according to claim 48 in the manufacture of a pharmaceutical.

51. A bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47, or a pharmaceutical composition according to claim 48, for use in the treatment of cancer.

52. Use of a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 for the treatment of cancer in subjects requiring cancer treatment.

53. Use of a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 for treating cancer in a subject requiring cancer treatment.

54. A method for treating cancer in a subject, comprising administering to the subject an effective amount of a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or a pharmaceutical composition according to claim 48.

55. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, use, or method according to any one of claims 51 to 54, wherein the cancer is selected from the group consisting of bladder cancer, blastoma, hematological cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer.

56. Use of a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 to deplete regulatory T cells.

57. A method for depleting regulatory T cells in the tumor microenvironment of a subject having cancer, comprising administering to the subject a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 in an effective amount sufficient to deplete the regulatory T cells in the tumor microenvironment.

58. A method for depleting regulatory T cells outside the tumor microenvironment in a subject having cancer, comprising administering to the subject a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 in an effective amount sufficient to deplete the regulatory T cells outside the tumor microenvironment.

59. The use or method according to claim 56 or 57, wherein the regulatory T cells present in the tumor microenvironment of cancer are depleted.

60. The use or method according to claim 56 or 58, wherein the regulatory T cells outside the tumor microenvironment of cancer are depleted.

61. An in vitro method for depleting regulatory T cells from a population of cancer cells, comprising contacting the cell population with a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 in an amount sufficient to deplete the regulatory T cells from the cell population.

62. Use of a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 to reduce CCR8 mRNA expression.

63. A method for reducing CCR8 mRNA expression in the blood of a subject, comprising administering to the subject a bispecific antigen-binding molecule according to any one of claims 1 to 41 and 47 or the pharmaceutical composition according to claim 48 in an effective amount sufficient to reduce CCR8 mRNA expression in the blood.

64. A bispecific antigen-binding molecule for use, a pharmaceutical composition for use, a method of use, or a method of use according to any one of claims 51 to 63, further comprising administering an additional therapeutic agent to a target.

65. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method of use according to claim 64, wherein the additional therapeutic agent is an anticancer agent.

66. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method of use according to claim 65, wherein the anticancer agent is selected from the group consisting of microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapies, kinase inhibitors, receptor antagonists, tumor cell apoptosis activators, anti-angiogenic agents, immunomodulators, cell adhesion inhibitors, cytotoxic or cell proliferation inhibitors, cell apoptosis activators, agents that increase the sensitivity of cells to apoptosis-inducing substances, cytokines, anticancer vaccines or oncolytic viruses, Toll-like receptor (TLR) agents, bispecific antibodies, cell therapies, and immune cell engagers.

67. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, use or method according to claim 65 or 66, wherein the anticancer agent is a PD-L1-binding antagonist.

68. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method of use according to claim 67, wherein the PD-L1-binding antagonist is atezolizumab.

69. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method for use according to claim 64, wherein the additional therapeutic agent is tocilizumab or a corticosteroid.

70. The use of a bispecific antigen-binding molecule or a pharmaceutical composition containing the bispecific antigen-binding molecule for depleting regulatory T cells, wherein the bispecific antigen-binding molecule is (a) A first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) A first antigen-binding domain comprising CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) Use comprising a second antigen-binding domain that binds to an activated T cell antigen.

71. A method for depleting regulatory T cells in the tumor microenvironment of a subject with cancer, comprising administering to the subject a sufficient amount to deplete the regulatory T cells in the tumor microenvironment, wherein the bispecific antigen-binding molecule is (a) A first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) A first antigen-binding domain comprising CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) A method comprising a second antigen-binding domain that binds to an activated T cell antigen.

72. A method for depleting regulatory T cells outside the tumor microenvironment in a subject with cancer, comprising administering to the subject a sufficient amount to deplete the regulatory T cells outside the tumor microenvironment, wherein the bispecific antigen-binding molecule is (a) A first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) A first antigen-binding domain comprising CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) A method comprising a second antigen-binding domain that binds to an activated T cell antigen.

73. The use or method according to claim 70 or 71, wherein the regulatory T cells present in the tumor microenvironment of cancer are depleted.

74. The use or method according to claim 70 or 72, wherein the regulatory T cells outside the tumor microenvironment of cancer are depleted.

75. An in vitro method for depleting regulatory T cells from a population of cancer cells, comprising contacting the cell population with a bispecific antigen-binding molecule or a pharmaceutical composition containing the bispecific antigen-binding molecule in an amount sufficient to deplete the regulatory T cells from the cell population, wherein the bispecific antigen-binding molecule (a) A first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) A first antigen-binding domain comprising CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) An in vitro method comprising a second antigen-binding domain that binds to an activated T cell antigen.

76. The use of a bispecific antigen-binding molecule or a pharmaceutical composition containing the bispecific antigen-binding molecule for reducing CCR8 mRNA expression, wherein the bispecific antigen-binding molecule is (a) A first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) A first antigen-binding domain comprising CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) Use comprising a second antigen-binding domain that binds to an activated T cell antigen.

77. A method for reducing CCR8 mRNA expression in the blood of a subject, comprising administering to the subject a sufficient amount of a bispecific antigen-binding molecule or a pharmaceutical composition containing the bispecific antigen-binding molecule to reduce CCR8 mRNA expression in the blood, wherein the bispecific antigen-binding molecule is (a) A first antigen-binding domain that binds to CCR8, comprising the following six CDRs: (i) CDR-H1 containing the amino acid sequence TYAMG (SEQ ID NO: 1); (ii) CDR-H2 containing the amino acid sequence LIHRSGRTYYATWAKG (SEQ ID NO: 2); (iii) CDR-H3 containing the amino acid sequence SYPDYSATASI (SEQ ID NO: 3); (iv) CDR-L1 containing the amino acid sequence QASENIANALA (SEQ ID NO: 4); (v) CDR-L2 containing the amino acid sequence GASNLAS (SEQ ID NO: 5); and (vi) A first antigen-binding domain comprising CDR-L3 containing the amino acid sequence QQAYYGNSFVEGT (SEQ ID NO: 6); and (b) A method comprising a second antigen-binding domain that binds to an activated T cell antigen.

78. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, use or method according to any one of claims 70 to 77, wherein the activated T cell antigen is CD3.

79. A bispecific antigen-binding molecule for use, a pharmaceutical composition for use, a method of use, or a method of use according to any one of claims 70 to 78, further comprising administering an additional therapeutic agent to a target.

80. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method of use according to claim 79, wherein the additional therapeutic agent is an anticancer agent.

81. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method of use according to claim 80, wherein the anticancer agent is selected from the group consisting of microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapies, kinase inhibitors, receptor antagonists, tumor cell apoptosis activators, anti-angiogenic agents, immunomodulators, cell adhesion inhibitors, cytotoxic or cell proliferation inhibitors, cell apoptosis activators, agents that increase the sensitivity of cells to apoptosis-inducing substances, cytokines, anticancer vaccines or oncolytic viruses, TLR agents, bispecific antibodies, cell therapies, and immune cell engagers.

82. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, use or method according to claim 80 or 81, wherein the anticancer agent is a PD-L1-binding antagonist.

83. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method for use according to claim 82, wherein the PD-L1-binding antagonist is atezolizumab.

84. The bispecific antigen-binding molecule for use, pharmaceutical composition for use, or method of use according to claim 79, wherein the additional therapeutic agent is tocilizumab or a corticosteroid.

85. An isolated polynucleotide or set of isolated polynucleotides containing a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of the nucleic acid sequences of sequence numbers 85-89.

86. An isolated polynucleotide or set of isolated polynucleotides containing one nucleic acid sequence from sequence numbers 85 to 89.

87. A set of isolated polynucleotides, comprising an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO: 85, an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO: 86, an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO: 87, and an isolated polynucleotide containing the nucleic acid sequence of SEQ ID NO:

88.

88. A vector or set of vectors comprising an isolated polynucleotide or a set of isolated polynucleotides according to any one of claims 85 to 87.

89. (i) a host cell or a set of host cells comprising an isolated polynucleotide or a set of isolated polynucleotides according to any one of claims 85 to 87, or a vector or a set of vectors according to claim 88.

90. A method for producing a bispecific antigen-binding molecule that binds to CCR8 and CD3, comprising the step of culturing a host cell or set of host cells as described in claim 89 under conditions suitable for the expression of the bispecific antigen-binding molecule.

91. The method according to claim 90, further comprising recovering the bispecific antigen-binding molecule.

92. A bispecific antigen-binding molecule that binds to CCR8 and CD3, produced by the method described in claim 91.

93. The use or method according to any one of claims 52 to 54, 57, 58, 71, and 72, wherein the subject has reduced CCR8 mRNA expression.

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